Antifungal activity of the botanical compound rhein against Phytophthora capsici and the underlying mechanisms.

BACKGROUND: Phytophthora capsici is an extremely destructive phytopathogenic oomycete that causes huge economic losses. However, due to the drug resistance risk and environmental threat of chemical fungicides, it is necessary to develop environmentally friendly biocontrol alternatives. Rhein is a major medicinal ingredient of traditional Chinese herbs, and it is widely used in the medical field. However, its inhibitory effect against phytopathogens is unknown. Herein, the antifungal spectrum of rhein and its possible action mechanism against P. capsici were investigated. RESULTS: Rhein possessed broad-spectrum antifungal activity against phytopathogens, particularly P. capsici, Phytophthora infestans, Helminthosporium maydis, and Rhizoctonia solani. Rhein inhibited the mycelial growth as well as the spore germination of P. capsici with mean 50% effective concentration (EC50 ) values of 4.68 µg mL-1 and 6.57 µg mL-1 against 117 P. capsici isolates, respectively. Rhein effectively suppressed the occurrence and spread of Phytophthora blight and significantly destroyed the cell membrane permeability and integrity of P. capsici, corroded its cell wall integrity, and damaged its morphology and ultrastructure. Moreover, rhein caused a considerable reduction in the phospholipid and cellulose contents. Genome-wide transcriptional profiling of P. capsici in response to rhein indicated significant reduction in the expression levels of genes participating in glycerolipid metabolism and starch and sucrose metabolism. Additionally, rhein strengthened the disease defense system of pepper by enhancing related enzyme activities. CONCLUSION: This study demonstrated that rhein could effectively inhibit P. capsici using multiple mechanisms of action. Rhein has the potential to be an efficient alternative to control diseases caused by P. capsici. © 2023 Society of Chemical Industry.

Two lathyrane diterpenoid stereoisomers containing an unusual trans-gem-dimethylcyclopropane from the seeds of Euphorbia lathyris.

Two novel lathyrane-type diterpenoids, the Euphorbia factors L2a (1) and L2b (2), and their stereoisomer Euphorbia factor L2 (3) were obtained from seeds of Euphorbia lathyris. Both Euphorbia factors L2a and L2b possess an unprecedented trans-gem-dimethylcyclopropane as structural feature. Also, the Euphorbia factor L2a is the first example of a lathyrane diterpenoid with an endocyclic 12(Z)-double bond. The structures of the molecules and their absolute configurations were elucidated by comprehensive spectroscopic analyses, Cu-Kα radiation X-ray diffraction, and comparison with calculated electronic circular dichroism (ECD) data. The Euphorbia factor L2b exhibited an inhibitory effect against U937 cell line with an IC50 value of 0.87 µM.

Inhibitory Effects of the Natural Product Esculetin on Phytophthora capsici and Its Possible Mechanism.

Esculetin is an important plant-derived natural product that has multiple bioactivities and applications. Phytophthora capsici is a notorious plant pathogen capable of infecting a broad range of hosts. In this study, we evaluated the antifungal activity of esculetin against P. capsici. The baseline sensitivity of P. capsici to esculetin was established using 108 isolates collected from various geographical regions in the Jiangsu and Shandong Provinces of China. The median effective concentration (EC50) values for esculetin ranged from 2.08 to 16.46 µg/ml (mean, 6.87 ± 2.70 µg/ml) and were normally distributed. Furthermore, both zoospore production and germination were strongly inhibited by esculetin. Importantly, esculetin exhibited protective as well as curative activities against P. capsici on tomato and was capable of restricting the early infection of P. capsici on Nicotiana benthamiana. We found that the esculetin treatment led to cell membrane damage of P. capsici, as revealed by morphological observations and measurements of relative conductivity and malondialdehyde (MDA). Finally, our results also suggested that esculetin may adversely affect P. capsici by inhibiting its DNA and protein synthesis. These findings will contribute to the broader evaluation of the use of esculetin to control diseases caused by P. capsici and toward a better understanding of its mode of action as a potential fungicide.

Effects of camptothecin on the rice blast fungus Magnaporthe oryzae.

Rice blast caused by Magnaporthe oryzae B. Couch is one of the most devastating diseases on rice. Camptothecin (CPT), which was primarily isolated from Camptotheca acuminata, is well-known for its anti-tumor activities, and is also developed as a potential biological pesticide. We previously investigated the anti-microbial activities of CPT against 11 fungi, 3 oomycetes, and 4 bacteria, and found that CPT was strongly effective against M. oryzae, indicating its potential as a lead for developing fungicide against rice blast. However, the anti-fungal effects of CPT on M. oryzae need further elucidation. In this study, the anti-fungal activities of CPT against M. oryzae were further investigated, which revealed that CPT was effective against M. oryzae both in vitro and in vivo. The transcriptome of M. oryzae was analyzed after CPT treatment, which showed that CPT had a strong inhibitory effect on 'translation' and 'carbohydrate metabolism/energy metabolism' of M. oryzae. Some physiology characteristics of M. oryzae were also assayed, which confirmed that CPT inhibited RNA synthesis, protein synthesis, and carbohydrate metabolism/energy metabolism of M. oryzae, and caused membrane damage. The molecular simulation result showed that CPT binds to the interface of DNA-topoisomerase I complex of M. oryzae. In conclusion, CPT is a promising lead for developing fungicide against rice blast. CPT may bind to DNA-topoisomerase I complex of M. oryzae, thus affecting 'translation' and 'carbohydrate metabolism/energy metabolism', leading to cell death.

Antifungal activity of zedoary turmeric oil against Phytophthora capsici through damaging cell membrane.

Phytophthora capsici is a plant oomycete pathogen, which causes many devastating diseases on a broad range of hosts. Zedoary turmeric oil (ZTO) is a kind of natural plant essential oil that has been widely used in pharmaceutical applications. However, the antifungal activity of ZTO against phytopathogens remains unknown. In this study, we found ZTO could inhibit P. capsici growth and development in vitro and in detached cucumber and Nicotiana benthamiana leaves. Besides, ZTO treatment resulted in severe damage to the cell membrane of P. capsici, leading to the leakage of intracellular contents. ZTO also induced a significant increase in relative conductivity, malondialdehyde concentration and glycerol content. Furthermore, we identified 50 volatile organic compounds from ZTO, and uncovered Curcumol, ß-elemene, curdione and curcumenol with strong inhibitory activities against mycelial growth of P. capsici. Overall, our results not only shed new light on the antifungal mechanism of ZTO, but also imply a promising alternative for the control of phytophthora blight caused by P. capsici.

Teratogenic jervine increases the activity of doxorubicin in MCF-7/ADR cells by inhibiting ABCB1.

Jervine is a natural teratogenic compound isolated from Veratrum californicum. In this study, for the first time, we revealed a novel activity of jervine in sensitizing the anti-proliferation effect of doxorubicin (DOX). We demonstrated that the synergistic mechanism was related to the intracellular accumulation of DOX via modulating ABCB1 transportation. Jervine did not affect the expression of ABCB1 in mRNA nor protein levels. However, jervine increased the ATPase activity of ABCB1 and possibly served as a substrate of ABCB1. The molecular docking results indicated that jervine was bound to a closed ABCB1 conformation and blocked drug entrance to the central binding site at the transmembrane domain. The present study identifies jervine acts as a substrate of ABCB1, and has potential to be developed as a novel and potent chemotherapy sensitizer used for patients developing multidrug resistance.

Fungicidal activity of 10-deacetylbacatin III against Phytophthora capsici via inhibiting lysine biosynthesis.

10-deacetyl-bacatin III (10-DAB) is a natural plant-derived taxane diterpene, whose antimicrobial activity against phytopathogens remains unknown. In this study, we demonstrated the antimicrobial effect of 10-DAB on plant-pathogenic oomycetes. Our results revealed that 10-DAB exhibited significant antimicrobial activities against test oomycetes, especially against Phytophthora capsici, with a median effective concentration (EC50) of 1.46 µg/mL, but had no effect on test fungi. Under 10-DAB treatment, mycelia of P. capsici were contorted with an increased number of top branches, and the production and germination of zoospores were inhibited and delayed, respectively. In addition, 10-DAB had favorable protective and curative activities with control efficacies of 63.90% and 74.81% at 200 µg/mL on detached pepper leaves. Furthermore, 10-DAB caused a significant decrease in soluble protein, lysine, and α, ε-diaminopimelic acid content of P. capsici, which suggested that 10-DAB inhibited the lysine biosynthesis. On the contrary, treatment with exogenous lysine effectively counteracted 10-DAB's inhibition activity on P. capsici. Moreover, relative expression of four key lysine biosynthesis-related genes of P. capsici were decreased upon 10-DAB treatment. Taken together, our findings suggest a lysine biosynthesis inhibiting-dependent antimicrobial activity of 10-DAB against P. capsici, which contributes to accelerating the application of 10-DAB for successful management of phytophthora blight disease in agricultural production.

Crucial role of oxidative stress in bactericidal effect of parthenolide against Xanthomonas oryzae pv. oryzae.

BACKGROUND: Xanthomonas oryzae pv. oryzae (Xoo) causes rice bacterial blight, which is one of the most devastating diseases on rice. Parthenolide (PTL) is a sesquiterpene lactone possessing multiple bioactivities. In the preliminary study, we found PTL can totally inhibit the growth of Xoo at 10 mg L-1 in vitro. In this study, we aim to further evaluate the anti-bacterial activity of PTL against Xoo and discern the role of oxidative stress in its bactericidal effect. RESULTS: PTL was effective against Xoo both in vitro and in vivo. PTL induced reactive oxygen species (ROS) accumulation in Xoo, leading to cell death, while exogenous catalase can fully abolish its bactericidal effect. PTL sensitivity of catalase deletion mutants of Xoo increased significantly compared with that of wild-type Xoo strain. In addition, PTL treatment increased glutathione peroxidase activity and decreased glutathione (GSH) reductase activity in Xoo, but had no effect on its catalase and superoxide dismutase activities. Interestingly, PTL dramatically reduced the GSH level in Xoo, resulting in disturbed GSH/GSSG balance. Moreover, PTL rapidly reacted with GSH by a nucleophilic addition reaction. CONCLUSION: PTL is a promising lead compound for developing bactericide against Xoo. PTL rapidly reacts with GSH, resulting in disturbed GSH/GSSG balance in Xoo, which causes ROS accumulation, leading to cell death. Oxidative stress plays a critical role in the bactericidal effect of PTL against Xoo. © 2018 Society of Chemical Industry.

Synthesis and Cytotoxicity against K562 Cells of 3-O-Angeloyl-20-O-acetyl Ingenol, a Derivative of Ingenol Mebutate.

Ingenol mebutate possesses significant cytotoxicity and is clinically used to treat actinic keratosis. However, ingenol mebutate undergoes acyl migration which affects its bioactivity. Compound 3-O-angeloyl-20-O-acetyl ingenol (AAI, also known as 20-O-acetyl-ingenol-3-angelate or PEP008) is a synthetic derivative of ingenol mebutate. In this work, we report the AAI synthesis details and demonstrate AAI has higher cytotoxicity than ingenol mebutate in a chronic myeloid leukemia K562 cell line. Our data indicate that the increased activity of AAI originates from the improved intracellular stability of AAI rather than the increased binding affinity between AAI and the target protein protein kinase Cδ (PKCδ). AAI inhibits cell proliferation, induces G2/M phase arrest, disrupts the mitochondrial membrane potential, and stimulates apoptosis, as well as necrosis in K562 cells. Similar to ingenol mebutate, AAI activates PKCδ and extracellular signal regulated kinase (ERK), and inactivates protein kinase B (AKT). Furthermore, AAI also inhibits JAK/STAT3 pathway. Altogether, our studies show that ingenol derivative AAI is cytotoxic to K562 cells and modulates PKCδ/ERK, JAK/STAT3, and AKT signaling pathways. Our work suggests that AAI may be a new candidate of chemotherapeutic agent.

Macranthoside B Induces Apoptosis and Autophagy Via Reactive Oxygen Species Accumulation in Human Ovarian Cancer A2780 Cells.

Macranthoside B (MB), a saponin compound in Lonicera macranthoides, can block cell proliferation and induce cell death in several types of cancer cells; however, the precise mechanisms by which MB exerts its anticancer effects remain poorly understood. MB blocked A2780 human ovarian carcinoma cell proliferation both dose- and time-dependently. MB induced apoptosis, with increased poly (ADP-ribose) polymerase (PARP) and caspase-3/9 cleavage. MB also caused autophagy in A2780 cells, with light chain 3 (LC3)-II elevation. Inhibiting MB-induced autophagy with the autophagy inhibitor 3-methyladenine (3-MA) significantly decreased apoptosis, with a reduction of growth inhibition; inhibiting MB-induced apoptosis with the pan-caspase inhibitor Z-VAD-FMK did not decrease autophagy but elevated LC3-II levels, indicating that MB-induced autophagy is cytotoxic and may be upstream of apoptosis. Furthermore, MB increased intracellular reactive oxygen species (ROS) levels, with activated 5' adenosine monophosphate-activated protein kinase (AMPK), decreased mammalian target of rapamycin (mTOR) and P70S6 kinase phosphorylation, and increased PARP and caspase-3/9 cleavage, and LC3-II elevation; treatment with the ROS scavenger N-acetyl cysteine and the AMPK inhibitor Compound C diminished this effect. Therefore, the ROS/AMPK/mTOR pathway mediates the effect of MB on induction of apoptosis via autophagy in human ovarian carcinoma cells.

13-Oxyingenol dodecanoate, a cytotoxic ingenol derivative, induces mitochondrial apoptosis and caspase-dependent Akt decrease in K562 cells.

13-Oxyingenol dodecanoate (13OD) is an ingenol derivative prepared from Chinese traditional medicine Euphorbia kansui without any report about its bioactivity. The present study demonstrated for the first time that 13OD displayed potent cytotoxicity against chronic myeloid leukemia K562 cells in vitro. 13OD inhibited proliferation, induced G2/M phase arrest, and exhibited potent apoptotic activity in K562 cells. In K562 cells, 13OD disrupted the mitochondrial membrane potential and induced high level of ROS, which played an indispensable role in 13OD-induced apoptosis. Further investigations on the molecular mechanisms revealed that total Akt protein level was decreased in a caspase-dependent way after treatment with 13OD; in addition, ERK was activated by 13OD, and this activation played a protective role in 13OD stimulation. Altogether, these results revealed that the cytotoxic ingenol derivative 13OD induced apoptosis with novel mechanisms for the proapoptotic function in cancer cells, and suggested that 13OD may serve as a lead template for rational drug design and for future anticancer agent development.

Isocyclopamine, a novel synthetic derivative of cyclopamine, reverts doxorubicin resistance in MCF-7/ADR cells by increasing intracellular doxorubicin accumulation and downregulating breast cancer stem-like cells.

Cyclopamine (CPM) showed promise as a human cancer chemotherapy agent. However, limitations such as stomach acid instability and low solubility impair its clinical application. In this study, we synthesized a novel CPM analogue, isocyclopamine (ICPM), which had comparative bioactivity with CPM and improved stability and solubility. ICPM reversed doxorubicin resistance and had potent synergy with doxorubicin in MCF-7/ADR cells. We further demonstrated that the synergistic mechanism was related to the increased intracellular accumulation of doxorubicin in the cells and the downregulation of the cancer stem-like cells via modulation on both ABCB1 and ABCG2 transporters with independence of Smoothened. The present study identified ICPM as a novel derivative of CPM with better stability and solubility, which provided a useful tool for the biological and medicinal studies, as well as a novel agent for the development of new cancer chemotherapy with improved efficacy.

Cytotoxicity of the compounds isolated from Pulsatilla chinensis saponins and apoptosis induced by 23-hydroxybetulinic acid.

CONTEXT: The rizoma of Pulsatilla chinensis (Bunge) Regel has been used as a traditional Chinese medicinal herb for thousands of years. Total saponins from P. chinensis can induce the apoptosis of solid cancer cells; however, their activity on chronic myeloid leukemia and the mechanisms remains unknown. OBJECTIVE: To study the activity of total saponins and the main active fractions from P. chinensis saponins on chronic myeloid leukemia, and to illustrate the mechanisms underlying the anticancer activities. MATERIALS AND METHODS: The cytotoxic activity were assayed by MTT; cell cycle arrest and apoptosis were tested by flow cytometry system; changes in the mitochondrial membrane potential were determined using JC-1; and the apoptosis signaling pathway was determined by western blotting. RESULTS: We demonstrated that total P. chinensis saponin displayed cytotoxic activity against K562 cell line. In addition, we identified 23-hydroxybetulinic acid (HBA), pulchinenoside A (PA), and anemoside B4 (AB4) from the total saponins, with the most cytotoxic compound HBA. Glycosylation at C3 and C28 of HBA significantly reduces its cytotoxicity. HBA could promote cell cycle arrest at S phase and induce apoptosis via intrinsic pathway. HBA disrupts mitochondrial membrane potential significantly (p < 0.01) and selectively downregulates the levels of Bcl-2, survivin and upregulates Bax, cytochrome C, cleaved caspase-9 and -3. DISCUSSION AND CONCLUSION: Total saponins from P. chinensis may be effective natural products against human chronic myelogenous leukemia; HBA is one of the bioactive components responsible for its anticancer activity, and could be further investigated as an alternative therapeutic drug for leukemia.

[Study on the coumarin glucosides of Angelica dahurica].

OBJECTIVE: To study the coumarin glucosides of Angelica dahurica. METHODS: Fresh roots and rhizomes of Angelica dahurica were extracted with ethanol at room temperature. Repeated column chromatography and preparative HPLC were used to isolate and purify the compounds. Their structures were elucidated on the basis of chemical evidence and spectral analysis. RESULTS: Twenty-six coumarin glucosides were isolated from Angelica dahurica, while here we reported 7 of them: sec.-O-beta-D-Galactopyranosyl-(R)-byakangelicin (I);8-O-beta-D-Galactopyranosyl-xanthotoxol (II); 7-O-beta-D-Apiofuranosyl-(1-->6)-beta-D-Glucopyranosyl-peucedanol (III); (R)-peucedanol-7-O-beta-D-Glucopyranoside (IV); sec.-O-beta-D-Glucopyranosyl-(R)-Oxypeucedaninhydrate (V); 7-O-beta-D-Galactopyranosyl-Sco-poletin( VI); Aesculin (VII). CONCLUSION: Compound V is a new compound, Compound VII is isolated from Umbellifera for the first time, compounds III, IV are isolated from Angelica for the first time,while compound I, II and VI are isolated from this plant for the first time.

[Studies on the chemical constituents from stems and leaves of Lonicera macranthoides].

OBJECTIVE: To research the chemical constituents from stems and leaves of Lonicera macranthoides. METHODS: Various column chromatographies were employed to isolate and purify the constituents. Their structures were elucidated by spectral analysis (IR, MS, 1H-NMR, 13 C-NMR) and chemical evidence. RESULTS: Nine constituents were obtained and identified as loganin (I), loganic acid (II), morroniside (III),7-O-ethyl-morroniside (IV), scopoletin (V), caffeic acid (VI), chlorogenic acid (VII), beta-sitosterol (VIII), daucosterol (IX). CONCLUSION: Compounds I-VI are isolated from the plant for the first time. All the compounds are found for the first time from the stems and leaves of Lonicera macranthoides.

Apoptosis and membrane permeabilisation induced by macranthoside B on HL-60 cells.

Triterpene saponins are throught to be potential anti-tumour agents in many cell types. This study aims to evaluate the cytotoxic activity and mechanism of a triterpene saponin, macranthoside B (MB), isolated from Lonicera macranthoides Hand.-Mazz. (Caprifoliaceae). A cell viability assay showed that MB inhibited cell growth of a panel of six cancer cell lines, especially in human acute promyelocytic leukaemia HL-60 cells, with an IC50 value of 3.8 µmol. A hypodiploid cells assay and an annexin-V-FITC/PI double staining assay showed a significant increase of apoptosis in a dose-dependent manner on HL-60 cells both 24 and 48 h after MB treatment. MB-induced apoptosis was through the caspase-mediated pathway, by activation of caspase-3. Furthermore, a lactate dehydrogenase (LDH) release test suggested that an MB-cholesterol interaction led to the rearrangement of the lipid bilayer and to subsequent cell membrane impairment. Taken together, these findings demonstrate that MB may exhibit cytotoxic activity against HL-60 cells by inducing apoptosis via caspase-dependent pathways and also membrane permeabilisation.

[Study on the chemical constituents from the bulbs of Zephyranthes candida].

OBJECTIVE: To study the chemical constituents from the bulbs of Zephyranthes candida. METHODS: The chemical constituents were separated and purified by means of several chromatographies and their structures were elucidated by spectroscopic methods. RESULTS: Six compounds were isolated and identified as trisphaeridine (I), p-hydroxybenzaldehyde (II), sucrose (III), (-)-amarbellisine (IV), lycorine (V), (+)-haemanthamine (VI). CONCLUSION: Compounds I - IV are obtained from this genus for the first time.

Macranthoside B, a hederagenin saponin extracted from Lonicera macranthoides and its anti-tumor activities in vitro and in vivo.

Macranthoside B (MB) is a hederagenin saponin extracted from the flower bud of Lonicera macranthoides. In this study, we defined the anticancer effect of MB both in vitro and in vivo using cell proliferation assays and xenograft tumor growth assays. Our data indicate that MB inhibits the proliferation of various kinds of cancer cells with IC(50) values in the range of 10-20 microM. Moreover, the volume and weight of xenograft tumors in nude mice treated with 5mg/kgMB were decreased remarkably compared to those of the vehicle control group. Furthermore, DAPI staining and flow cytometry analysis with Annexin V/PI double staining revealed that more apoptotic cells were observed following MB treatment. In addition, degradation of PARP (poly-ADP-ribose polymerase), and activation of the caspase cascade for intrinsic pathways were observed. We also found that the expression of Bcl-2 protein decreased and the protein level of Bax increased which leading to an increase of the Bax/Bcl-2 ratio. Our results showed that MB exhibited strong anti-tumor effect and mitochondrion-mediated apoptosis induction involved in it.

Elevated CO2 accelerates flag leaf senescence in wheat due to ear photosynthesis which causes greater ear nitrogen sink capacity and ear carbon sink limitation.

It was anticipated that wheat net photosynthesis would rise under elevated CO2, and that this would alter the progress of senescence due to the unbalance of carbohydrates and nitrogen. Our study showed that ear carbon sink was limited, and sugar was accumulated, hexokinase activities and levels of phosphorylated sugar were increased within the flag leaves, grain nitrogen sink capacity was enhanced, and flag leaf senescence was accelerated under elevated CO2. However, if the ear of the main stem was covered, these responses to elevated CO2 were absent, and the senescence of flag leaf was not accelerated by elevated CO2. Thus, it appeared that elevated CO2 accelerated the rate of flag leaf senescence, depending on ear photosynthesis. The ears have far higher enhancement of net photosynthesis than flag leaves, and the role of the flag leaf relative to the ear was declined in supplying C assimilation to grain under elevated CO2. This indicates that as CO2 rises, the grain sink needs the N more than C assimilate from flag leaf, so the declining rates of N% and soluble proteins concentration were markedly accelerated under elevated CO2 conditions. This suggests that, the large increase in ear net photosynthesis accelerated grain filling, accelerated remobilising N within flag leaf as the result of the greater grain nitrogen sink capacity. In addition, as the result of grain carbon sink limitation, it limited the export of flag leaf sucrose and enhanced sugar cycling, which was the signal to accelerate leaf senescence. Hence, elevated CO2 subsequently accelerates senescence of flag leaf.

[Studies on the chemical constituents of Lonicera macranthoides].

OBJECTIVE: To study the chemical constituents of flower buds of Lonicera macranthoides. METHODS: The 90% EtOH extract of Lonicera macranthoides. was successively partitioned with petroleum ether and ethyl acetete. RESULTS: Repeated column chromatography of the ethyl acetete fraction afforded the following compounds (1-9): ginnol (1), triacontanol (2), ursolic acid (3), beta-sitosterol (4), triacontane (5), palmitic acid (6), beta-daucosterol (7), 3-decyl-3-octyldocosan-1-ol (8), 3-dodecyl-3-nonyldocosan-1-ol (9). All compounds except 4 are isolated from this plant for the first time while compounds 2, 3, 5, 8 and 9 are their first time been isolated from genus Lonicera.