[Researc h progress on chemical constituents, pharmacological effects, toxicity,and heavy metal enrichment characteristics of Phytolacca plants].

In China, the dry roots of Phytolacca acinosa or P. americana in the Phytolacca plants are listed as the traditional Chinese medicine(TCM) Phytolaccae Radix. Phytolacca plants contain triterpenoids, triterpenoid saponins, polysaccharides, antiviral proteins, flavonoids, and other chemical components. The most important compounds are triterpenoids and triterpenoid saponins.Phytolacca plants have anti-inflammatory, anti-tumor, antibacterial, antifungal, antimalarial, deworming, and other activities. In this paper, the chemical constituents, pharmacological effects, toxicity, and heavy metal enrichment characteristics of Phytolacca plants were summarized to provide a theoretical basis for the study of active ingredients and drug development of Phytolacca plants.

Development of an ultra-high-performance liquid chromatography coupled with triple quadrupole mass spectrometry method for comparative pharmacokinetics of six triterpenoids in rat plasma and application to different forms of Phytolacca acinosa.

Phytolacca acinosa is an herb for treatment of ascites and tumor. Two forms of P. acinosa, i.e. raw and vinegar-processed herb, have been used in clinic. However, pharmacokinetic difference between the two forms of P. acinosa has not been fully understood. Herein, a comparative pharmacokinetic method based on liquid chromatography with tandem mass spectrometry was developed for quantification of six bioactive triterpenoids, including esculentoside H, esculentoside T, esculentoside A, esculentoside B, phytolaccagenic acid, and phytolaccagenin in rat plasma after oral administration of different forms of P. acinosa. Separation was performed on an Acquity BEH C18 column (1.7 µm, 2.1 mm × 50 mm). The method was validated over a linear range of 2.0-5000 ng/mL. Intraday and interday bias were within ±5%. Besides, all triterpenoids were stable in plasma during different storage conditions. The described method was successfully applied to a comparative pharmacokinetic study of raw and vinegar-processed P. acinosa in rats. Notably, double peak phenomenon for six triterpenoids of P. acinosa was observed for the first time. AUC0→t and Cmax values of esculentoside H, esculentoside T, phytolaccagenic acid, and phytolaccagenin were significantly lower in vinegar-processed group than that of raw group, indicating the oral bioavailability of the four triterpenoids was decreased after vinegar processing.

Chemical composition and enzyme inhibition of Phytolacca dioica L. seeds extracts.

Phytolacca, which belongs to the family of Phytolaccaceae, are known for their use in popular medicine. Bioactivity of five extracts from Phytolacca dioica seeds were evaluated in four bioassays. A selected group of compounds from the extract that displayed the best bioactivity was analysed. The ethyl acetate extract (EAE) possessed the highest content of phenolics, the highest inhibitory activity on the tyrosinase and xanthine oxidase enzymes and showed a high antioxidant activity. HPLC-DAD-MS was employed to identify the phenolics profile of the most active one (EAE). HSCCC analysis of the EAE led to the isolation of phytolaccoside B and a mixture of 4 isomers, isoamericanol B1, B2, C1 and C2. These isoamericanol isomers presented activity against tyrosinase and xanthine oxidase. Our results revealed for the first time an interesting biological activity of the extract and isolated compounds from P. dioica seeds, which could be considered as a source of bioactive molecules.

Novel bioactive peptides from PD-L1/2, a type 1 ribosome inactivating protein from Phytolacca dioica L. Evaluation of their antimicrobial properties and anti-biofilm activities.

Antimicrobial peptides, also called Host Defence Peptides (HDPs), are effectors of innate immune response found in all living organisms. In a previous report, we have identified by chemical fragmentation, and characterized the first cryptic antimicrobial peptide in PD-L4, a type 1 ribosome inactivating protein (RIP) from leaves of Phytolacca dioica L. We applied a recently developed bioinformatic approach to a further member of the differently expressed pool of type 1 RIPs from P. dioica (PD-L1/2), and identified two novel putative cryptic HDPs in its N-terminal domain. These two peptides, here named IKY31 and IKY23, exhibit antibacterial activities against planktonic bacterial cells and, interestingly, significant anti-biofilm properties against two Gram-negative strains. Here, we describe that PD-L1/2 derived peptides are able to induce a strong dose-dependent reduction in biofilm biomass, affect biofilm thickness and, in the case of IKY31, interfere with cell-to-cell adhesion, likely by affecting biofilm structural components. In addition to these findings, we found that both PD-L1/2 derived peptides are able to assume stable helical conformations in the presence of membrane mimicking agents (SDS and TFE) and intriguingly beta structures when incubated with extracellular bacterial wall components (LPS and alginate). Overall, the data collected in this work provide further evidence of the importance of cryptic peptides derived from type 1 RIPs in host/pathogen interactions, especially under pathophysiological conditions induced by biofilm forming bacteria. This suggests a new possible role of RIPs as precursors of antimicrobial and anti-biofilm agents, likely released upon defensive proteolytic processes, which may be involved in plant homeostasis.

[Triterpenoid saponins from roots of Phytolacca acinosa].

A new triterpenoid saponin named esculentoside U(1), along with the five known compounds, was isolated and characterized from the roots of Phytolacca acinosa, a commonly used traditional Chinese medicine with anti-inflammatory and anti-rheumatoid activities. The structure of the new saponin was elucidated as 3-O-[ß-D-glucopyranosyl-(1→4)]-ß-D-xylopyranosyl]-2, 23-dihydroxyolean-11, 13(18)-diene-28, 29-dioic acid 29-methyl ester(1). The assignment of all NMR signals of 1 was performed by means of 2D-NMR experiments.

Biological and antipathogenic activities of ribosome-inactivating proteins from Phytolacca dioica L.

BACKGROUND: The species from the genus Phytolacca constitute one of the best sources of ribosome-inactivating proteins (RIPs) that have been used both in the therapy against virus and tumors and in the construction of transgenic plants resistant to virus, bacteria, fungi and insects. Here we investigate new activities of three representative RIPs from Phytolacca dioica (dioicin 2, PD-S2 and PD-L4). RESULTS: The three RIPs displayed, in addition to already reported activities, rRNA N-glycosylase activities against plant, bacterial and fungal ribosomes. Additionally dioicin 2 and PD-L4 displayed endonuclease activity on a supercoiled plasmid DNA, and dioicin 2 and PD-S2 arrested the growth of the fungus Penicillium digitatum. Furthermore, dioicin 2 induced caspase activation and apoptosis in cell cultures. CONCLUSIONS: The different activities of the RIPs from Phytolacca dioica may explain the antipathogenic properties attributed to these RIPs in plants and their antiviral and antitumoral effects. In spite of the similarity in their rRNA N-glycosylase and DNA polynucleotide:adenosine glycosylase activities, they differed in their activities against viral RNA, plasmid DNA, fungi and animal cultured cells. This suggests that the presence of isoforms might optimize the response of the plant against several types of pathogens. GENERAL SIGNIFICANCE: RIPs from Phytolacca can induce plant resistance or tumor cell death not only by means of ribosome inactivation but also by the activities found in this report. Furthermore, the induction of cell death by different mechanisms turns these RIPs into more useful tools for cancer treatment rendering the selection of RIP-resistant mutants impossible.

Novel Flavones from the Root of Phytolacca acinosa Roxb.

Two new flavones, 6,7-methylenedioxy-4-hydroxypeltogynan-7'-one (1), cochliophilin B (2), as well as two known ones, cochliophilin A (3) and 6-methoxy-7-hydroxy flavone (4), were isolated from the ethanol extract of the root of Phytolacca acinosa Roxb. Compound 1 is a flavanol framework with one δ-lactone unit, which is rather rare in nature. The structures of the new compounds were determined on the basis of extensive spectroscopic (IR, MS, 1D- and 2D-NMR) analyses, the absolute configuration of 1 was established by comparing experimental and calculated electronic circular dichroism spectra. The structures of known compounds were fixed by comparison with literatures data. Compounds 2 and 4 showed modest inhibitory activities against BEL-7402 cell line, with IC50 values of 28.22 and 39.16 µmol/L, respectively.

[Intestinal toxicity of n-BuOH fraction from Phytolacca Radix before and after being processed with vinegar].

To research the intestinal toxicity of n-BuOH fraction in Phytolacca Radix before and after being processed with vinegar. Toxic n-BuOH fractions were separated from Phytolacca Radix. In the animal model, the level of intestinal edema, water content of intestine and stool, IC50 values of HT-29 and IEC-6 were detected with MTT method to compare the changes in toxicity of n-BuOH fractions from Phytolacca Radix before and after being processed with vinegar. n-BuOH fractions of Phytolacca Radix could cause intestinal edema in mice, increase the edema of duodenum, jejunum and the water content in stool, inhibit the proliferation of HT-29 cells and IEC-6 cells, indicating its intestinal toxicity, with HT-29 IC50 at 14.59 mg•L⁻¹ and IEC-6 IC50 at 43.77 mg•L⁻¹. After being processed with vinegar, the level of intestinal edema, edema of duodenum and jejunum and the water content in stool and inhibition ratio of cells line were reduced, with HT-29 IC50 at 58.51 mg•L⁻¹ and IEC-6 IC50 at 84.37 mg•L⁻¹. After being processed with vinegar, the toxicity of n-BuOH fractions from Phytolacca Radix decreased obviously.

Structural characterization of dioicin 1 from Phytolacca dioica L. gains novel insights into phylogenetic relationships of Phytolaccaceae type 1 RIPs.

Ribosome-inactivating proteins are plant cytotoxic enzymes, also present in fungi, algae and bacteria, mainly known for their ability to inhibit protein synthesis. We previously purified and structurally characterized three type 1 RIPs (PD-S1-3) from Phytolacca dioica seeds and four type 1 RIPs (PD-L1-4) from adult plant leaves. Two additional RIPs, named dioicin 1 and dioicin 2, were isolated from leaves of young plants and developing leaves of adult plants. The evidence that P. dioica synthesizes and accumulates these RIPs isoforms suggests that these proteins have been conserved during evolution. Though several aspects of P. dioica type 1 RIP characterization have been studied, some important questions remain to be answered especially with respect to Phytolaccaceae RIP evolution. One of the major problems encountered in approaching RIPs phylogeny concerns the availability of their sequences. In this study, we report the characterization of biological and structural properties of dioicin 1, including the determination of its primary structure by using a combined approach based on Edman degradation, de novo sequencing by ESI-Q-TOF-MS/MS and peptide mapping by MALDI-TOF MS. Knowledge of dioicin 1 primary structure provide us a mean to deepen Phytolaccaceae's RIPs phylogeny. We speculate that both dioicins 1 and 2 share common ancestors with PAP-II and PAP icos-II and that dioicin 1 is not closely related to other members of this clade, thus shedding lights on evolutionary relationships among type 1 RIPs from Phytolaccaceae.

Physiological and biochemical mechanisms of allelopathy mediated by the allelochemical extracts of Phytolacca latbenia (Moq.) H. Walter.

In allelopathy, one plant suppresses the growth and development of other plant/plants by negatively affecting a variety of physiological and biochemical reactions. We checked the effects of methanolic extracts (allelochemical extracts) of Phytolacca latbenia (Moq.) H. Walter on antioxidant enzyme activities such as peroxidases (PODs), super oxide dismutases (SODs) and catalase (CAT) and on total protein contents (TPC), cellular injury (CI), and malondialdehyde (MDA) in the germinating seeds of Brassica napus L. (dicot) and Triticum aestivum L. (monocot). Both the crude methanolic extract root (CMER) and crude methanolic extract aerial (CMEA) of P. latbenia at 10000 ppm significantly reduced the POD activity in both the test seeds. The activity of SODs was significantly decreased by both CMER and CMEA in B. napus germinating seeds. A linear increase in the activity of CAT, CI, and MDA contents was found in both the test seeds with the increasing concentrations of CMEA and CMER, while TPC of the germinating seeds was found decreased. It is inferred that both the CMEA and CMER inhibited/delayed the seed germination, reduced the seedling growth by affecting a variety of biochemical and physiological attributes, and also caused cellular membrane injury in the germinating seeds of both the monocot and dicot seeds.

Phytotoxicity evaluation and phytochemical analysis of three medicinally important plants from Pakistan.

This work examines the crude methanolic extracts of three medicinally important plants native to Pakistan for potent phytotoxic activities and important phytochemicals. These plants include Euphorbia wallichii, Bergenia ciliata and Phytolacca latbenia. The phytotoxic effects were checked at 10,000, 1000, and 100 µg/ml against two economically important standard target species, Triticum aestivum (monocot representative) and Brassica napus (dicot representative). The phytotoxicity effects on seed germination, seedling growth and seedling weight were checked. A simple, cost-effective in vitro phytotoxicity assay (that uses petri plates) was used to evaluate the allelopathic properties of crude extracts. At highest concentration, extracts from all the three plants showed phytotoxic activities such that P. latbenia > E. wallichii > B. ciliata. In seedling growth, root length was affected more than shoot length, whereas among the target species B. napus was found to be more sensitive towards extracts when compared with T. aestivum. Phytochemical analysis showed that P. latbenia is rich in saponins and terpenoids, while E. wallichii and B. ciliata are rich in tannins, terpenoids and cardiac glycoside. P. latbenia also carries a moderate amount of cardiac glycosides.

Phytolacca Dodecandra (L' Herit) (Phytolaccaceae) Methanol Root Extract Protects Liver from Acetaminophen-Induced Injury in Rats.

Phytolacca dodecandra (L' Herit), or 'Endod', is one of the widely known medicinal plants in Ethiopia. Berries of the endod have been used as a detergent for centuries. The present study was aimed to test the hepatoprotective effects of the plant against acetaminophen (APAP)-induced liver injury in rats. Mice of either sex were used for oral acute toxicity tests and APAP-induced lethality tests. Hepatoprotective experiments were done on male rats using 2 g/kg of APAP to induce liver damage. Liver enzymes, total bilirubin (TB), and lipid profile were determined. Liver tissues were also examined histopathologically to see a morphologic change in the control and experiment groups. The protective effect of the plant extract was also tested through sodium pentobarbital (SPB)-induced sleeping time. A significant increase in serum levels of liver enzymes, TB, low-density lipoprotein (LDL), and triglycerides (TGs) was seen from oral administration of 2 g/kg APAP. Total cholesterol (TC) and high-density lipoprotein (HDL) levels were decreased. Serum levels of all parameters were reversed to normal after administration of silymarin 100 mg/kg and, 100, 200, and 400 mg/kg doses of the extract. A significant dose-dependent hepatoprotective effect of Phytolacca dodecandra Methanol Root Extract (PDME) was seen in terms of LDL. Histopathological investigations and SPB-induced sleeping time confirmed the findings of biochemical analysis. The findings of the present study indicate that PDME protected the liver from APAP injury.

Mechanistic insights into xanthomicrol as the active anti-HCC ingredient of Phytolacca acinosa Roxb.: A network pharmacology analysis and transcriptomics integrated experimental verification.

ETHNOPHARMACOLOGICAL RELEVANCE: Phytolacca acinosa Roxb. (PAR) is a Traditional Chinese Medicinal (TCM) plant with a broad global distribution encompassing 35 species, four of which are found in the People's Republic of China. It occupies a significant role in both Oriental and American traditional medicine, employed in treating a range of conditions such as edema, inflammation, dermatitis, and rheumatism. PAR is also used as a molluscicide and for addressing tumors and bronchitis. The plant is documented in the Chinese Pharmacopoeia and has a longstanding history in TCM, particularly for its diuretic properties and in treating ailments such as edema, swelling, and ulcers. Notably, PAR has demonstrated potent inhibitory effects against the A549 human lung cancer cell line, underscoring its potential in contributing to the development of novel cancer therapeutics. AIM OF THE STUDY: The research aims to elucidate the active components of PAR and their mechanisms in treating hepatocellular carcinoma (HCC). MATERIALS AND METHODS: Employing network pharmacology, this study predicted the principal active compounds and key targets of PAR. A holistic methodology incorporating biological network analysis, transcriptomics sequencing, molecular docking, and molecular dynamics (MD) simulations was utilized to forecast the effects of PAR on HCC, with empirical evidence supporting these findings. RESULTS: Network pharmacology identified xanthomicrol as the foremost active compound in PAR. The tumor-suppressive functions of PAR, as indicated by KEGG pathway analysis and transcriptomics sequencing, predominantly occur via the PI3K/AKT pathway. Molecular docking and dynamics simulations demonstrated the high affinity of xanthomicrol towards TNF, MMP9, PPARG, KDR, and MMP2. In vivo experiments verified the efficacy of xanthomicrol in curtailing HCC tumor growth, while in vitro assessments revealed its substantial impact on the proliferation and apoptosis of HepG2 and HCCLM3 cells. Moreover, the study indicates that xanthomicrol may modulate the expression of TNF, MMP9, PPARG, KDR, and MMP2 in HCC cells and inhibit the activation of the PI3K/AKT pathway. CONCLUSIONS: Xanthomicrol, a principal active component of PAR, has been identified to impede the growth of HCC by targeting the PI3K/Akt/MMP9 pathway. This insight could enhance therapeutic approaches for HCC.

Phytolacca tetramera berries extracts and its main constituents as potentiators of antifungal drugs against Candida spp.

BACKGROUND: Extensive antifungal drug use has enhanced fungal resistance, resulting in persistent mycoses. Combining antifungal plant extracts/compounds with these drugs offers good alternatives to increase the activity of both partners, minimize side effects, and overcome drug resistance. In our previous study, Phytolacca tetramera berries extracts demonstrated activity against Candida spp., correlating with the amount of the main constituent phytolaccoside B and its genin, phytolaccagenin. The extracts and phytolaccagenin altered the fungal plasma membrane by binding to ergosterol, whereas phytolaccoside B increased chitin synthase activity. However, the presence of triterpenoid saponins in Phytolacca spp. has been linked to acute toxicity in humans. PURPOSE: This study aimed to evaluate combinations of P. tetramera berries extracts, phytolaccoside B and phytolaccagenin, together with commercial antifungals [amphotericin B, fluconazole, itraconazole, posaconazole, and caspofungin] against Candida albicans and Candida glabrata, to find synergistic effects with multi-target actions, in which the doses of both partners are reduced, and therefore their toxicity. Additionally, we intended to explore their anti-virulence capacity, thereby hindering the development of drug-resistant strains. METHODS: The effects of these combinations were evaluated using both the checkerboard and isobologram methods. Fractional Inhibitory Concentration Index and Dose Reduction Index were calculated to interpret the combination results. To confirm the multi-target effect, studies on mechanisms of action of synergistic mixtures were performed using ergosterol-binding and quantification assays. The ability to inhibit Candida virulence factors, including biofilm formation and eradication from inert surfaces, was also evaluated. Quantification of active markers was performed using a validated UHPLC-ESI-MS method. RESULTS: Eight synergistic combinations of P. tetramera extracts or phytolaccagenin (but not phytolaccoside B) with itraconazole or posaconazole were obtained against C. albicans, including a resistant strain. These mixtures acted by binding to ergosterol, decreasing its whole content, and inhibiting Candida biofilm formation in 96-well microplates and feeding tubes in vitro, but were unable to eradicate preformed biofilms. CONCLUSIONS: This study demonstrated the synergistic and anti-virulence effects of P. tetramera berries extracts and phytolaccagenin with antifungal drugs against Candida spp., providing novel treatment avenues for fungal infections with reduced doses of both natural products and commercial antifungals, thereby mitigating potential human toxicity concerns.

[Comparative study on toxicity of extracts from Phytolaccae Radix before and after being processed with vinegar].

OBJECTIVE: To extract and separate toxic components from Phytolaccae Radix, and to comare the changes in toxicity of Phytolaccae Radix before and after being processed with vinegar. METHOD: The mucous membrane irritation response, mouse peritoneal inflammation model and in vitro macrophages release NO model were applied to compared the changes in inflammatory toxicity of toxic components from Phytolaccae Radix before and after being processed with vinegar. RESULT: Toxic components of Phytolacca Radix had significant inflammatory toxicity, which could cause conjunctival edema in rabbits, and increase of PGE2 and macrophages release NO content in peritoneal exudate in mice. After being processed with vinegar, they showed reduced irritation, which resulted in decrease of PGE2 and macrophages release NO content in peritoneal exudate in mice. CONCLUSION: After being processed with vinegar, the toxicity of toxic components from Phytolacca Radix decreased obviously.

Two new triterpene glycosides with antiproliferative activities on HepG2 from Phytolacca acinosa fruit fermentation broth.

Two new oleanane-type triterpene glycosides, phytolasides A (1) and B (2), and six known ones (3-8), were isolated from Phytolacca acinosa fruit fermentation broth. Their structures were elucidated by HR-ESI-MS and 1 D- and 2 D-NMR spectroscopic methods. Antiproliferation of compounds 1 and 2 against HepG2 cells was examined by using CCK8 assays.

Rapid green synthesis of silver nanoparticles from silver nitrate by a homeopathic mother tincture Phytolacca Decandra.

OBJECTIVE: To examine if a homeopathic mother tincture (Phytolacca Decandra) is capable of precipitating silver nanoparticles from silver nitrate (AgNO(3)) and to characterize the biosynthesized nanoparticles for evaluating their biological activities. METHODS: A total of 100 mg of AgNO(3) was added to 20mL of Milli-Q water and stirred vigorously. Then 5mL of the homeopathic mother tincture of Phytolacca Decandra (ethanolic root extract of Phytolacca decandra) was added and stirred continuously. Reduction took place rapidly at 300K and completed in 10 min as shown by stable light greenish-yellow color of the solution which gave colloid of silver nanoparticles. The colloid solution was then centrifuged at 5000×g to separate the nanoparticles for further use. The nanoparticles were characterized by spectroscopic analysis, particle size analysis and zeta potential measurements, and morphology was analyzed by atomic force microscopy. The drug-DNA interaction was determined by circular dichroism spectrophotometry and melting temperature profiles by using calf thymus DNA as the target. The biological activities were determined using a cancer cell line A549 in vitro and using bacteria Escherichia coli and fungus Saccharomyces cerevisiae as test models. RESULTS: Phytolacca Decandra precipitated silver nanoparticles in ambient conditions. The nanoparticles had 91 nm particle size, with polydispersity index of 0.119 and zeta potential of -15.6 mV. The silver nanoparticles showed anticancer and antibacterial properties, but no clear antifungal properties. CONCLUSION: This could be a novel environment-friendly method to biosynthesize silver nanoparticles using a cost-effective, nontoxic manner. The homeopathic mother tincture may utilize this property of nano-precipitation in curing diseases or disease symptoms.

Rapid Screening and Characterization of Triterpene Saponins from the Root of Phytolacca acinosa Roxb by High-Performance Liquid Chromatography Coupled to Electrospray Ionization and Quadrupole Time-of-Flight Mass Spectrometry.

Triterpene saponins (TSs) are important bioactive constituents with structural diversity widely distributed in many plants. The root of Phytolacca acinosa Roxb (RPa) has been used as a traditional Chinese medicine. However, TSs as the main active ingredients in RPa have not been fully characterized. Here, we profiled TSs from RPa by high-performance liquid chromatography coupled to electrospray ionization and quadrupole time-of-flight mass spectrometry (HPLC-ESI-QTOF-MS/MS). We tentatively identified 29 TSs, including 13 that had not been reported previously from this plant. This study indicates that HPLC-ESI-QTOF-MS/MS is an effective and rapid method for the characterization of complicated TSs in herbal extracts.

Synthesis of novel derivatives of esculentoside A and its aglycone phytolaccagenin, and evaluation of their haemolytic activity and inhibition of lipopolysaccharide-induced nitric oxide production.

A series of 46 compounds derived from esculentoside A and its aglycone were synthesized and characterized. The effect of these compounds on lipopolysaccharide (LPS)-induced NO production, haemolytic activity, and cell viability was evaluated. Structure-activity relationship was established by comparing the derivatives of esculentoside A with its aglycone derivatives. Both the aglycone and its derivatives showed higher inhibitory effects on LPS-induced NO production, and lower haemolytic activities than esculentoside A and its derivatives.

[Comparative study of mucosa irritation of crude and processed Radix Phytolaccae].

OBJECTIVE: To study the changes of mucosa irritation of crude and processed Radix Phytolaccae. METHOD: Gastrointestinal experiments on rats and mice and the rabbits' eyes irritation experiments were applied to investigate the changes of mucosa irritation of crude and processed Radix Phytolaccae. RESULT: The mucosa irritation of processed Radix Phytolaccae decreased obviously compared to crude one. CONCLUSION: After being processed with vinegar, the mucosa irritation on Radix Phytolaccae decreased obviously.