Selective Antitumor Effect and Lower Toxicity of Mitochondrion-Targeting Derivatization of Triptolide.

Triptolide has a significant antitumor activity, but its toxicity limits its clinical application. As the mitochondrion-targeting strategy showed an advantage in selective antitumor effect based on the higher mitochondrial membrane potential (MMP) in tumor cells than normal cells, the lipophilic cations triphenylphosphonium and E-4-(1H-indol-3-yl vinyl)-N-methylpyridinium iodide (F16) were selected as targeting carriers for structural modification of triptolide. The derivatives bearing F16 generally retained most antitumor activities, overcame its inhibition plateau phenomena, and enhanced its selective antitumor effect in lung cancer. The representative derivative F9 could accumulate in the mitochondria of NCI-H1975 cells, inducing apoptosis and a dose-dependent increase in intracellular reactive oxygen species and reducing MMP. Moreover, no effects were observed in normal cells BEAS-2B. In vivo studies showed that the developmental, renal, and liver toxicities of F9 to zebrafish were significantly lower than those of triptolide. This study provides a promising idea to relieve the toxicity problem of triptolide.

Mitochondrial targeting derivatives of honokiol enhanced selective antitumor activity in NCI-H446 cells and decreased in vivo toxicity in Caenorhabditis elegans.

Mitochondria, responsible for ATP production and apoptosis regulation, play a key role in cancer cells. Honokiol regulates apoptosis through the endogenous mitochondrial pathway but does not specifically target tumor cells. We designed 28 novel derivatives of honokiol using triple-function delocalized lipophilic cations such as berberine and F16 as mitochondrion-targeting carriers. While all derivatives exhibited enhanced cytotoxicity toward tumor cells compared to honokiol, the derivative 2E-3-F16 exhibited a substantial tumor cell selectivity between NCI-H446 cancer cells and HBE cells by one order of magnitude and enhanced the sensitivity of A549 cells to cisplatin. Mechanistically, it targeted mitochondria and induced apoptosis by preventing tumor cells from entering the G0/G1 phases as well as inducing an abnormal elevation of reactive oxygen species, thereby decreasing the mitochondrial membrane potential level. It also showed lower toxicity toward Caenorhabditis elegans than honokiol. This study provides a possible method for developing mitochondrion-targeting antitumor drugs with high efficiency and low toxicity based on natural products.

Synthesis of 3-O-Acetyl-11-keto-ß-boswellic Acid (AKBA)-Derived Amides and Their Mitochondria-Targeted Antitumor Activities.

In this study, we synthesized a series of amide and mitochondria-targeted derivatives with 3-O-acetyl-11-keto-ß-boswellic acid (AKBA) as the parent structure and an ethylenediamine moiety as the link chain. Compound 5e, a mitochondrial-targeting potential derivative, showed significantly stronger antitumor activity than that of AKBA, and it could induce vacuolization of A549 cells and stimulate the production of reactive oxygen species (ROS) in a time- and concentration-dependent manner. The antioxidant N-acetylcysteine (NAC) could inhibit the ROS level but could not suppress vacuolization and cell death induced by 5e. Further studies demonstrated that 5e caused abnormal opening of mitochondrial permeability transition pore (MPTP) and a decrease of mitochondrial membrane potential; additionally, it caused cell cycle arrest in G0/G1 but did not induce apoptosis. 5e represented a compound with improved antiproliferative effects for cancer therapy working through new mechanisms.

Antitumor and toxicity study of mitochondria-targeted triptolide derivatives using triphenylphosphine (TPP+) as a carrier.

Based on the higher mitochondrial membrane potential (Δψm) of tumor cells than normal cells, a mitochondria-targeting strategy using delocalized lipophilic cations as carriers is a promising way to improve the antitumor effect of small molecules and to reduce toxicity. Triptolide (TP) has a strong antitumor effect but is limited in the clinic due to high systemic toxicity. Mitochondria-targeted TP derivatives were designed and synthesized using triphenylphosphine cations as carriers. The optimal derivative not only maintained the antitumor activity of TP but also showed a tumor cell selectivity trend. Moreover, the optimal derivative increased the release of lactate dehydrogenase and the production of ROS, decreased Δψm, and arrested HepG2 cells in G0/G1 phase. In a zebrafish HepG2 xenograft tumor model, the inhibitory effect of the optimal derivative was comparable to that of TP, while it had no obvious toxic effect on multiple indicators in zebrafish at the test concentrations. This work provided some evidence to support the mitochondria-targeting strategy.

Genus Sapium (Euphorbiaceae): A review on traditional uses, phytochemistry, and pharmacology.

ETHNOPHARMACOLOGICAL RELEVANCE: Genus Sapium, belonging to Euphorbiaceae family, has a wide distribution in Asia and in temperate and tropical regions of Africa and America. The various parts of Sapium species have been used in traditional Chinese herbal medicine for the treatment of edema, skin-related diseases, bacterial infections, cancers, diabetes, and other ailments. AIM OF THE STUDY: A comprehensive and updated review on the phytochemistry, pharmacology, and traditional medicinal uses of Sapium has been summarized and discussed to facilitate further exploitation of the therapeutic values of Sapium species. MATERIALS AND METHODS: The relevant information of Sapium species was collected by scientific search engines including Elsevier, Google Scholar, Scifinder, and CNKI (China national knowledge infrastructure), and Master's dissertations and Summon from Shandong University Library. RESULTS: Phytochemical studies revealed that approximately 259 compounds including terpenoids, phenylpropanoids, flavonoids, tannins, steroids, alkaloids, etc. have been isolated and identified from Sapium species, among which terpenoids, phenylpropanoids and tannins are the main constituents. Pharmacological in vitro and in vivo studies revealed that the extracts and pure compounds possessed significant antibacterial, antiinflammatory, antioxidant, antihypertensive effects, cytotoxicity, antidiabetic, molluscicidal effects. Terpenoids, phenylpropanoids, tannins, flavonoids, and alkaloids may be responsible for these activities. CONCLUSIONS: The traditional uses, phytochemistry, and pharmacology described in this article demonstrated that the plants of Sapium genus possess many different types of compounds exhibiting wide range of biological activities, and they have high medicinal value and potential in the treatment of a variety of diseases. Detailed phytochemical studies have been conducted on only twelve species in the literature. More wide-ranging studies are still needed to explore this genus. Most of the existing bioactivity-related studies were implemented on crude extracts. More in-depth studies are necessary to reveal the links between the traditional uses and bioactivity in the future.

Anticancer Effects of Honokiol via Mitochondrial Dysfunction Are Strongly Enhanced by the Mitochondria-Targeting Carrier Berberine.

Mitochondrion is a favorable therapeutic target in cancer, given its regulation of bioenergetics and cell death. Honokiol exhibits antiproliferative effects through mitochondria-mediated death signaling. To enhance its anticancer potential and selectivity, we conjugated honokiol to berberine, a mitochondria-targeting carrier. All designed derivatives displayed 1 order of magnitude increased cytotoxicity compared with the parent compounds, especially with massive cytoplasmic vacuoles. Biological evaluation demonstrated the representative compound 6b localized within the mitochondria, and mitochondrial dilation resulted in vacuolization. 6b induced vacuolation-associated cell death and apoptosis with obvious mitochondrial dysfunction, as demonstrated by booming reactive oxygen species generation, opening mitochondrial permeability transition pore, and reducing mitochondrial membrane potential. The targeting property also conferred 6b with selectivity for tumor cells compared to normal cells. 6b inhibited cancer cell proliferation in the zebrafish xenograft model. These results demonstrate that berberine-linked honokiol derivatives open up a direction for novel mitochondrial-targeting antitumor agents.

CLG from Hemp Seed Inhibits LPS-Stimulated Neuroinflammation in BV2 Microglia by Regulating NF-κB and Nrf-2 Pathways.

The healthy benefits of hemp (Cannabis sativa L.) seed have often been attributed to its oils and proteins. Recent studies reveal that hemp seed phenylpropionamides could also show various bioactivities. Continuation of our study on hemp seed provided a phenylpropionamide, coumaroylaminobutanol glucopyranoside (CLG). This work investigated the neuroprotective effect of CLG and its underlying mechanism using lipopolysaccharide-induced BV2 microglia. Our study demonstrated that CLG increased adenosine monophosphate-activated protein kinase (AMPK) expression, suppressed the nuclear factor-kappa B (NF-κB) signaling pathway by inhibiting the phosphorylation of IκBα and NF-κB p65 and decreased proinflammatory cytokine levels in a concentration-dependent manner. Furthermore, CLG reduced the production of cellular reactive oxygen species and stimulated the nuclear factor erythroid 2-related factor 2 (Nrf-2) signaling pathway. Collectively, these results suggested that CLG effectively and simultaneously inhibited inflammatory responses and oxidative stress through the NF-κB and Nrf-2 signaling pathways. AMPK was also involved in the anti-inflammatory effect of CLG. This study provides new insights into the diverse bioactive constituents of hemp seed.

New coumarins and monoterpene galloylglycoside from the stem bark of Sapium baccatum.

Sapium baccatum has been traditionally used as therapeutic remedies. To support its medicinal benefits, our current phytochemical investigation attempted to further discover novel bioactive compounds from S. baccatum. Eight new phenolic compounds, namely, seven coumarins (1-7) and one monoterpene galloylglycoside (8), together with 23 (9-31) known compounds were isolated. Their structures were determined by extensive spectroscopic methods and comparison with literatures. The three pairs of enantiomers of 1, 2 and 7 were confirmed on the basis of HPLC chiral analysis, electronic circular dichroism data and optical rotations. Two coumarins (1-2) were proven to be artifacts through HPLC analysis. The inhibitory effects on TNF-α secretion were examined biologically in LPS-induced BV2 microglia cells and all of the tested compounds exhibited significant inhibitory activity, especially new compound 1 possessed stronger inhibitory effects compared to the positive control quercetin. In addition, compounds 14 and 15 showed weak antifungal activity against Candida albicans SC5314 with MIC values both at 64 µg/mL. The results laid a solid foundation for additional research on S.baccatum related to its anti-inflammatory and antifungal medicinal value.

Cannabisin F from Hemp (Cannabis sativa) Seed Suppresses Lipopolysaccharide-Induced Inflammatory Responses in BV2 Microglia as SIRT1 Modulator.

Hemp seed (Fructus cannabis) is rich in lignanamides, and initial biological screening tests showed their potential anti-inflammatory and anti-oxidative capacity. This study investigated the possible effects and underlying mechanism of cannabisin F, a hempseed lignanamide, against inflammatory response and oxidative stress in lipopolysaccharide (LPS)-stimulated BV2 microglia cells. Cannabisin F suppressed the production and the mRNA levels of pro-inflammatory mediators such as interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α) in a concentration-dependent manner in LPS-stimulated BV2 microglia cell. Furthermore, cannabisin F enhanced SIRT1 expression and blocked LPS-induced NF-κB (Nuclear factor kappa B) signaling pathway activation by inhibiting phosphorylation of IκBα (Inhibit proteins of nuclear factor kappaB) and NF-κB p65. And the SIRT1 inhibitor EX527 significantly inhibited the effect of cannabisin F on pro-inflammatory cytokines production, suggesting that the anti-inflammatory effects of cannabisin F are SIRT1-dependent. In addition, cannabisin F reduced the production of cellular reactive oxygen species (ROS) and promoted the expression of Nrf2 (Nuclear factor erythroid-2 related factor 2) and HO-1 (Heme Oxygenase-1), suggesting that the anti-oxidative effects of cannabisin F are related to Nrf2 signaling pathway. Collectively, these results suggest that the neuro-protection effect of cannabisin F against LPS-induced inflammatory response and oxidative stress in BV2 microglia cells involves the SIRT1/NF-κB and Nrf2 pathway.

Hemp (Cannabis sativa L.) Seed Phenylpropionamides Composition and Effects on Memory Dysfunction and Biomarkers of Neuroinflammation Induced by Lipopolysaccharide in Mice.

Hempseed has achieved a growing popularity in human nutrition, particularly regarding essential amino acids and fatty acids. The multiple positive attributes of hempseed have led to the further study of its constituents. In this study, hempseed extract containing phenylpropionamides (TPA) was obtained and its chemical profile and content were obtained using high-performance liquid chromatography technology based on previous study. The anti-neuroinflammatory effect of TPA extract was evaluated using a lipopolysaccharide (LPS)-induced mouse model. Fourteen phenylpropionamides (TPA) were identified in the obtained extract with a total content of 233.52 ± 2.50 µg/mg extract. In mice, TPA prevented the learning and spatial memory damage induced by LPS. Increased brain levels of IL-1ß, IL-6, and TNF-α in the LPS-induced mice were reduced by TPA treatment. Furthermore, TPA attenuated LPS-induced hippocampal neuronal damage in mice. This study demonstrates the nutraceutical potential of hempseed from a neuroprotective perspective.

Bioactive constituents from cinnamon, hemp seed and polygonum cuspidatum protect against H2O2 but not rotenone toxicity in a cellular model of Parkinson's disease.

Mitochondrial dysfunction and oxidative stress are two factors that are thought to contribute to the pathogenesis of Parkinson's disease (PD), a debilitating progressive neurodegenerative disorder that results in the loss of catecholamine producing cells throughout specific regions of the brain. In this study we aimed to compare the effects of hydrogen peroxide (H2O2) and rotenone (a pesticide and mitochondrial complex 1 inhibitor) on cell viability and the expression of tyrosine hydroxylase (TH) in a cellular model of PD. We also sought to investigate the potential neuroprotective benefits of bioactive constituents from cinnamon, hemp seed and polygonum cuspidatum. To create a model, SH-SY5Y cells transfected with human TH isoform 1 were treated with varying concentrations of H2O2 and rotenone, in the presence or absence of bioactive constituents. The effect of these toxins and constituents on cell viability, apoptosis and protein expression was assessed using MTT viability assays and western blotting. Rotenone treatment caused a significant decrease in cell viability but a significant increase in TH in the remaining cells. H2O2 treatment caused a significant decrease in cell viability but had no significant effect on TH expression. Curcumin, cinnamaldehyde, caffeoyltyramide (hemp seed extract) and piceatannol glucoside (polygonum cuspidatum extract) were unable to attenuate rotenone induced cell death, however they were able to provide protection against H2O2 induced cell death. This is the first study to demonstrate the neuroprotective properties of cinnamaldehyde, caffeoyltyramide and piceatannol glucoside in a dopaminergic cell line in response to H2O2.

Mitochondria-Targeted Lupane Triterpenoid Derivatives and Their Selective Apoptosis-Inducing Anticancer Mechanisms.

Betulin and betulinic acid have been widely studied for their anticancer activities. However, their further development is limited due to low bioavailability, poor aqueous solubility, and limited intracellular accumulation. In the present study, a triphenylphosphonium cation moiety was linked to betulin and betulinic acid to specifically target them to cancer cell mitochondria. Biological characterization established that uptake of mitochondria-targeted compound 1a in the mitochondria of cancer cells was increased compared to betulin. The mitochondria-targeted derivatives of betulin and betulinic acid showed stronger cytotoxicity than their parent drugs and exhibited more cytotoxic effects in cancer cells than normal cells. The mechanisms may involve the mitochondrial apoptotic pathway, probably caused by the induction of reactive oxygen species production and reducing mitochondrial membrane potential. More importantly, 1a significantly inhibited cancer cell proliferation and migration in an in vivo zebrafish xenograft model. Collectively, these results encourage further study of 1a analogs as anticancer agents.

Anti-neuroinflammatory effects of grossamide from hemp seed via suppression of TLR-4-mediated NF-κB signaling pathways in lipopolysaccharide-stimulated BV2 microglia cells.

Grossamide, a representative lignanamide in hemp seed, has been reported to possess potential anti-inflammatory effects. However, the potential anti-neuroinflammatory effects and underlying mechanisms of action of grossamide are still unclear. Therefore, the present study investigated the possible effects and underlying mechanisms of grossamide against lipopolysaccharide (LPS)-induced inflammatory response in BV2 microglia cells. BV2 microglia cells were pre-treated with various concentrations of grossamide before being stimulated with LPS to induce inflammation. The levels of pro-inflammatory cytokines were determined using the enzyme-linked immunoassay (ELISA) and mRNA expression levels were measured by real-time PCR. The translocation of nuclear factor-kappa B (NF-κB) and contribution of TLR4-mediated NF-κB activation on inflammatory effects were evaluated by immunostaining and Western blot analysis. This study demonstrated that grossamide significantly inhibited the secretion of pro-inflammatory mediators such as interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α), and decreased the level of LPS-mediated IL-6 and TNF-α mRNA. In addition, it significantly reduced the phosphorylation levels of NF-κB subunit p65 in a concentration-dependent manner and suppressed translocation of NF-κB p65 into the nucleus. Furthermore, grossamide markedly attenuated the LPS-induced expression of Toll-like receptor 4 (TLR4) and myeloid differentiation factor 88 (MyD88). Taken together, these data suggest that grossamide could be a potential therapeutic candidate for inhibiting neuroinflammation in neurodegenerative diseases.

Ring A-modified Derivatives from the Natural Triterpene 3-O-acetyl-11-keto-ß-Boswellic Acid and their Cytotoxic Activity.

BACKGROUND: Natural triterpene boswellic acids (BAs) have attracted much interest due to their anticancer activity, but more chemical modification is necessary to explore their pharmacological value. In addition to subtle functionalization, transformations that alter the triterpene skeleton are viewed as an alternative approach. OBJECTIVE: In this study, transformations altering ring A of 3-O-acetyl-11-keto-ß-boswellic acid (AKBA) were performed to obtain A-lactone, A-lactam, A-seco and A-contracted derivatives. METHOD: Thirty-two new derivatives were synthesized, and their structures were confirmed by NMR and MS. Their anticancer activity against human cancer cell lines K562, PC3, A549 and HL60 was screened. RESULTS: Biological evaluation indicated that the ring A cleavage or contraction transformations themselves did not significantly enhance the cytotoxic activity, but most of the derivatives based on these ring A-modified skeletons exhibited good cytotoxic activity. Significantly improved cytotoxicity was discovered for the esterified analogues of the A-lactone and A-lactam series and the amidated analogues of the A-seco and ring A contracted series, especially those bearing two nitrogen-containing substituents. Among them, compounds 6a, 11b, 12k and 18e showed strong cytotoxic activity, with IC50 values of 5.0~3.5 µM against K562 cells, almost ninefold stronger than that of AKBA. Further study proposed that the antiproliferative activities of 6a, 11b, 12k and 18e may be due to apoptosis induction. CONCLUSION: The transformations of the ring A skeleton of AKBA provide new platforms to discover anticancer candidates.

Hapmnioides A-C, Rearranged Labdane-Type Diterpenoids from the Chinese Liverwort Haplomitrium mnioides.

Many exceptional labdane-type diterpenoids have been exclusively found in liverworts, which serve as taxonomic molecules or play important ecological roles in interactions among organisms. Three unprecedented labdane-type diterpenoids hapmnioides A (1), B (2), and C (3) formed through cascade rearrangement from the Chinese liverwort Haplomitrium mnioides are reported. Their structures were established by comprehensive spectroscopic analysis coupled with single-crystal X-ray diffraction, and their anti-inflammatory activities were also preliminarily tested.

Preparative Scale MS-Guided Isolation of Bioactive Compounds Using High-Resolution Flash Chromatography: Antifungals from Chiloscyphus polyanthos as a Case Study.

In natural product research, the efficient purification of molecules from large amounts of complex extracts is a key element. In this regard, an integrative strategy for efficient MS-guided isolation of antifungal compounds has been developed. First, off-line HPLC antifungal activity-based profiling and HPLC-PDA-MS profiling were used to localize the compounds of interest on the analytical scale. Then, the analytical gradient was geometrically transferred to the flash chromatographic level. Finally, an MS-triggered isolation of the localized bioactive molecules was realized using high-resolution flash chromatographic columns (15 µm spherical particles) coupled to a single quadrupole mass spectrometer via a splitter system. This isolation strategy was applied for the MS-targeted purification of antifungal principles from the liverwort Chiloscyphus polyanthos. This rational methodology has high potential for the targeted large-scale purification of bioactive compounds, avoiding the need to repeat a given bioassay at each isolation step. Seven sesquiterpene lactones were isolated, of which five were found to be bioactive and one was reported as a new compound. The absolute configuration of some compounds was established for the first time by electronic circular dichroism spectroscopy.

ent-Eudesmane-Type Sesquiterpenoids from the Chinese Liverwort Chiloscyphus polyanthus var. rivularis.

Seven new ent-eudesmane-type sesquiterpenoids (1-7) and six known analogues (8-13) were isolated from the Chinese liverwort Chiloscyphus polyanthus var. rivularis. Their structures were determined from analysis of MS and NMR spectroscopic data and single-crystal X-ray diffraction. A cytotoxic evaluation showed that compound 1 exhibited weak inhibitory activity against the A549 cancer cell line with an IC50 value of 27.7 µM.

Metabolite identification of the antimalarial piperaquine in vivo using liquid chromatography-high-resolution mass spectrometry in combination with multiple data-mining tools in tandem.

Artemisinin-based combination therapy is widely used for the treatment of uncomplicated Plasmodium falciparum malaria, and piperaquine (PQ) is one of important partner drugs. The pharmacokinetics of PQ is characterized by a low clearance and a large volume of distribution; however, metabolism of PQ has not been thoroughly investigated. In this work, the metabolite profiling of PQ in human and rat was studied using liquid chromatography tandem high-resolution LTQ-Orbitrap mass spectrometry (HRMS). The biological samples were pretreated by solid-phase extraction. Data processes were carried out using multiple data-mining techniques in tandem, i.e., isotope pattern filter followed by mass defect filter. A total of six metabolites (M1-M6) were identified for PQ in human (plasma and urine) and rat (plasma, urine and bile). Three reported metabolites were also found in this study, which included N-oxidation (M1, M2) and carboxylic products (M3). The subsequent N-oxidation of M3 resulted in a new metabolite M4 detected in urine and bile samples. A new metabolic pathway N-dealkylation was found for PQ in human and rat, leading to two new metabolites (M5 and M6). This study demonstrated that LC-HRMS(n) in combination with multiple data-mining techniques in tandem can be a valuable analytical strategy for rapid metabolite profiling of drugs. Copyright © 2016 John Wiley & Sons, Ltd.

Characterization of Lignanamides from Hemp (Cannabis sativa L.) Seed and Their Antioxidant and Acetylcholinesterase Inhibitory Activities.

Hemp seed is known for its content of fatty acids, proteins, and fiber, which contribute to its nutritional value. Here we studied the secondary metabolites of hemp seed aiming at identifying bioactive compounds that could contribute to its health benefits. This investigation led to the isolation of 4 new lignanamides, cannabisin M (2), cannabisin N (5), cannabisin O (8), and 3,3'-demethyl-heliotropamide (10), together with 10 known lignanamides, among which 4 was identified for the first time from hemp seed. Structures were established on the basis of NMR, HR-MS, UV, and IR as well as by comparison with the literature data. Lignanamides 2, 7, and 9-14 showed good antioxidant activity, among which 7, 10, and 13 also inhibited acetylcholinesterase in vitro. The newly identified compounds in this study add to the diversity of hemp seed composition, and the bioassays implied that hemp seed, with lignanamides as nutrients, may be a good source of bioactive and protective compounds.

Polyacetylated labdane-type diterpenoids, ptychantins P-R from Chinese liverwort Ptychanthus striatus.

Three new polyacetylated labdane diterpenoids ptychantins P-R (1-3) and four known compounds (4-7) were isolated from an EtOH extract of the Chinese liverwort Ptychanthus striatus (Lehm. & Lindenb.) Nees. Their structures were established by extensive analysis of spectroscopic data (IR, UV, HRESIMS, 1D NMR, and 2D NMR).