Electrochemiluminescence sensor for point-of-care detection of pyrrolizidine alkaloids.

Pyrrolizidine alkaloids (PAs) and PA N-oxides are hepatotoxic natural products, produced by over 6000 plant species worldwide. However, an unmet need remains for confirmative measurement of PAs in routine clinical tests. Here, we develop a visual, easy-to-use, and economic mesoporous silica-electrochemiluminescence (MPS-ECL) sensor for point-of-care (POC) testing of PAs, utilizing MPS's amplification effect on positive ions. The relationship between PAs' different structures and corresponding Ru(bpy)32+ ECL activity shows that reaction mechanism, stability of intermediate, molecular geometry and alternative anodic reactivity significantly affect the ECL activity. The ECL intensity varies among different PAs: monocrotaline ˃ senecionine N-oxide ˃ retrorsine ˃ senkirkine. The POC sensors possess excellent linearity (0.9993 > R2 > 0.9944), low detection limits (0.02 µM-0.07 µM), and good recoveries (90.12%-105.93%), indicating good accuracy and practicability. The portable and low-cost sensor is user-friendly, which holds promise to be applied to POC testing of PAs in drugs, food products, and clinical samples, which is promising for initial assessments of PA-induced health risk.

Nrf2-mediated liver protection by 18ß-glycyrrhetinic acid against pyrrolizidine alkaloid-induced toxicity through PI3K/Akt/GSK3ß pathway.

BACKGROUND: Misusage of pyrrolizidine alkaloid (PA)-containing plants or unaware intake of PA-contaminated foodstuffs causes thousands of PA poisoning cases in humans. PA intoxication is accompanied by oxidative stress and subsequent extensive hepatocellular damage. Our previous study has demonstrated that 18ß-glycyrrhetinic acid (GA), a bioactive constituent of liquorice, prevented PA-induced hepatotoxicity in rats, however the underlying mechanisms remain unclear. OBJECTIVE: This study aims to explore the mechanisms underlying the hepato-protective effect of GA in combating retrorsine (RTS, a representative toxic PA)-induced liver injury. METHODS: Histological and biochemical assessments were employed to evaluate the protective effect of GA on RTS-induced hepatotoxicity in rats. Sulforhodamine B assay, real-time PCR, western blotting, and immunostaining were used to explore the underlying mechanisms in human hepatocytes and rats. RESULTS: Our findings demonstrated that GA alleviated RTS-induced elevation of serum ALT and bilirubin levels, as well as hepatocytes necrosis and sinusoidal endothelial cells (SECs) damage in rats. GA also enhanced the activities and expressions of several antioxidant enzymes through upregulating nuclear factor-erythroid 2-related factor2 (Nrf2). Moreover, inhibition of Nrf2 blocked the hepatoprotective effect of GA against RTS intoxication. Mechanistically, GA increased the phosphorylation of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) and enhanced glycogen synthase kinase 3 beta (GSK3ß) inhibitory phosphorylation at serine 9, thus promoting the nuclear accumulation of Nrf2 and activating its downstream targets. CONCLUSION: This study for the first time demonstrated that GA exerted protective effects against RTS-induced liver injury by potentiating the Nrf2-mediated antioxidant system through PI3K/Akt/GSK3ß pathway. The findings indicated that GA may serve as a potential candidate drug for the treatment of PA intoxication.

Neuroprotective Properties of Quinone Reductase 2 Inhibitor M-11, a 2-Mercaptobenzimidazole Derivative.

The ability of NQO2 to increase the production of free radicals under enhanced generation of quinone derivatives of catecholamines is considered to be a component of neurodegenerative disease pathogenesis. The present study aimed to investigate the neuroprotective mechanisms of original NQO2 inhibitor M-11 (2-[2-(3-oxomorpholin-4-il)-ethylthio]-5-ethoxybenzimidazole hydrochloride) in a cellular damage model using NQO2 endogenous substrate adrenochrome (125 µM) and co-substrate BNAH (100 µM). The effects of M-11 (10-100 µM) on the reactive oxygen species (ROS) generation, apoptosis and lesion of nuclear DNA were evaluated using flow cytometry and single-cell gel electrophoresis assay (comet assay). Results were compared with S29434, the reference inhibitor of NQO2. It was found that treatment of HT-22 cells with M-11 results in a decline of ROS production triggered by incubation of cells with NQO2 substrate and co-substrate. Pre-incubation of HT-22 cells with compounds M-11 or S29434 results in a decrease of DNA damage and late apoptotic cell percentage reduction. The obtained results provide a rationale for further development of the M-11 compound as a potential neuroprotective agent.

Chemical Constituents and Biological Properties of Genus Doronicum (Asteraceae).

The genus Doronicum, belonging to tribe Senecioneae (Fam. Asteraceae), is found mainly in the Asia, Europe and North Africa. This genus of plant has always been used in traditional medicinal treatments due to the many biological properties shown such as killing parasitic worms and for relieving constipation, as well as to improve heart health, to alleviate pain and inflammation, to treat insect bites, etc. According to the World Flora the genus Doronicum contains 39 subordinate taxa.[1-3] The purpose of this article, which covers data published from 1970 to 2021 with more than 110 articles, aims to carry out a complete and critical review of the Doronicum genus, examining traditional uses and reporting the antioxidant, antimicrobial, anti-inflammatory and antitumor activity shown from crude extracts or essential oils, and from single isolated compounds. Furthermore, critical considerations of the published data have been highlighted by comparing them with the results obtained from species of other genus belonging to the Asteraceae family.

Active Transport of Hepatotoxic Pyrrolizidine Alkaloids in HepaRG Cells.

1,2-unsaturated pyrrolizidine alkaloids (PAs) are secondary plant metabolites occurring as food contaminants that can cause severe liver damage upon metabolic activation in hepatocytes. However, it is yet unknown how these contaminants enter the cells. The role of hepatic transporters is only at the beginning of being recognized as a key determinant of PA toxicity. Therefore, this study concentrated on assessing the general mode of action of PA transport in the human hepatoma cell line HepaRG using seven structurally different PAs. Furthermore, several hepatic uptake and efflux transporters were targeted with pharmacological inhibitors to identify their role in the uptake of the PAs retrorsine and senecionine and in the disposition of their N-oxides (PANO). For this purpose, PA and PANO content was measured in the supernatant using LC-MS/MS. Also, PA-mediated cytotoxicity was analyzed after transport inhibition. It was found that PAs are taken up into HepaRG cells in a predominantly active and structure-dependent manner. This pattern correlates with other experimental endpoints such as cytotoxicity. Pharmacological inhibition of the influx transporters Na+/taurocholate co-transporting polypeptide (SLC10A1) and organic cation transporter 1 (SLC22A1) led to a reduced uptake of retrorsine and senecionine into HepaRG cells, emphasizing the relevance of these transporters for PA toxicokinetics.

Current Knowledge and Perspectives of Pyrrolizidine Alkaloids in Pharmacological Applications: A Mini-Review.

Pyrrolizidine alkaloids (PAs) are a widespread group of secondary metabolites in plants. PAs are notorious for their acute hepatotoxicity, genotoxicity and neurological damage to humans and animals. In recent decades, the application of PAs for beneficial biological activities to cure disease has drawn greater attention. Here, we review the current knowledge regarding the pharmacological properties of PAs and discuss PAs as promising prototypes for the development of new drugs.

Fortuneicyclidins A and B, Pyrrolizidine Alkaloids with a 7-Azatetracyclo[5.4.3.0.02,8]tridecane Core, from Cephalotaxus fortunei.

Fortuneicyclidins A (1) and B (2), a pair of epimeric pyrrolizidine alkaloids containing an unprecedented 7-azatetracyclo[5.4.3.0.02,8]tridecane core, were isolated from the seeds of Cephalotaxus fortunei, along with two biogenetically relative known analogues, 3 and 4. The structures were determined by multiple spectral techniques and chemical derivatization methods. Compound 1 showed inhibitory activity against α-glucosidase.

Genotoxicity of selected pyrrolizidine alkaloids in human hepatoma cell lines HepG2 and Huh6.

INTRODUCTION: Pyrrolizidine alkaloids (PAs) are found in many plant species as secondary metabolites which affect humans via contaminated food sources, herbal medicines and dietary supplements. Hundreds of compounds belonging to PAs have been identified. PAs undergo hepatic metabolism, after which they can induce hepatotoxicity and carcinogenicity. Many aspects of their mechanism of carcinogenicity are still unclear and it is important for human risk assessment to investigate this class of compounds further. MATERIAL AND METHODS: Human hepatoma cells HepG2 were used to investigate the genotoxicity of different chemical structural classes of PAs, namely europine, lycopsamine, retrorsine, riddelliine, seneciphylline, echimidine and lasiocarpine, in the cytokinesis-block micronucleus (CBMN) assay. The different ester type PAs europine, seneciphylline, and lasiocarpine were also tested in human hepatoma Huh6 cells. Six different PAs were investigated in a crosslink comet assay in HepG2 cells. RESULTS: The maximal increase of micronucleus formation was for all PAs in the range of 1.64-2.0 fold. The lowest concentrations at which significant induction of micronuclei were found were 3.2 µM for lasiocarpine and riddelliine, 32 µM for retrorsine and echimidine, and 100 µM for seneciphylline, europine and lycopsamine. Significant induction of micronuclei by lasiocarpine, seneciphylline, and europine were achieved in Huh6 cells at similar concentrations. Reduced tail formation after hydrogen peroxide treatment was found in the crosslink comet assay for all diester type PAs, while an equimolar concentration of the monoesters europine and lycopsamine did not significantly reduce DNA migration. CONCLUSION: The widely available human hepatoma cell lines HepG2 and Huh6 were suitable for the assessment of PA-induced genotoxicity. Selected PAs confirmed previously published potency rankings in the micronucleus assay. In HepG2 cells, the crosslinking activity was related to the ester type, which is a first report of PA mediated effects in the comet assay.

Lycopsamine inhibits the proliferation of human lung cancer cells via induction of apoptosis and autophagy and suppression of interleukin-2.

PURPOSE: Lycopsamine is an active pyrrolizidine alkaloid that shows significant bioactivity. Herein, lycopsamine was evaluated for the first time for its anti-lung cancer activity. Its effects on cellular apoptosis, autophagy, cell cycle and IL-2 gene were also examined. METHODS: The human lung cancer A549 and normal MRC5 cells were used in the study. MTT assay was used to determine the cytotoxicity of lycopsamine. Transmission electron microscopy (TEM) and western blotting were implemented for analyzing autophagy. DAPI staining, Annexin V/FITC/Propidium iodide (PI) and western blotting assays were used to study cellular apoptosis. Cell cycle was examined through flow cytometry. The expression of IL-2 gene was monitored by western blotting. RESULTS: Lycopsamine targeted the proliferation rate and reduced it remarkably in a dose-dependent manner. On searching for underlying mechanism, the antiproliferative effect of lycopsamine was due to autophagy and the expressions of pro-autophagy proteins (LC3-I, LC3-II, Beclin-1) increased on drug exposure. Furthermore, the antiproliferative effects were also found to be mediated via apoptosis induction and were associated with increased Bax and decreased Bcl-2 levels. Next, flow cytometry showed that lycopsamine inhibited cell cycle progression at G2/M-check point in lung cancer cells. Furthermore, the expressions of IL-2 gene decreased after lycopsamine treatment of these cells. In conclusion, on testifying the current designed hypothesis, lycopsamine showed significant antiproliferative effects in A549 lung cancer cells in a dose reliant manner. The antiproliferative effects of lycopsamine were associated with its autophagy inducing, apoptosis inducing, and inhibiting IL-2 expression, potential. CONCLUSION: Taken together, lycopsamine is a potent anti-lung cancer agent and can be a lead molecule in lung cancer treatment.

Antitumor activity of nervosine VII, and the crosstalk between apoptosis and autophagy in HCT116 human colorectal cancer cells.

Nervosine VII is one of the known saturated pyrrolizidine alkaloids isolated from the plant of Liparis nervosa. This is first study to investigate the antitumor activity of nervosine VII in vitro, and the results indicated that nervosine VII induced autophagy and apoptosis in HCT116 human colorectal cancer cells. Mechanistic studies showed that nervosine VII-induced apoptosis was associated with the intrinsic pathway by the activation of caspase-9, -3 and -7. Autophagy induced by nervosine VII was characteristic with the regulation of autophagic markers including the increase of LC3-II and beclin 1 proteins, and the decrease of p62 protein. Nervosine VII simultaneously induced autophagy and apoptosis by activated MAPKs signaling pathway including JNK, ERK1/2 and p38, suppressing the p53 signaling pathway.

Expansion, in vivo-ex vivo cycling, and genetic manipulation of primary human hepatocytes.

Primary human hepatocytes (PHHs) are an essential tool for modeling drug metabolism and liver disease. However, variable plating efficiencies, short lifespan in culture, and resistance to genetic manipulation have limited their use. Here, we show that the pyrrolizidine alkaloid retrorsine improves PHH repopulation of chimeric mice on average 10-fold and rescues the ability of even poorly plateable donor hepatocytes to provide cells for subsequent ex vivo cultures. These mouse-passaged (mp) PHH cultures overcome the marked donor-to-donor variability of cryopreserved PHH and remain functional for months as demonstrated by metabolic assays and infection with hepatitis B virus and Plasmodium falciparum mpPHH can be efficiently genetically modified in culture, mobilized, and then recultured as spheroids or retransplanted to create highly humanized mice that carry a genetically altered hepatocyte graft. Together, these advances provide flexible tools for the study of human liver disease and evaluation of hepatocyte-targeted gene therapy approaches.

Development of a two-layer transwell co-culture model for the in vitro investigation of pyrrolizidine alkaloid-induced hepatic sinusoidal damage.

Pyrrolizidine alkaloids (PAs) are hepatotoxic and specifically damage hepatic sinusoidal endothelial cells (HSECs) via cytochrome P450 enzymes (CYPs)-mediated metabolic activation. Due to the lack of CYPs in HSECs, currently there is no suitable cell model for investigating PA-induced HSEC injury. This study aimed to establish a two-layer transwell co-culture model that mimics hepatic environment by including HepaRG hepatocytes and HSECs to evaluate cytotoxicity of PAs on their major target HSECs. In this model, PAs were metabolically activated by CYPs in HepaRG hepatocytes to generate reactive pyrrolic metabolites, which react with co-cultured HSECs leading to HSEC damage. Three representative PAs, namely retrorsine, monocrotaline, and clivorine, induced significant concentration-dependent cytotoxicity in HSECs in the co-culture model, but did no cause obvious cytotoxicity directly in HSECs. Using the developed co-cultured model, further mechanism studies of retrorsine-induced HSEC damage demonstrated that the reactive pyrrolic metabolite generated by CYP-mediated bioactivation in HepaRG hepatocytes caused formation of pyrrole-protein adducts, reduction of GSH content, and generation of reactive oxygen species in HSECs, leading to cell apoptosis. The established co-culture model is reliable and applicable for cytotoxic assessment of PA-induced HSEC damage and offers a novel platform for screening toxicity of different PAs on their target cells.

Characterizing the selectivity of ER α-glucosidase inhibitors.

The endoplasmic reticulum (ER) contains both α-glucosidases and α-mannosidases which process the N-linked oligosaccharides of newly synthesized glycoproteins and thereby facilitate polypeptide folding and glycoprotein quality control. By acting as structural mimetics, iminosugars can selectively inhibit these ER localized α-glycosidases, preventing N-glycan trimming and providing a molecular basis for their therapeutic applications. In this study, we investigate the effects of a panel of nine iminosugars on the actions of ER luminal α-glucosidase I and α-glucosidase II. Using ER microsomes to recapitulate authentic protein N-glycosylation and oligosaccharide processing, we identify five iminosugars that selectively inhibit N-glycan trimming. Comparison of their inhibitory activities in ER microsomes against their effects on purified ER α-glucosidase II, suggests that 3,7a-diepi-alexine acts as a selective inhibitor of ER α-glucosidase I. The other active iminosugars all inhibit α-glucosidase II and, having identified 1,4-dideoxy-1,4-imino-D-arabinitol (DAB) as the most effective of these compounds, we use in silico modeling to understand the molecular basis for this enhanced activity. Taken together, our work identifies the C-3 substituted pyrrolizidines casuarine and 3,7a-diepi-alexine as promising "second-generation" iminosugar inhibitors.

In vitro ovicidal effect of a Senecio brasiliensis extract and its fractions on Haemonchus contortus.

BACKGROUND: Haemonchosis affects sheep husbandry and its treatment is often compromised due to the development of anthelminthic resistance. Plant-derived bioactive compounds have been studied as alternative to control Haemonchus contortus. The objective of this study was to evaluate the effect of Senecio brasiliensis extracts on H. contortus egg hatching and infective larvae migration. RESULTS: The aqueous extract from dried and fresh plant and alkaloid-enriched fraction of the previously dried leaves of S. brasiliensis inhibited H. contortus egg hatching. The main plant compound in alkaloid fraction was integerrimine, a pyrrolizidine alkaloid (PA). However, the aqueous extract from dried plant displayed higher efficacy when compared to their alkaloid enriched or non-polar fractions, meaning that, although PAs contributed to the ovicidal effect, other compounds in the plant can also contribute to their effect. Furthermore, the aqueous extract from dried plant also had higher efficacy than aqueous extract from fresh plant in larvae migration inhibition. Finally, extract from dried plant presented low in vitro cytotoxic effect. CONCLUSION: Taken together our results suggest a good anthelmintic effect of S. brasiliensis, especially when aqueous extract is prepared from dried plant. Further in vivo studies should be performed focused on forms of administration of this extract in rearing sheep.

Sensitization of Human Liver Cells Toward Fas-Mediated Apoptosis by the Metabolically Activated Pyrrolizidine Alkaloid Lasiocarpine.

SCOPE: Pyrrolizidine alkaloids (PAs) are common phytotoxins. Intoxication can lead to liver damage. Previous studies showed PA-induced apoptosis in liver cells. However, the exact role of the extrinsic apoptotic pathway has not been investigated yet. This study aims to analyze whether the PA representative lasiocarpine sensitizes human liver cells toward extrinsic Fas-mediated apoptosis. METHODS AND RESULTS: HepG2 cells with limited xenobiotic metabolic activity are used to analyze metabolism-dependent effects. External in vitro metabolism is simulated using rat or human liver enzymes. Additionally, metabolically competent HepaRG cells are used to confirm the observed effects in a human liver cell system with internal xenobiotic metabolism. Metabolized lasiocarpine decreases cell viability and induces Fas receptor gene expression in both cell lines. Increased Fas receptor protein expression on the cell surface is demonstrated by flow cytometry. The addition of a Fas ligand-simulating antibody induces apoptosis. Induction of extrinsic Fas-mediated apoptosis is verified by Western blotting for cleaved caspase 8, the initiator caspase of extrinsic apoptosis. All effects are dependent on lasiocarpine metabolism. CONCLUSION: The results demonstrate that metabolically metabolized lasiocarpine sensitizes human liver cells toward Fas-mediated apoptosis. They broaden our knowledge on the hepatotoxic molecular mechanisms of PA as widely distributed food contaminants.

Corrected Structure of Natural Hyacinthacine C1 via Total Synthesis.

Hyacinthacines C1 and C4 are natural products that were isolated from Hyacinthoides non-scripta and Scilla socialis in 1999 and 2007, respectively. Despite their different 1H NMR and 13C NMR spectroscopic data, these compounds have been assigned the same structures, including absolute configurations. This work details the total synthesis of natural (+)-hyacinthacine C1, whose structure is confirmed as being the C-6 epimer of that reported. The synthetic strategy focused on inverting the configuration at C-1 of the final hyacinthacines via operating the inversion at the corresponding carbon atom in three previously synthesized intermediates. To do this, the advanced intermediates were subjected to Swern oxidation, followed by a stereoselective reduction with L-Selectride. This approach led to the synthesis of (+)-5 -epi-hyacinthacine C1 (15), the corrected structure for (+)-hyacinthacine C1 (19), (+)-6,7-di- epi-hyacinthacine C1 (23), and (+)-7- epi-hyacinthacine C1 (29). Glycosidase inhibition assays revealed that (+)-hyacinthacine C1 (19) proved the most active, with IC50 values of 33.7, 55.5, and 78.2 µM, against the α-glucosidase of rice, human lysosome, and rat intestinal maltase, respectively.

Pyrrolizidine Alkaloids: Biosynthesis, Biological Activities and Occurrence in Crop Plants.

Pyrrolizidine alkaloids (PAs) are heterocyclic secondary metabolites with a typical pyrrolizidine motif predominantly produced by plants as defense chemicals against herbivores. They display a wide structural diversity and occur in a vast number of species with novel structures and occurrences continuously being discovered. These alkaloids exhibit strong hepatotoxic, genotoxic, cytotoxic, tumorigenic, and neurotoxic activities, and thereby pose a serious threat to the health of humans since they are known contaminants of foods including grain, milk, honey, and eggs, as well as plant derived pharmaceuticals and food supplements. Livestock and fodder can be affected due to PA-containing plants on pastures and fields. Despite their importance as toxic contaminants of agricultural products, there is limited knowledge about their biosynthesis. While the intermediates were well defined by feeding experiments, only one enzyme involved in PA biosynthesis has been characterized so far, the homospermidine synthase catalyzing the first committed step in PA biosynthesis. This review gives an overview about structural diversity of PAs, biosynthetic pathways of necine base, and necic acid formation and how PA accumulation is regulated. Furthermore, we discuss their role in plant ecology and their modes of toxicity towards humans and animals. Finally, several examples of PA-producing crop plants are discussed.

The History of the Glycosidase Inhibiting Hyacinthacine C-type Alkaloids: From Discovery to Synthesis.

BACKGROUND: The inherent glycosidase inhibitory activity and potentially therapeutic value of the polyhydroxylated pyrrolizidine alkaloids containing a hydroxymethyl substituent at the C-3 position have been well documented. Belonging to this class, the naturally occurring hyacinthacine C-type alkaloids are of general interest among iminosugar researchers. Their selective micromolar α -glycosidase inhibitory ranges (10 - 100 µM) suggest that these azasugars are potential leads for treating type II diabetes. However, the structures of hyacinthacine C1, C3 and C4 are insecure with hyacinthacine C5 being recently corrected. OBJECTIVE: This review presents the hyacinthacine C-type alkaloids: their first discovery to the most recent advancements on the structures, biological activities and total synthesis. CONCLUSION: The hyacinthacine C-type alkaloids are of exponentially increasing interest and will undoubtedly continue to be reported as synthetic targets. They represent a challenging but rewarding synthetic feat for the community of those interested in accessing biologically active iminosugars. Since 2009, ten total syntheses have been employed towards accessing similarly related products but only three have assessed the glycosidase inhibitory activity of the final products. This suggests the need for an accessible and universal glycosidase inhibitory assay so to accurately determine the structure-activity relationship of how the hyacinthacine C-type alkaloids inhibit specific glycosidases. Confirming the correct structures of the hyacinthacine C-type alkaloids as well as accessing various analogues continues to strengthen the foundation towards a marketable treatment for type II diabetes and other glycosidase related illnesses.

S29434, a Quinone Reductase 2 Inhibitor: Main Biochemical and Cellular Characterization.

Quinone reductase 2 (QR2, E.C. 1.10.5.1) is an enzyme with a feature that has attracted attention for several decades: in standard conditions, instead of recognizing NAD(P)H as an electron donor, it recognizes putative metabolites of NADH, such as N-methyl- and N-ribosyl-dihydronicotinamide. QR2 has been particularly associated with reactive oxygen species and memory, strongly suggesting a link among QR2 (as a possible key element in pro-oxidation), autophagy, and neurodegeneration. In molecular and cellular pharmacology, understanding physiopathological associations can be difficult because of a lack of specific and powerful tools. Here, we present a thorough description of the potent, nanomolar inhibitor [2-(2-methoxy-5H-1,4b,9-triaza(indeno[2,1-a]inden-10-yl)ethyl]-2-furamide (S29434 or NMDPEF; IC50 = 5-16 nM) of QR2 at different organizational levels. We provide full detailed syntheses, describe its cocrystallization with and behavior at QR2 on a millisecond timeline, show that it penetrates cell membranes and inhibits QR2-mediated reactive oxygen species (ROS) production within the 100 nM range, and describe its actions in several in vivo models and lack of actions in various ROS-producing systems. The inhibitor is fairly stable in vivo, penetrates cells, specifically inhibits QR2, and shows activities that suggest a key role for this enzyme in different pathologic conditions, including neurodegenerative diseases.

Quinolizidine and Pyrrolizidine Alkaloid Chemical Ecology - a Mini-Review on Their Similarities and Differences.

This mini-review summarizes over 40 years of research on quinolizidine (QAs) and pyrrolizidine alkaloids (PAs). Emphasis is on the chemical ecology of both groups of alkaloids, which serve as general defense compounds against herbivores for the plants producing them. For QAs and PAs, a number of insects (aphids, moths, beetles) have acquired tolerance. These specialists store the alkaloids and use them as defense chemicals against predators. In some PA sequestering moths, the adaptation is even more intricate and advanced. PAs can function as a morphogen to induce the formation of male coremata, inflatable organs that dissipate pheromones. In these insects, PAs are additionally used as a precursor for male pheromones. Female moths utilize their own PAs and those obtained from males via the spermatophore as nuptial gift, to transfer them to the eggs that thus become chemically protected. Novel genomic technologies will allow deeper insights in the molecular evolution of these two classes of alkaloids in plant-insect interactions.