The growth-inhibitory effects of pawpaw (Asimina triloba [L.] Dunal) roots, twigs, leaves, and fruit against human gastric (AGS) and cervical (HeLa) cancer cells and their anti-inflammatory activities.

BACKGROUND: The pawpaw tree has several beneficial effects. However, no studies have been conducted to address the mechanisms underlying the cytotoxic effects of pawpaw extracts against cancer cells, and no study has investigated the anti-inflammatory effects. Hence, in this study, the growth-inhibitory effects of pawpaw (Asimina triloba [L.] Dunal) extracts against gastric (AGS) and cervical (HeLa) cancer cells and the inhibitory effects of pawpaw extracts against inflammatory factors (NO, TNF-α, IL-6, iNOS, and COX-2) were investigated. METHODS AND RESULTS: The viability of AGS and HeLa cells, the analysis of cell cycle, and the expression of apoptosis marker protein were determined using MTT assay, FACS, western blotting, and TUNEL assays. The inflammatory factors were determined using Griess method, ELISA assay kit, and RAW 264.7 cells. The IC50 values of twig and unripe fruit extracts for AGS cells were 82.01 and 100.61 µg/mL, respectively. For HeLa cells, pawpaw twig extracts exhibited the strongest ability to inhibit cervical cancer cell growth (IC50 = 97.73 µg/mL). Analysis of the cell cycle phase distribution and expression of the apoptosis regulatory proteins BCL-2, BAX, caspase-3, and PARP showed that pawpaw twig, root, and unripe fruit extracts induced Sub G1 cell cycle arrest and apoptosis of AGS and HeLa cells. In addition, the twig, root, and unripe fruit extracts of pawpaw effectively inhibited the inflammatory makers NO, TNF-α, IL-6, and iNOS. Particularly, the twig, root, and unripe fruit extracts at concentrations of 50 µg/mL exhibited > 50% inhibition of TNF-α. CONCLUSIONS: These findings indicate that pawpaw extracts are natural therapeutic agents that may be used for the prevention and treatment of gastric and cervical cancers, and encourage further studies on the anti-inflammatory potential of the pawpaw tree.

Identification and analysis of cell wall glycan epitopes and polyphenol oxidase in pawpaw (Asimina triloba [L.] Dunal) fruit pulp as affected by high pressure processing and refrigerated storage.

This research explores the cell wall composition and polyphenol oxidase activity of two pawpaw (Asimina triloba) fruit varieties, Susquehanna and Green River Belle, that were subjected to high pressure processing and 45 days of refrigerated storage. We hypothesize that high pressure processing may inhibit enzymatic action responsible for pawpaw's deleterious postharvest tissue softening and browning. Glycome profiling uses mAb groupings that recognize 19 groups of glycan epitopes present in most major classes of cell wall glycans and was used to determine cell wall composition. Results show that both varieties have typical type I primary cell walls of flowering dicots. However, differences in the fine cell wall structure between the varieties can be inferred and the varieties behaved differently during refrigerated storage, likely indicating of a difference in cell wall-modifying enzymes present in the primary cell walls. High pressure processing treatment does not seem to be effective at eliminating polyphenol oxidase activity.

Isoquinoline alkaloids from Asimina triloba.

A new aporphine glycoside, (-)-anolobine-9-O-ß-D-glucopyranoside was isolated from the twigs of pawpaw (Asimina triloba) along with 7 known alkaloids including five aporphine alkaloids (anolobine, nornuciferine, norushinsunine, liriodenine, and lysicamine), a proaporhine alkaloid (stepharine), and a tetrahydrobenzylisoquinoline alkaloid (coclaurine). Among these compounds, nornuciferine, lysicamine, stepharine, and coclaurine are reported for the first time from this plant. The structure of the new compound was elucidated by spectroscopic methods, including 1 D, 2 D NMR, and HRESI-MS. The absolute configuration of compounds 1, 2, 7 and 8 was determined by CD experiment.

Phenolic Profiles, Antioxidant and Antimicrobial Activities of Pawpaw Pulp (Asimina triloba [L.] Dunal) at Different Ripening Stages.

In this study, the phenolic components, as well as the antioxidant and antimicrobial activities, of the ripe and unripe fruit of pawpaw (Asimina triloba [L.] Dunal) extracted using five different solvents (distilled water, 95% methanol, 80% methanol, 95% ethanol, and 80% ethanol) were analyzed. The total phenolic content and total flavonoid content were the highest in the 95% ethanol (149.50 mg CAE/g) and 80% ethanol (5.62 mg RE/g) extracts of the unripe fruit, respectively. Analysis of 17 phenolic compounds in pawpaw extracts revealed that epigallocatechin, epicatechin, and p-coumaric acid were the as major compounds, and the amounts of all components significantly decreased with the ripening (P < 0.05). In all antioxidant assays, the 95% ethanol extract of the unripe fruit showed the highest antioxidant activity (EC50 0.22 to 0.93 mg/mL). The pawpaw extracts were more sensitive against Corynebacterium xerosis and Clostridium perfringens. In particular, the 95% ethanol extract of the ripe fruit notably inhibited C. xerosis growth, with minimum inhibitory concentration of 1.56 mg/mL. These results showed that the unripe fruit of pawpaw has abundant phenolic compounds and superior antioxidant activity, and that the 95% ethanol extract of the ripe fruit shows strong inhibitory activity against various microorganisms. Therefore, pawpaw fruit can be utilized as an attractive source of nutrients and therapeutic agents. PRACTICAL APPLICATION: In this study, we identified that the unripe fruit of pawpaw is rich in phenolic compounds and shows strong antioxidant activities. The 95% ethanol extract of the ripe fruit showed strong high inhibitory effect against various microorganisms. These results suggest that pawpaw fruit can serve as a source of antioxidants and delay the aging process. In addition, the fruit could also potentially be utilized as a potential antimicrobial agent.

Targeted and non-targeted analysis of annonaceous alkaloids and acetogenins from Asimina and Annona species using UHPLC-QToF-MS.

In current work, targeted and non-targeted analysis of alkaloids and acetogenins from methanolic extracts of Asimina, Annona species and dietary supplements have been performed using UHPLC-QToF in positive ion mode. Thirty-five standard compounds (twelve alkaloids and twenty-three acetogenins) were used for the analysis. The fragment ions produced by collision induced dissociation (CID) revealed the characteristic cleavage and provided structural information. Aporphine alkaloids and acetogenins are the major groups found in Asimina and Annona species. An untargeted analysis based on high-resolution mass spectrometry was carried out to profile the alkaloids and acetogenins from Asimina species (As. triloba, As. parviflora). Magnoflorine, being a major alkaloid from twigs of As. triloba samples, was used as an example to discuss the fragmentation patterns. In (+)-ESI-MS, magnoflorine gave [M]+ ions at m/z 342.1705. The fragment ions at m/z 297.1127 [M-(CH3)2NH]+, 282.0886 [M-(CH3)3NH]+, 265.0865 [M-(CH3)2NH-CH3OH]+, 237.0916 [M-(CH3)2NH-CH3OH-CO]+, and 222.0681 [M-(CH3)2NH-CH3OH-CO-CH3]+ resulted from the [M]+ molecular ion. One dietary supplement claiming to contain paw paw (As. triloba) was also analyzed and showed a similar profile to twigs of As. triloba. A total of 131 compounds including standard compounds were identified from the different parts of As. triloba and As. parviflora samples. These compounds can be used to distinguish Asimina species. However, for definite identification of these unknown components, further investigation is required. This may provide a model for the rapid screening and structural characterization of bioactive constituents from plant extracts in a single analysis.

Antiangiogenic Activity and Chemical Derivatization of the Neurotoxic Acetogenin Annonacin Isolated from Asimina triloba.

Annonacin (1) was isolated from the North American pawpaw ( Asimina triloba), as reported earlier from these laboratories. Natural 1 was submitted to the rat aortic ring bioassay for evaluation of antiangiogenic activity and was found to inhibit microvessel growth (IC50 value of 3 µM). 4,10,15,20-Tetraazido derivatives of 1 were prepared by permesylation followed by azide displacement or by iodination followed by azide displacement. The tetraazide derived from mesylation/azidation was antiangiogenic, while that derived from iodination/azidation exhibited no appreciable activity. The membrane permeability of natural 1 was evaluated using the parallel artificial membrane permeability assay and was found to be marginally permeable as compared to several clinically relevant compounds.

Correlation Between Acetogenin Content and Antiproliferative Activity of Pawpaw (Asimina triloba [L.] Dunal) Fruit Pulp Grown in Korea.

Pawpaw (Asimina triloba [L.] Dunal) is widely cultivated in Korea for its fruit, which contains bioactive compounds, such as acetogenins. In this study, we investigated the acetogenin content and antiproliferative activity of pawpaw fruit pulp against various cancer cell lines and evaluated the relationship between these two variables at different maturation stages. Unripe fruit had higher antiproliferative activity than ripe fruit, and the activity level depended on acetogenin content. In addition, the presence of specific acetogenins was related to inhibition of certain cancer cell types. The unripe fruit methanol and ethanol extracts (URFM and URFE, respectively) that were rich in acetogenins strongly inhibited the growth of HT-1080, HeLa, and AGS cells by >50% at concentrations of less than 115 µg/mL. These findings indicate that URFM and URFE have therapeutic potential for the treatment of cancer, and our study establishes a basis for further mechanistic studies of the antiproliferative activity of pawpaw fruit. However, it is necessary to further study the anticancer activity of acetogenins from pawpaw fruit using in vivo activity approaches. PRACTICAL APPLICATION: Pawpaw (Asimina triloba) contains acetogenins that can inhibit the growth of cancer cells. In our study, we demonstrate that the antiproliferative activity is higher in unripe than in ripe fruit and depends on acetogenin content. Our results indicate that the extract of unripe pawpaw fruit has value not only as a functional food, but has therapeutic potential for the treatment of cancer as a naturally derived substance that may be less toxic than conventional chemotherapy drugs.

Valorization of underutilized North American pawpaw (Asimina triloba): investigation as a lipid oxidation inhibitor in turkey homogenate model system.

BACKGROUND: The objective of this study was to characterize the ability of extracts from nine varieties of pawpaw pulp standardized to the phenolics level of 0.1% grape seed extract (GSE) on inhibition of the formation of thiobarbituric reactive substances (TBARS) in a turkey model system. The antioxidant activity of the extracts was also determined using four common assays. RESULTS: Over the 240 min sampling time, the standardized pawpaw extracts from all nine varieties were as effective as GSE in inhibiting TBARS formation in turkey muscle homogenate compared to the untreated control. Extracts from all pawpaw varieties and GSE began to inhibit TBARS formation at 60 min of incubation, and by 240 min TBARS were reduced from 35 µmol malondialdehyde kg-1 tissue in the homogenate to which no antioxidant was added to 4-18 µmol malondialdehyde kg-1 tissue in the antioxidant-enriched extracts. There does not appear to be a clear relationship between inhibition of TBARS and any of the antioxidant capacity measurements (ORAC, DPPH inhibition, reducing potential as measured by FRAP assay, or pyrogallol red bleaching). CONCLUSION: The results of this research indicate that there is potential to add value to pawpaw as a functional food source of natural antioxidants, particularly in meat products. © 2017 Society of Chemical Industry.

Antioxidant Activities and Phenolic Compounds of Several Tissues of Pawpaw (Asimina triloba [L.] Dunal) Grown in Korea.

Pawpaw (Asimina triloba [L.] Dunal) possesses antioxidant compounds and strong inhibitors of cancer cells, and is widely cultivated in North America, Canada, and Korea. We analyzed the total phenolic and total flavonoid contents (TPC and TFC, respectively) of pawpaw plants grown in Korea and the antioxidant activities of their roots, twigs, leaves, and fruit with respect to 1,1-diphenyl-2-picryl-hydrazyl (DPPH) radical scavenging activity, 2,2'-azino-bis diammonium salt (ABTS) radical scavenging activity, ferrous (Fe2+ ) chelating ability, and nitrite scavenging activity. Pearson's correlation analyses revealed a linear correlation between TPC and antioxidant activities (r2 >0.69). Root methanol extracts had higher TPC and antioxidant activities than other extracts, which was also consistent with those from the phenolic compounds found in those extracts. Therefore, antioxidant activities seem to depend on the TPC of each pawpaw tissue and pawpaw roots might be useful as a natural source of natural antioxidants.

Phytochemical analysis of ten varieties of pawpaw (Asimina triloba [L.] Dunal) fruit pulp.

Pawpaw (Asimina triloba [L.] Dunal) is a tree fruit with the potential to become a high-value fruit crop, however, its rapid perishability is a significant obstacle. The objective was to determine the phytochemical content and quality characteristics of pawpaw pulp from ten varieties. This study reports for the first time the mass spectral characterization of phenolic acids and flavonoids of pawpaw, which indicated that the predominant polyphenolic compounds were three phenolic acids, protocatechuic acid hexoside, p-coumaroyl hexoside, and 5-O-p-coumaroylquinic acid, and flavonols, particularly (-)-epicatechin, B-type procyanidin dimers and trimers. The relationship between the polyphenolics identified in the current study and future work on polyphenolic oxidase activity will help the process of assessing whether pawpaws should be selected based on potential health benefits, i.e. high polyphenolic content, or increased shelf life in the form of decreased browning that may be afforded pawpaws containing low polyphenolic levels via decreased action of polyphenol oxidase.

Comparative study of chromatographic medium-associated mass and potential antitumor activity loss with bioactive extracts.

Natural product drug discovery programs often rely on the use of silica (Si) gel, reversed-phase media, or size-exclusion resins (e.g., RP-C18, Sephadex LH-20) for compound purification. The synthetic polymer-based sorbent Diaion HP20SS (cross-linked polystyrene matrix) is used as an alternative to prepare purified natural product libraries. To evaluate the impact of chromatographic media on the isolation of biologically active, yet chromatographically unstable natural products, Diaion HP20SS was evaluated side-by-side with normal-phase sorbents for irreversible binding of extract constituents and their effects on bioactivity. An array of chemically diverse natural product-rich extracts was selected as a test panel, and a cell-based reporter assay for hypoxia-inducible factor-1 (HIF-1) was employed to monitor potential change(s) in bioactivity. Silica gel caused significant irreversible binding of three out of 10 extracts. Curcuma longa, Saururus cernuus, and Citrus reticulata extracts showed decreased HIF-1 inhibitory activity after elution through Si gel. An additional nonpolar column wash of HP20SS with EtOAc retained considerable bioactivities of active extracts. In general, Si gel produced the greatest loss of bioactivity. However, HP20SS elution reduced significantly HIF-1 inhibitory activity of certain extracts (e.g., Asimina triloba).

Annonacin in Asimina triloba fruit: implication for neurotoxicity.

INTRODUCTION: The acetogenin, annonacin, from the tropical annonaceous plant Annona muricata, is a lipophilic, mitochondrial complex I inhibitor reported to be more toxic than rotenone to mesencephalic neurons. The temperate annonaceous plant Asimina triloba (pawpaw) is native to the Eastern United States and products are available online. This study determined whether annonacin is in the pawpaw fruit pulp and whether it or the crude ethyl acetate extract is toxic to cortical neurons. METHODS: Pawpaw extract was prepared by pulp extraction with methanol and liquid-liquid partitioning with ethyl acetate (EtOAc). Annonacin was isolated from the crude EtOAc extract via column chromatography using a gradient solvent system of increasing polarity. Mass spectroscopy, nuclear magnetic resonance and infrared spectroscopy were used to compare isolated material with synthetic annonacin data and a natural annonacin sample. Toxicity of isolated annonacin and the total EtOAc extract was determined in primary rat cortical neurons using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. RESULTS: The average concentration of annonacin in the fruit pulp was 0.0701±0.0305mg/g. Purified annonacin (30.07µg/ml) and crude EtOAc extract (47.96µg/ml) induced 50% death of cortical neurons 48h post treatment. Annonacin toxicity was enhanced in the presence of crude extract. DISCUSSION: Pawpaw fruit contains a high concentration of annonacin, which is toxic to cortical neurons. Crude fruit extract also induced neurotoxicity, highlighting the need for additional studies to determine the potential risks of neurodegeneration associated with chronic exposure to pawpaw products.

In vitro trematocidal effects of crude alcoholic extracts of Artemisia annua, A. absinthium, Asimina triloba, and Fumaria officinalis: trematocidal plant alcoholic extracts.

Trematode infections negatively affect human and livestock health, and threaten global food safety. The only approved human anthelmintics for trematodiasis are triclabendazole and praziquantel with no alternative drugs in sight. We tested six crude plant extracts against adult Schistosoma mansoni, Fasciola hepatica, and Echinostoma caproni in vitro. Mortality was best achieved by ethanolic extracts of Artemisia annua (sweet Annie), Asimina triloba (paw-paw), and Artemisia absinthium (wormwood) which, at 2 mg/mL, killed S. mansoni and E. caproni in 20 h or less (except for wormwood), F. hepatica between 16 and 23 h (sweet Annie), or 40 h (paw-paw). Some extracts were active at 0.2 mg/mL and 20 µg/mL, although more time was required to kill trematodes. However, aqueous A. annua and methanol extracts of Fumaria officinalis had no activity. Chromatographic analysis of the three best extracts established that A. annua and A. triloba extracts contained bioactive artemisinin and acetogenins (asimicin and bullatacin), respectively. The anthelmintic activity of our extracts at such low doses indicates that their anthelmintic activity deserves further testing as natural alternative controls for parasites of both animals and humans. Our results also support recent evidence that synergistic effects of multiple bioactive compounds present in crude plant extracts is worth exploring.

The alternative medicine pawpaw and its acetogenin constituents suppress tumor angiogenesis via the HIF-1/VEGF pathway.

Products that contain twig extracts of pawpaw (Asimina triloba) are widely consumed anticancer alternative medicines. Pawpaw crude extract (CE) and purified acetogenins inhibited hypoxia-inducible factor-1 (HIF-1)-mediated hypoxic signaling pathways in tumor cells. In T47D cells, pawpaw CE and the acetogenins 10-hydroxyglaucanetin (1), annonacin (2), and annonacin A (3) inhibited hypoxia-induced HIF-1 activation with IC(50) values of 0.02 microg/mL, 12 nM, 13 nM, and 31 nM, respectively. This inhibition correlates with the suppression of the hypoxic induction of HIF-1 target genes VEGF and GLUT-1. The induction of secreted VEGF protein represents a key event in hypoxia-induced tumor angiogenesis. Both the extract and the purified acetogenins blocked the angiogenesis-stimulating activity of hypoxic T47D cells in vitro. Pawpaw extract and acetogenins inhibited HIF-1 activation by blocking the hypoxic induction of nuclear HIF-1alpha protein. The inhibition of HIF-1 activation was associated with the suppression of mitochondrial respiration at complex I. Thus, the inhibition of HIF-1 activation and hypoxic tumor angiogenesis constitutes a novel mechanism of action for these anticancer alternative medicines.

Identification of annonaceous acetogenins in the ripe fruit of the North American pawpaw ( Asimina triloba ).

The North American pawpaw [ Asimina triloba (L.) Dunal] is a tree fruit in the early stages of commercial production in the United States. This plant contains annonaceous acetogenins in the twigs, unripe fruit, seeds, roots, and bark tissues, which display antitumor, pesticidal, antimalarial, anthelmintic, piscicidal, antiviral, and antimicrobial effects, suggesting many potentially useful applications. However, commercial development of these compounds, based on twig extracts, has been problematic due to limited availability of biomass for extraction. Additionally, acetogenin compounds contained in fruit of pawpaw relatives (soursop or Annona muricata ) and tea made from the leaves of these plants may lead to an increased risk of atypical Parkinsonism later in life with overconsumption of these compounds. Therefore, the objectives of this study were (1) to determine if extracts of ripe pawpaw fruit pulp displayed acetogenin activity, (2) to identify potential acetogenin compounds in the fruit tissue, and (3) to determine if the acetogenin activity varied in diverse pawpaw genotypes and closely related Annona species. Extracts of ripe fruit had total extract weights and bioactivity using the brine shrimp bioassay similar to those from 'NC-1' pawpaw twig tissue. Pulp from soursop, cherimoya, and several additional pawpaw cultivars ('Mitchell', 'Overleese', 'NC-1','Zimmerman', 'Wells', and 'Sunflower') also displayed bioactivity, but peach or banana pulp did not. Ripe pawpaw pulp extract subjected to HPLC-MS analysis identified three prominent acetogenins: asimicin, bullatacin, and bullatalicin. This study points to pawpaw fruit pulp serving as a new biomass source for the extraction of acetogenin compounds for product development. An assessment of the potential human health risk of overconsumption of fruit and acetogenin bioavailability and degradation studies should be pursued.

Paw paw and cancer: annonaceous acetogenins from discovery to commercial products.

Extracts of paw paw ( Asimina triloba, Annonaceae) are among the most potent of the 3500 species of higher plants screened for bioactive compounds in our laboratories at Purdue University. The paw paw is a small tree native to eastern North America; its edible fruits (sometimes referred to as "Indiana Bananas") have nurtured mankind for centuries. Activity-directed fractionation of the paw paw extracts, using the brine shrimp lethality bioassay, led to the isolation and molecular characterization of over 50 unique annonaceous acetogenins. Fractionation of extracts from related species resulted in the identification of over 150 additional acetogenins. The annonaceous acetogenins are derivatives of long-chain (C32 or C34) fatty acids. They are potent inhibitors of mitochondrial (complex I) as well as cytoplasmic (anaerobic) production of adenosine triphosphate (ATP) and the related nucleotides. The powerful cytotoxicity, in vivo antitumor, pesticidal, antimalarial, anthelmintic, piscicidal, antiviral, and antimicrobial effects indicated a myriad of potentially useful applications. Commercial development of these compounds uses natural mixtures of active components, incorporated into pesticidal, topical, and dietary supplement products. Successful applications and commercial products include a shampoo, highly effective in treating infestations of head lice, fleas, and ticks; a series of pesticidal sprays, which protects host plants against a diversity of pests; and an ointment for treatment of oral herpes (HSV-1) and other skin afflictions. The extract (in capsule form) enhances a mixture of natural anthelmintics. In addition, an encapsulated extract has been effectively used by certain cancer patients as a botanical supplement product.

[Study on the extraction method and constituents of papaw seed oil].

The extraction method and the condition of supersonic extraction to papaw seed were studied. The result showed that supersonic extraction was a good method with less time and high extraction rate. And the orthogonal experiment proved that the best condition was that the papaw seed was extracted 3 time; the proportion of dissolvent and the seed was 1: 10, and the time for once was 20 min. The methyl esterification method and the condition of GC for the fatty acid of oil and its content: C15H31COOH (17.3%), C17 H35COOH (5.22%), C19H39COOH (1.12%), C17H33COOH (69.25%), C17H31COOH (4.31%), C18H35COOH (1.68%), C19H37 COOH (0.75%), C20H39COOH (0.33%). The content of unsatisfied fatty acid was 75.92%.

[Preparation and purification of polyclonal antibody against chymopapain].

OBJECTIVE: To prepare and purify polyclonal antibody against chymopapain, and to make a foundation for establishing an immunossay for chymopapain. METHODS: New Zealand rabbit was immunized with chymopapain. Antiserum was purified by Protein A and analyzed by ELISA. RESULTS: The titer of the antiserum obtained in this experiment by ELISA was up to 1:380000 and the purity was proved to be high by SDS-PAGE.

Asimitrin and 4-hydroxytrilobin, new bioactive annonaceous acetogenins from the seeds of Asimina triloba possessing a bis-tetrahydrofuran ring.

Bioactivity-directed fractionation of the seeds of Asimina triloba resulted in the isolation of asimitrin (1) and 4-hydroxytrilobin (2). Compound 1 represents an adjacent ring-hydroxylated bis-tetrahydrofuran (THF) acetogenin. Compound 2 has an adjacent bis-THF ring with two flanking hydroxyl groups and a alpha,beta-unsaturated gamma-lactone with a 4-hydroxyl group. Compounds 1 and 2 showed cytotoxic selectivity, with 100-10 000 times the potency of adriamycin against prostate adenocarcinoma (PC-3) and colon adenocarcinoma (HT-29) cell lines.

[Purification of chymopapain and its effect on nucleus pulposus in vitro].

OBJECTIVE: To establish a simple and effective procedure for purification of chymopapain and study about its effect on nucleus pulposus in vitro. METHODS: Chymopapain was purified by ion exchange chromatograph and tested its effect on nucleus pulposus in vitro. RESULTS: A simple and effective procedure for purification of chymopapain was established and the chymopapain did degrade nucleus pulposus. CONCLUSION: The ion exchange chromatograph was a simple and effective procedure for purification of chymopapain. It is necessary to study its effect on nucleus pulposus in vivo.