Determination of huperzine A in formulated products by reversed-phase-liquid chromatography using diode array and electrospray ionization mass spectrometric detection.

A precise and selective high-performance liquid chromatographic (HPLC) method with diode-array detection for quantifying huperzine A in formulated products was developed and validated. A liquid chromatographic-mass spectrometric (LC/MS) procedure was devised to confirm the HPLC method. Huperzine A was dissolved in 1,2-dichloroethane, chromatographed on a YMCBasic C18 column, and detected at 308 nm. A gradient mobile phase of 10 mM ammonium acetate (pH = 3.5)--methanol was used. Identification was based on retention time, UV spectra and mass spectra by comparison with a commercial standard. The UV peak areas were used for quantitation of huperzine A content. The correlation coefficient (R2) of the calibration curve was 1 over the range 0.8-11.6 microg/ml. Overall recovery of huperzine A was 103.9% +/- 1.8 (mean +/- SD). Relative standard deviations for intra- and interday precision were < 2%.

Development of a paw paw herbal shampoo for the removal of head lice.

The development and clinical testing of an herbal lice removal shampoo, containing a standardized extract of paw paw, thymol, and tea tree oil, are described. All of these ingredients were selected for their ability to deplete adenosine triphosphate (ATP) levels and, thus, prevent ATP-dependent pesticide resistance. Optimum concentrations of the ingredients, treatment times, and dosing schedules were established through in vitro tests with head lice. In addition to pilot studies involving 21 participants, a final clinical trial, using the optimum shampoo formulation in 16 participants, demonstrated 100% effectiveness in removing head lice and nits.

Robinlin: a novel bioactive homo-monoterpene from Robinia pseudoacacia L. (Fabaceae).

A bioactivity-directed fractionation of the ethanolic extracts of Robinia pseudoacacia L. (Fabaceae) afforded robinlin (1), a novel homo-monoterpene. The structure of 1 was elucidated by spectral analyses of the parent compound as well as its derivatives; 1 showed strong bioactivity in the brine shrimp lethality test (BST).

MtrC, an outer membrane decahaem c cytochrome required for metal reduction in Shewanella putrefaciens MR-1.

Shewanella putrefaciens is a facultative anaerobe that can use metal oxides as terminal electron acceptors during anaerobic respiration. Two proteins, MtrB and Cct, have been identified that are specifically involved in metal reduction. Analysis of S. putrefaciens mutants deficient in metal reduction led to the identification of two additional proteins that are involved in this process. MtrA is a periplasmic decahaem c-type cytochrome that appears to be part of the electron transport chain, which leads to Fe(III) and Mn(IV) reduction. MtrC is an outer membrane decahaem c-type cytochrome that appears to be required for the activity of the terminal Fe(III) reductase. Membrane fractions of mutants deficient in MtrC exhibited a decreased level of Fe(III) reduction compared with the wild type. We suggest that MtrC may be a component of the terminal reductase or may be required for its assembly.

Bioactive flavonoids from the black locust tree, robinia pseudoacacia.

Five flavonoids, acacetin ( 1 ), secundiflorol I ( 2 ), mucronulatol ( 3 ), isomucronulatol ( 4 ), and isovestitol ( 5 ) were isolated, with the fractionation being guided by the brine shrimp lethality test (BST), from the ethanolic extract of the whole plant of Robinia pseudoacacia (Fabaceae). The structures of 1 - 5 were identified by spectral analyses. Compounds 2 - 5 are, for the first time, reported from this species. Corrections have been made for the previous literature assignments of the 13 C NMR resonances of compounds 1 - 3 . Bioactivities in BST and cytotoxicities against a panel of six solid human tumor cell lines were determined, and 1 was significantly cytotoxic in the prostate cell line (PC-3).

Annocherin and (2,4)-cis- and trans-annocherinones, monotetrahydrofuran Annonaceous acetogenins with a C-7 carbonyl group from Annona cherimolia seeds.

The new cytotoxic monotetrahydrofuran Annonaceous acetogenins, annocherin (1) and a mixture of (2,4)-cis- and trans-annocherinones (2 and 3), were isolated from the bioactive methanolic extract of Annonacherimolia seeds. Compounds 1-3 each possess an unusual 7-carbonyl group. Their structures were established on the basis of chemical and spectral evidence. Compounds 1-3 showed significant toxicity in the brine shrimp lethality test and cytotoxicity for six human solid tumor cell lines, with selectivity for the renal cell line (A-498) at potencies equivalent to Adriamycin.

Asitrilobins A and B: cytotoxic mono-THF annonaceous acetogenins from the seeds of Asimina triloba.

The seeds of Asimina triloba have yielded two novel cytotoxic mono-tetrahydrofuran (THF) Annonaceous acetogenins, asitrilobins A (1) and B (2). In addition, annonacin, asimin and asiminacin, which are known, and annomontacin and xylomaticin, which are known but are new in this species, were obtained. Compounds 1 and 2 have a relative stereochemical relationship of erythro/cis/threo across the mono-THF ring with its two flanking hydroxyls and they, thus, represent a new type of acetogenin. Their structures were established on the basis of chemical and spectral evidence. 1 and 2 showed potent bioactivities in the brine shrimp lethality test (BST) and among six human solid tumor cell lines with notable selectivity for the pancreatic cell line (MIA PaCa-2) at ten to one-hundred times the potency of adriamycin.

Two novel acetogenins, annoglaxin and 27-hydroxybullatacin, from Annona glabra.

Two new bioactive Annonaceous acetogenins, annoglaxin (1) and 27-hydroxybullatacin (2), have been isolated from the fractionated ethanolic extracts of the leaves of Annona glabra, directing the fractionation with the brine shrimp lethality test (BST). The structures of 1 and 2 were elucidated on the basis of spectroscopic and chemical methods, and the absolute stereochemistries were determined by the advanced Mosher ester method. 1 presents unusual features of an OH at C-8 and a carbonyl at C-12 and, while less potent than 2, shows interesting selectivity for the human breast carcinoma (MCF-7) cell line. Compound 2 was at least 100 000 times more potent than adriamycin against the human kidney carcinoma (A-498), prostate carcinoma (PC-3), and pancreatic carcinoma (PACA-2) cell lines in our panel of six human solid tumor cell lines.

Two bioactive mono-tetrahydrofuran acetogenins, annoglacins A and B, from Annona glabra.

Two new bioactive mono-THF Annonaceous acetogenins, annoglacins A and B, have been isolated from the fractionated ethanolic extracts of the leaves of Annona glabra, directing the fractionation with the brine shrimp lethality test (BST). Their structures were elucidated based on spectroscopic and chemical methods and the absolute stereochemistries were determined by the advanced Mosher ester method. Annoglacins A and B were selectively 1000 and 10,000 times, respectively, more potent than adriamycin against the human breast carcinoma (MCF-7) and pancreatic carcinoma (PACA-2) cell lines in our panel of six human solid tumor cell lines.

Annonaceous acetogenins: recent progress.

The Annonaceous acetogenins are promising new antitumor and pesticidal agents that are found only in the plant family Annonaceae. Chemically, they are derivatives of long-chain fatty acids. Biologically, they exhibit their potent bioactivities through depletion of ATP levels via inhibiting complex I of mitochondria and inhibiting the NADH oxidase of plasma membranes of tumor cells. Thus, they thwart ATP-driven resistance mechanisms. This review presents the progress made in the chemistry, biology, and development of these compounds since December 1995.

Chemical defense in the zebra swallowtail butterfly, Eurytides marcellus, involving annonaceous acetogenins.

Few herbivores feed on the foliage of the North American paw paw tree, Asimina triloba; notable exceptions are the larvae of the zebra swallowtail butterfly, Eurytides marcellus. Toxic annonaceous acetogenins, produced by A. triloba, are responsible for the relative unpalatability of the leaves. Acetogenins found in A. triloba extracts are potent pesticidal and antineoplastic agents and have emetic activity in vertebrates. In this study, partitioned aqueous MeOH fractions of the bioactive CH2Cl2 extracts, of freeze-dried and pulverized larvae, and of mature butterflies revealed acetogenin content through the use of HPLC coupled to tandem MS (LC-MS/MS). This sensitive technique provides an uncomplicated method for the detection of trace compounds and, in this instance, has confirmed tissue presence of acetogenins that serve a probable role as chemical defense agents against bird predation in zebra swallowtail larvae and adults.

Goniotriocin and (2,4-cis- and -trans)-xylomaticinones, bioactive annonaceous acetogenins from Goniothalamus giganteus.

Goniotricin (1) and a mixture of (2,4-cis and -trans)-xylomaticinones (2), new bioactive annonaceous acetogenins, were isolated from the bark of Goniothalamus giganteus (Annonaceae) by activity-directed fractionation using the brine shrimp lethality test. Compound 1 is the first nonadjacent ring-hydroxylated bis-tetrahydrofuran (THF) acetogenin to be reported. Compound 2 is the cis- and trans-ketolactone mixture of xylomaticin, a known compound whose absolute stereochemistry has not been previously determined. The absolute stereochemistry of 2 was determined by the advanced Mosher's ester method. Both 1 and 2 showed significant and selective cytotoxicities among the six tumor cell lines in our seven-day MTT human solid tumor panel. In the yellow fever mosquito larvae microtiter assay, 1 was quite active, with an ED50 value of 3. 5 microg/mL.

Annojahnin from Annona jahnii: a possible precursor of mono-tetrahydrofuran acetogenins.

A new cytotoxic Annonaceous acetogenin, annojahnin (1), was isolated from the twigs of Annona jahnii (Annonaceae) by bioactivity-directed fractionation using lethality to brine shrimp. Compound 1 represents an unusual type of C-37 Annonaceous acetogenin, lacking either tetrahydrofuran (THF) or epoxide rings, bearing a keto group at C-10, and possessing a double bond located two methylenes away from a vicinal diol. The structure and absolute configuration of 1 were elucidated by 1H and 13C NMR, COSY, and single-relayed COSY and from chemical derivatives. 4-Deoxy-18/21-trans-annomontacin 10-one (4) and 4-deoxy-18/21-cis-annomontacin-10-one (5), two semisynthetic mono-THF acetogenins, were prepared from 1 by reactions that mimic the biogenetic pathways. These acetogenins showed selective cytotoxicities, comparable or superior to adriamycin, among six human solid tumor cell lines. Reduction of the 10-keto of 1, to the racemic 10-OH derivative (3), retained the bioactivities as did the conversion of 1 to 4 and 5.

Mono-tetrahydrofuran acetogenins from Goniothalamus giganteus.

Goniotetracin and a mixture of (2,4-cis and trans)-gonioneninone, new, bioactive, mono-tetrahydrofuran (THF) gamma-lactone and ketolactone acetogenins, were isolated from the bark of Goniothalamus giganteus (Annonaceae) by activity-directed fractionation using the brine shrimp lethality test (BST). The structures were elucidated based on spectroscopic and chemical methods. Their absolute stereochemistries were determined by the advanced Mosher ester method. Both showed selective and significant cytotoxicities, comparable to the potency of adriamycin, to the human pancreatic tumour cell line (PACA-2), while goniotetracin and goniothalamicin were comparable to the activity of rotenone against yellow fever mosquito larvae.

Muricoreacin and murihexocin C, mono-tetrahydrofuran acetogenins, from the leaves of Annona muricata.

Bioactivity-directed fractionation of the leaves of Annona muricata L. (Annonaceae) resulted in the isolation of two new Annonaceous acetogenins, muricoreacin (1) and murihexocin C (2). Compounds 1 and 2 showed significant cytotoxicities among six human tumor cell lines with selectivities to the prostate adenocarcinoma (PC-3) and pancreatic carcinoma (PACA-2) cell lines.

Glabracins A and B, two new acetogenins from Annona glabra.

Two new bioactive bis-THF Annonaceous acetogenins, glabracins A (1) and B (2), and two previously known acetogenins, javoricin (3) and bullatanocin (4), have been isolated from the leaves of Annona glabra by activity-directed fractionation using the brine shrimp lethality test (BST). The structures of 1 and 2 were elucidated based on spectroscopic and chemical methods, and the absolute stereochemistries were partially determined by the advanced Mosher ester method. 1 and 2 showed selective cytotoxicities to certain human tumor cell lines, and 1 was significantly more potent although 1 and 2 differ only in the stereochemistry of their vicinal diols at C-23/24.

Essential structural factors of annonaceous acetogenins as potent inhibitors of mitochondrial complex I.

The annonaceous acetogenins are the most potent of the known inhibitors of bovine heart mitochondrial complex I. These inhibitors act, at the terminal electron transfer step of the enzyme, in a similar way to the usual complex I inhibitors, such as piericidin A and rotenone; however, structural similarities are not apparent between the acetogenins and these known complex I inhibitors. A systematic set of isolated natural acetogenins was prepared and examined for their inhibitory actions with bovine heart mitochondrial complex I to identify the essential structural factors of these inhibitors for the exhibition of potent activity. Despite their very potent activity, the structural requirements of the acetogenins are not particularly rigid and remain somewhat ambiguous. The most common structural units, such as adjacent bis-tetrahydrofuran (THF) rings and hydroxyl groups in the 4- and/or 10-positions, were not essential for exhibiting potent activity. The stereochemistry surrounding the THF rings, surprisingly, seemed to be unimportant, which was corroborated by an exhaustive conformational space search analysis, indicating that the model compounds, with different stereochemical arrangements around the THF moieties, were in fairly good superimposition. Proper length and flexibility of the alkyl spacer moiety, which links the THF and the alpha, beta-unsaturated gamma-lactone ring moieties, were essential for the potent activity. This probably results from some sort of specific conformation of the spacer moiety which regulates the two ring moieties to locate into an optimal spatial position on the enzyme. It is, therefore, suggested that the structural specificity of the acetogenins, required for optimum inhibition, differs significantly from that of the common complex I inhibitors in which essential structural units are compactly arranged and conveniently defined. The structure-activity profile for complex I inhibition is discussed in comparison with those for other biological activities.

Annonaceous acetogenins as natural pesticides: potent toxicity against insecticide-susceptible and -resistant German cockroaches (Dictyoptera: Blattellidae).

Six compounds, representing the mono-tetrahydrofuran (THF) (gigantetrocin A, annomontacin), adjacent bis-THF (asimicin, parviflorin), and nonadjacent bis-THF (sylvaticin, bullatalicin) classes of annonaceous acetogenins, were compared with technical grades of synthetic amidinohydrazone (hydramethylnon), carbamate (propoxur, bendiocarb), organophosphate (chlorpyrifos), and pyrethroid (cypermethrin) insecticides to determine their dietary toxicities to insecticide-resistant and insecticide-susceptible strains of the German cockroach, Blattella germanica (L.). Differential susceptibility occurred among B. germanica nymphs of both strains to this variety of the acetogenins and the 5 conventional synthetic insecticides. The speed of kill (LT50) values against insecticide-susceptible and insecticide-resistant 2nd and 5th instars permitted ranking of all 11 compounds. The adjacent bis-THF acetogenins showed the highest potency among the 3 acetogenin classes. The acetogenins caused high percentages of mortality and delays in development of the 5th instars of both strains. Insecticide-susceptible nymphal development was mainly affected by gigantetrocin A and annomontacin, whereas insecticide-resistant nymphal development was mainly affected by gigantetrocin A and bullatalicin. Most tested acetogenins performed better than the conventional insecticides against both stages of both strains. No growth regulation effects were caused by any of the compounds tested. Low resistance ratios were obtained for most compounds (except chlorpyrifos). Low resistance ratios values for 2nd instars ranged from 0.9 to 2.2 with the natural acetogenins and from 1.0 to 3.8 with the synthetic compounds; the 5th instars ranged from 0.2 to 3.9 with the natural acetogenins and from 0.6 to 8.0 with the synthetic compounds. Insecticidal properties and characteristics of acetogenins and the possible use of acetogenins in baits for cockroach control are discussed.

Glacins A and B: two novel bioactive mono-tetrahydrofuran acetogenins from Annona glabra.

Two new bioactive mono-THF Annonaceous acetogenins, glacins A (1) and B (2), have been isolated, from the fractionated ethanolic extracts of the leaves of Annona glabra, directing the fractionation with the brine shrimp lethality test (BST). The structures of 1 and 2 were elucidated on the basis of spectroscopic and chemical methods, and the absolute stereochemistries were solved by preparing their respective per-Mosher ester derivatives. 1 and 2 showed potent and selective in vitro cytotoxicities among several human solid tumor cell lines.

Three new bioactive bis-adjacent THF-ring acetogenins from the bark of Annona squamosa.

Continuing work on the bioactivity-directed fractionation of the bark of Annona squamosa has resulted in the discovery of three new Annonaceous acetogenins, (2,4-cis and trans)-squamolinone (1), (2,4-cis and trans)-9-oxoasimicinone (2), and bullacin B (3). Compounds 1-3 are all adjacent bis-THF ring acetogenins with 2 representing the first bis-ring acetogenin to contain a carbonyl along its aliphatic chain. Compound 3 was selectively cytotoxic in a panel of six human tumor cell lines with a potency of nearly a million times that of adriamycin against the MCF-7 (human breast adenocarcinoma) cell line.