Overview of piperlongumine analogues and their therapeutic potential.

Natural products have long been an important source for discovery of new drugs to treat human diseases. Piperlongumine (PL) is an amide alkaloid isolated from Piper longum L. (long piper) and other piper plants and has received widespread attention because of its diverse biological activities. A large number of PL derivatives have been designed, synthesized and assessed in many pharmacological functions, including antiplatelet aggregation, neuroprotective activities, anti-diabetic activities, anti-inflammatory activities, anti-senolytic activities, immune activities, and antitumor activities. Among them, the anti-tumor effects and application of PL and its derivatives are most extensively studied. We herein summarize the development of PL derivatives, the structure and activity relationships (SARs), and their therapeutic potential on the treatments of various diseases, especially against cancer. We also discussed the challenges and future directions associated with PL and its derivatives in these indications.

In vitro anthelmintic activity of the crude hydroalcoholic extract of Piper cubeba fruits and isolated natural products against gastrointestinal nematodes in sheep.

This study describes the in vitro anthelmintic activity of a hydroalcoholic extract from the fruit of Piper cubeba and its major isolated components against the eggs and larvae of gastrointestinal nematodes obtained from naturally-infected ovines. In vitro anthelmintic activity was evaluated using the egg hatch test (EHT), larval development test (LDT) and L3 migration inhibition test (LMT). The extract showed ovicidal and larvicidal activity, with an EC50 of 200 µg/mL and 83.00 µg/mL in the EHT and LDT, respectively. The extract inhibited 100% of larval migration at the lowest tested concentration (95 µg/mL). The crude extract was purified using successive silica gel chromatographic columns, which revealed the lignans hinokinin, cubebin and dihydrocubebin as the major compounds that were present, which were then used in in vitro tests. Cubebin, dihydrocubebin and hinokinin showed higher activity than the crude extract, with an EC50 for ovicidal activity of 150.00 µg/mL, 186.70 µg/mL and 68.38 µg/mL, respectively. In the LDT, cubebin presented an EC50 of 14.89 µg/mL and dihydrocubebin of 30.75 µg/mL. Hinokinin inhibited 100% the larval development at all concentrations evaluated. In the LMT, dihydrocubebin inhibited 100% the larval migration in all concentrations evaluated while cubebin and hinokinin showed EC50 values of 0.89 µg/mL and 0.34 µg/mL, respectively. P. cubeba extract is rich in several classes of active compounds, but here we demonstrate that the described anthelmintic activity may be related to the presence of these lignans, which are present in larger concentrations than other components of the extract. Our results demonstrate for first time the anthelmintic activity against gastrointestinal nematodes in sheep for this class of special metabolites that are present in P. cubeba fruit. However, future detailed studies are needed to evaluate the effectiveness of P. cubeba fruits extract and active lignans in in vivo tests.

HPLC-Guided Isolation, Purification and Characterization of Phenylpropanoid and Phenolic Constituents of Nutmeg Kernel (Myristica fragrans).

Many studies on the biological activities of nutmeg continue to appear in the literature. The most common targets include GIT, CNS, oxidative stress and inflammation. However, results obtained from most studies are often inconsistent due to the variability of utilized samples, lack of standardized nutmeg products or insufficient amounts of pure compounds for comprehensive follow-up investigation. To address the consistency and supply issue we utilized available technology to develop a reproducible procedure for preparation of specific extracts and isolation of the major phenolic constituents present in nutmeg kemel. A well-defined and reproducible sequence of extraction, fractionation and chromatographic purification was adopted and was guided by HPLC fingerprinting. Spectroscopic methods, mainly NMR, and mass spectrometry were utilized to identify each compound. Thirteen compounds were isolated in a pure form and identified as: elemicin (1), isoelemicin (2), myristicin (4), surinamensin (5), malabaricone C (6), 2-(3'-allyl-2',6'-dimethoxy-phenyloxy)-l- acetoxy-(3,4-dimethoxyphenyl)-propyl ester (7), methoxylicarin A (8), licarin A (9), malabaricone B (10), licarin C (11), 5'-methoxylicarin B (12), licarin B (13), and 2-(3'-allyl-2',6'-dimethoxy-phenyloxy)-l-methyl-5-methoxy-1,2-dihydrobenzofuran (3, a new compound). With repeated isolation runs, these pure compounds can be prepared in quantities sufficient for biological evaluation as needed. The availability of purified compounds will also allow the development of specific, accurate, and sensitive analytical procedures for pharmacokinetic studies and for quality control of nutmeg products. Both aspects are essential for nutmeg-focused drug discovery. The same approach can also be adapted to other medicinal plants of potential interest.

Two new peroxy fatty acids with antibacterial activity from Ophioglossum thermale Kom.

Two new peroxy fatty acids, thermalic acids A (1) and B (2), together with eight known compounds, (3ß)-methyl-3-hydroxy-urs-11-en-28 oate (3), luteolin (4), quercetin (5), 3-methoxyquercetin (6), ophioglonol (7), ophioglonol 4'-O-α-D-glucopyranoside (8), pedunculosumoside B (9), syringol (10), were isolated from the herba of Ophioglossum thermale Kom. The structures of 1 and 2 were identified by HRESIMS, EIMS, 1D and 2D NMR, and electronic circular dichroism (ECD) spectra. Both two acids exhibited potential antibacterial activities against Staphylococcus aureus, Bacillus subtilis, and Escherichia coli. This is the first report of peroxy fatty acids isolated from herbaceous plants of Ophioglossaceae.

[Chemical constituents from twigs of Piper hancei].

OBJECTIVE: To investigate the chemical constituents from the twigs of Piper hancei. METHODS: The chemical constituents were isolated and purified by means of chromatographic techniques including silica gel,Sephadex LH-20 and preparative RP-HPLC. Their structures were elucidated on the basis of physicochemical properties and spectral analysis. RESULTS: Eight compounds were isolated and identified as 4-allylpyrocatechol(I), piperlonguminine(II), d-sesamin(Ill), beta-sitosterol (IV), pellitorine(V), piperolactam A(VI) and piperolactam D(VII), respectively. CONCLUSION: Compound I, III, VI and VII are isolated from Piper hancei for the first time.

New phenyl derivatives from endophytic fungus Aspergillus flavipes AIL8 derived of mangrove plant Acanthus ilicifolius.

Two new aromatic butyrolactones, flavipesins A (1) and B (2), two new natural products (3 and 4), and a known phenyl dioxolanone (5) were isolated from marine-derived endophytic fungus Aspergillus flavipes. The structures of compounds 1-5 were elucidated by 1D- and 2D-NMR and MS analysis, the absolute configurations were assigned by optical rotation and CD data, and the stereochemistry of 1 was determined by X-ray crystallography analysis. 1 demonstrated lower MIC values against Staphylococcus aureus (8.0 µg/mL) and Bacillus subtillis (0.25 µg/mL). 1 also showed the unique antibiofilm activity of penetration through the biofilm matrix and kills live bacteria inside mature S. aureus biofilm.

Adipogenic effects of piperlonguminine in 3T3-L1 cells and plasma concentrations of several amide constituents from Piper chaba extracts after treatment of mice.

In our previous study, piperlonguminine from the fruit of Piper chaba was reported to promote adipogenesis in 3T3-L1 cells like the peroxisome proliferator-activated receptor-γ (PPARγ) agonist, troglitazone. In the present study, the mode of action of piperlonguminine in cells was examined. Piperlonguminine increased mRNA levels of adiponectin, glucose transporter 4, and fatty acid-binding protein (aP2). It also increased mRNA levels of PPARγ2 but, unlike troglitazone, piperlonguminine did not activate PPARγ directly in a nuclear receptor cofactor assay. Analyses of plasma from mice treated with piperlonguminine, piperine, and retrofractamide A, and an extract of the fruit, showed that concentrations of piperlonguminine were higher than those of piperine and retrofractamide A, and that the "area-under-the-curve" of piperine increased following in vivo administration of the extract.

In vitro inhibitory effect of piperlonguminine isolated from Piper longum on human cytochrome P450 1A2.

Piperlonguminine (PL), a major alkaloid isolated from Piper longum fruits, shows several biological activities including anti-tumor, anti-hyperlipidemic and anti-inflammatory effects. Although there have been studies of the biological effects of PL, the potential drug-interaction effect of PL following evaluation of inhibitory effects of cytochrome P450 (CYP) activities was not investigated. Here, to investigate the inhibitory effects of PL on the activities of CYP isoforms, CYP inhibition assays were conducted using a cocktail of probe substrates in pooled human liver microsome (HLMs) and human recombinant cDNA-expressed CYP. PL strongly inhibited CYP1A2-mediated phenacetin O-deethylation with an IC50 value of 8.8 µM, as NADPH-independent inhibition, while other CYPs were not significantly inhibited. A Lineweaver-Burk plot resulted in the inhibition mechanism of PL being divided into two different modes, reversible competitive inhibition in a low concentration range of 0-16 µM with a Ki value of 1.39 µM and uncompetitive inhibitory behavior at a high concentration range of 16-40 µM. In addition, PL only decreased CYP 1A2-catalyzed phenacetin O-deethylase activity with IC50 values of 10.0 µM in human recombinant cDNA-expressed 1A2, not 1A1. Overall, this is the first investigation of potential herb-drug interactions associated with PL conducted by identifying the competitive inhibitory effects of PL on CYP1A2 in HLMs.

GC/GC-MS analysis, isolation and identification of bioactive essential oil components from the Bhutanese medicinal plant, Pleurospermum amabile.

We have hydrodistilled the essential oil (EO) from the aerial parts of the Bhutanese medicinal plant, Pleurospermum amabile using a Clevenger apparatus and evaluated this EO by GC/GC-MS and NMR analysis followed by testing for bioactivity. The GC-MS analysis identified 52 compounds with (E)-isomyristicin as a major component (32.2%). Repeated purification yielded four compounds; (E)-isomyristicin (1), (E)-isoapiol (2), methyl eugenol (3) and (E)-isoelemicin (4). Compound 2 and the mother EO showed the best antiplasmodial activity against the Plasmodium falciparum strains, TM4/8.2 (chloroquine and antifolate sensitive) and K1CB1 (multidrug resistant). They exhibited mild antibacterial activity against Bacillus subtilis. None of the test samples showed cytotoxicity.

The natural compounds piperovatine and piperlonguminine induce autophagic cell death on Trypanosoma cruzi.

The currently available treatments for Chagas disease show limited therapeutic potential and are associated with serious side effects. Our group has been attempting to find alternative drugs isolated from natural products as a potential source of pharmacological agents against Trypanosoma cruzi. Here, we demonstrate the antitrypanosomal activity of the amides piperovatine and piperlonguminine isolated from Piper ovatum against epimastigotes and intracellular amastigotes. We also investigated the mechanisms of action of these compounds on extracellular amastigote and epimastigote forms of T. cruzi. These amides showed low toxicity to LLCMK(2) mammalian cells. By using transmission and scanning electron microscopy, we observed that the compounds caused severe alterations in T. cruzi. These alterations were mainly located in plasma membrane and mitochondria. Furthermore, the study of treated parasites labeled with Rh123, PI and MDC corroborate with our TEM data. These mitochondrial dysfunctions induced by the amides might trigger biochemical alterations that lead to cell death. Altogether, our data evidence a possible autophagic process.

Arylnaphthalene lignans from Taiwania cryptomerioides as novel blockers of voltage-gated K+ channels.

Lignans are natural phytochemicals which exhibit multiple pharmacological effects such as anti-inflammation, antivirus and anti-tumor activities. Whether they have effects on neural tissues and ion channels is still unknown. The effects of several arylnaphathalene lignans purified from Taiwania cryptomerioides on voltage-gated K(+) (Kv) channels in mouse neuroblastoma N2A cells were examined. These lignans included Taiwanin E, helioxanthin (HXT) and diphyllin. All lignans showed inhibitory effects on Kv channels and HXT was the most potent compound (IC(50)=1.7 µM). The mechanism of HXT block was further investigated. Its action was found to be extracellular but not intracellular. HXT accelerated current decay, caused a left-shift in steady-state inactivation curve but had no effect on voltage-dependence of activation. HXT block was unaffected by intracellular K(+) concentrations. Further, it did not affect ATP-sensitive K(+) channels. Our data therefore suggest that HXT is a potent and specific blocker of Kv channels, possibly with an inhibitory mechanism involving acceleration of slow inactivation.

[Studies on the alkaloids from roots of Corydalis impatiens].

OBJECTIVE: To study the alkaloids from Corydalis impatiens. METHODS: The alkaloids were isolated and purified by chromatography and their structures were identified by spectral data and others methods. RESULTS: Seven alkaloids were isolated and identified as bicuculline(1), ochotensine(2), ochotensimine(3), ochrobirine(4), tetrahydrothalifendine(5), norochotensimine(6), N-methylactinodaphnine(7). CONCLUSION: All these compounds are isolated from this plant for the first time.

Antihyperlipidemic compounds from the fruit of Piper longum L.

A bioassay-guided isolation of an ethanol extract of the fruit of Piper longum L. yielded piperlonguminine, piperine and pipernonaline, as the main antihyperlipidemic constituents. They exhibit appreciable antihyperlipidemic activity in vivo, which is comparable to that of the commercial antihyperlipidemic drug, simvastatin.

[New neolignan from seed of Myristica fragrans].

OBJECTIVE: To study the chemical constituents in the nutmeg (seed of Myristica fragrans). METHOD: The chemical constituents were isolated by various column chromatographic methods and structurally elucidated by IR, NMR and MS evidences. RESULT: Fifteen compounds were obtained and identified as myristicin (1), methyleugenol (2), safrole (3), 2, 3-dihydro-7-methoxy-2(3, 4-methylenedioxyphenyl)-3-methyl-5-(E) -propenyl-benzofuran (4), dehydrodiisoeugenol (5), 2, 3-dihydro-7-methoxy-2-(3-methoxy-4, 5-methylenedioxyphenyl) -3-methyl-5-(E)-propenyl-benzofuran (6), erythro-2-(4-allyl-2, 6-dimethoxyphenoxy)-1-(3, 4-dimetho- xyphenyl) propane (7), erythro-2-(4-allyl-2, 6-dimethoxyphenoxy)-1-(3, 4, 5-trimethoxyphenyl) propane (8), erythro-2-(4-allyl-2, 6-dimethoxyphenoxy)-1-(3, 4-dimethoxyphenyl) propan-1-ol acetate (9), erythro-2-(4-allyl-2, 6-dimethoxyphenoxy)-1-(3, 4-dimethoxyphenyl) propan-1-ol (10), erythro-2-(4-allyl-2, 6-dimethoxyphenoxy)-1-(3, 4, 5-trimethoxyphenyl) propan-1-ol (11), 5-methoxy-dehydrodiisoeugenol (12), erythro-2-(4-allyl-2, 6-dimethoxyphenoxy)-1-(4-hydroxy-3-methoxyphenyl)-propan-1-ol (13), guaiacin (14) and threo-2-(4-allyl-2, 6-dimethoxyphenoxy)-1-(3-methoxy-5-hydroxy-phenyl) propan-1-ol (15). CONCLUSION: Compound 15 is a new compound and named myrisisolignan. Compound 7 is isolated from the genus Myristica for the first time.

Perilla frutescens var. frutescens in northern Laos.

Twenty-eight samples of mericarps of Perilla frutescens var. frutescens were collected through fieldwork performed in Phongsali and Xieng Khouang provinces in northern Laos. No perilla samples were collected from Savannakhet province in the south although more than 20 sites were investigated. Perilla plants are mostly grown mixed with dry-paddy rice by slash-and-burn cultivation in Laos. The most popular local name for perilla mericarps in the area was "Ma Nga Chan". Weight of 1,000 grains and hardness of the mericarps were measured, and all mericarps were found to be large (weight of 1,000 grains around 2 g) and soft (limit load weight under 300 g), which were preferred for culinary use in Laos. The composition of the essential oils obtained from the herbaceous plants raised from the mericarps was divided into five types, perillaketone, elemicine plus myristicine, shisofuran, piperitenon, and myristicine, and GC-MS analysis of these Laotian perilla samples showed that they were similar to those of corresponding types of known Japanese perilla strains. One of the shisofuran-type perilla contained large amounts of putative alpha-naginatene, which is likely to be an intermediate of the biosynthesis of naginataketone. The farmers' indifference to the oil type of the leaf seems to leave Laotian perilla as a good genetic resource for studies of the biosynthesis of oil compounds.

[GC-MS analysis of essential oil from nutmeg processed by different traditional methods].

OBJECTIVE: To analyze the chemical components of the essential oil extracted from the seeds of Myristica fragrans (nutmeg) processed by different methods (steamed with water steam, roasted with flour, sauted with flour, roasted with talcum powder, roasted with loess, and roasted with bran) and to provide quality control foundations in the sciences. METHOD: The essential oil was extracted by steam distillation and separated with GC capillary column. The relative content of every compound was determined with area normalization method and the structures were elucidated by GC-MS technique. RESULT: Fifty-eight to one hundred and four of chromatographic peaks were detected, among them seventy-six compounds accounting for 98.32% to 99.99% of the total essential oil in nutmeg were identified, which were composed of 69.15% to 97.24% for monoterpenoids and 2.06% to 25.51% for aromatic compounds of the total essential oil, respectively. CONCLUSION: It was shown that monoterpenoids and their derivatives were main composition, and aromatic compounds were secondary composition in the total essential oil of nutmeg grows in Indonesia and processed by different traditional methods on the basis of theory of traditional Chinese medicine. In addition, it was suggested that we should be careful to use processed nutmeg owing to contain safrole and a-asarone induced genetoxicity in animals and mutagenicity in the Ames Salmonella assay, and myristicin and elemicin induced narcotism in human. The processed method roasted with bran for nutmeg may be better and will be developed.

Antibacterial principles from Myristica fragrans seeds.

Nutmeg (Myristica fragrans) is used in food preparations for its aromatic flavor. The present investigation was undertaken to evaluate the antibacterial activity of constituents of M. fragrans seeds. Seeds of M. fragrans were powdered and extracted with chloroform to obtain trimyristin, which on saponification yielded myristic acid. The mother liquor remaining after separation of trimyristin was concentrated and column-chromatographed with petroleum ether to separate myristicin. Antibacterial activity of these isolated constituents was evaluated by determination of minimum inhibitory concentration against selected Gram-positive and Gram-negative organisms. All the constituents isolated from nutmeg exhibited good antibacterial activity. This study shows the potential of natural compounds in replacement of synthetic preservatives.

Production of justicidin B, a cytotoxic arylnaphthalene lignan from genetically transformed root cultures of Linum leonii.

Callus and hairy root cultures of Linum leonii were established. The genetic transformation in hairy roots was proven by PCR analysis, which showed integration of rol A and rol C genes into the plant genome. Calli and hairy roots accumulate the arylnaphthalene lignan justicidin B as a major constituent. Hairy roots produce 5-fold higher yields of justicidin B (10.8 mg g(-1) DW) compared to calli. Justicidin B shows strong cytotoxicity on the chronic myeloid leukemia LAMA-8 and K-562 cell lines and on the chronic lymphoid leukemia SKW-3 cell line with IC(50) values of 1.11, 6.08, and 1.62 microM, respectively. Apoptotic properties of justicidin B are reported for the first time.

Complete assignments of 1H and 13C NMR data for seven arylnaphthalide lignans from Justicia procumbens.

Three new arylnaphthalide lignans named 6'-hydroxy justicidin A (1), 6'-hydroxy justicidin B (2) and 6'-hydroxy justicidin C (3) have been isolated from the whole plant of Justicia procumbens, together with four known ones, neojusticin A (4), chinensinaphthol methyl ester (5), isodiphyllin (6) and taiwanin C (7). The complete assignments of 1H and 13C NMR chemical shifts for the new lignans and the 13C NMR chemical shifts for the known lignans were obtained for the first time by means of 2D NMR techniques, including HSQC and HMBC.

A new lignan from the Jian-er syrup and its content determination by RP-HPLC.

The Jian-er syrup is a commonly used traditional Chinese medicinal preparation refined from two herbs, Metaplexis japonica (Thunb.) Makino and Justicia procumbens L. A new lignan, named 6'-hydroxy-Justicidin B, was isolated from the ethyl acetate extract of the Jian-er syrup and its structure was established on the basis of spectral analysis. The content of 6'-hydroxy-Justicidin B in the Jian-er syrup was determined by RP-HPLC for the first time. Chromatographic separation was achieved on an Agilent Zorbax SB-C18 column (150 mmx4.6 mm i.d., 5 microm) at 25 degrees C and the mobile phase was a mixture of acetonitrile and water (31:69 v/v) detected at 256 nm at a flow rate of 1.0 ml/min. This method was validated in terms of selectivity, linearity, precision, accuracy, limit of detection, limit of quantitation and solution stability. It can be used to control the quality of this preparation effectively.