Retrobisabolane A, a Novel Bisabolane-Derived Sesquiterpenoid Isolated from Deep-Sea-Derived Fungus Retroconis fusiformis MCCC 3A00792.

One novel bisabolane-derived sesquiterpenoid retrobisabolane A (1), featuring a methyl group location at the C-4 position instead of C-3 in the bisabolanes, and a known ester-substituted eremophilane-type sesquiterpenoid cryptosphaerolide (2), along with three known indole alkaloids (3-5) were discovered from the fermented cultures of a deep-sea-derived fungus Retroconis fusiformis MCCC 3A00792. The planar structure of new compound 1 was determined by extensive analysis of the NMR and HRESIMS spectra. The relative and absolute configurations of 1 were resolved by the coupling constant (J), calculation of ECD and NMR spectra, and the DP4+ probability analysis of the 1H and 13C NMR data. Interestingly, retrobisabolane A was the new subclass of bisabolanes bearing a methyl group linkage at C-4 instead of C-3 position. Three human cancer cell lines (Hela, AGS, and BIU-87) were subjected to evaluate the cytotoxic activities of compounds 1-5. As a result, compound 2 exhibited significant inhibitory activities against three cell lines with IC50 values ranging from 9.95 to 18.77 µM.

Malfilanol C, a new sesquiterpenoid isolated from the deep-sea-derived Aspergillus puniceus A2 fungus.

The chemical examination of the rice solid fermentation products of the deep-sea-derived fungus Aspergillus puniceus A2 resulted in the isolation of one new sesquiterpenoid malfilanol C (1), together with a rare analogue malfilanol B (2). The planar structure of 1 was resolved on the basis of the extensive analyses of the spectroscopic data (HRESIMS and NMR spectra), and its absolute configuration was assigned by quantum chemical calculation of the ECD data. Compound 1, featuring a bicyclo[5.4.0]-undecane nucleus skeleton, was the third example of this subclass sesquiterpenoids found from nature. Additionally, the subclass sesquiterpenoids 1 and 2 were discovered from marine-derived-fungi for the first time. All the isolated metabolites were evaluated the antibacterial activities against Staphylococcus aureus ATCC 29213 and Escherichia coli ATCC 25922. Compound 1 exhibited weak antibacterial activity against S. aureus ATCC 29213.

Phaeosphamides A and B, Cytotoxic Cyclodecadepsipeptides from the Mangrove-Derived Fungus Phaeosphaeriopsis sp. S296.

Chemical examination of the fermented broth of the mangrove-derived fungus Phaeosphaeriopsis sp. S296 resulted in the isolation of two new cyclodecadepsipeptides, namely phaeosphamides A (1) and B (2), as well as one known congener Sch 217048 (3). The structures of new metabolites, including absolute configurations, were established on the basis of extensive spectroscopic data analyses, chemical conversion, and Marfey's method. The 2-hydroxy-3-methylpentanoic acid (Hmp) moiety and pipecolic acid (Pip) unit in structures were rarely discovered in nature. Interestingly, compounds 1-3 are examples of peptides discovered from the fungal genus Phaeosphaeriopsis for the first time. All identified compounds were evaluated for their cytotoxicity against five tumor cell lines of AGS, BEL-7402, HepG2, B16, and BIU87. Among them, compound 1 showed inhibitory activities against these tumor cell lines with IC50 values ranging from 5.14 to 66.38 µM. A further mechanistic investigation found that 1 arrested AGS cells in the G2 phase and induced their apoptosis in a dose-dependent manner.

Effect of piperine on the mitigation of obesity associated with gut microbiota alteration.

An obese mouse model induced by high-fat diet (HFD) feeding was used to reveal the role of piperine in modulating gut microbiota (GM). Piperine was administrated at 20 and 40 mg/kg body weight every day. As a result, piperine at 40 mg/kg significantly decreased body weight, liver weight, perirenal fat weight, and lowered serum triglycerides, total cholesterol, low-density lipoprotein cholesterol, and glucose levels in HFD-fed mice. Additionally, piperine significantly attenuated fatty liver and modulated hepatic mRNA expressions of SREBP-1c, SREBP2, and HMGCR. In perirenal fat, FAS, C/EBPα, MCP1, and IL-6 expressions were significantly downregulated by piperine. 16S rRNA sequencing revealed that piperine elevated GM diversity. The relative abundance of Muribaculaceae and Ruminococcaceae were significantly elevated, while Dubosiella and Enterorhabdus genera were suppressed by piperine. The Pearson correlation analysis showed that the altered phylotypes were highly correlated with obesity phenotypes. These findings suggest that piperine modulates energy homeostasis and inflammation to alleviate obesity associated with GM regulation.

Phenolic Metabolites from a Deep-Sea-Derived Fungus Aspergillus puniceus A2 and Their Nrf2-Dependent Anti-Inflammatory Effects.

Four undescribed phenolic compounds, namely asperpropanols A-D (1-4), along with two known congeners 5 and 6, were isolated from Aspergillus puniceus A2, a deep-sea-derived fungus. The gross structures of the compounds were established by detailed analyses of the HRESIMS and NMR data, and their absolute configurations were resolved by modified Mosher's method and calculations of ECD data. Compounds 1-6 were found to have excellent anti-inflammatory effect on lipopolysaccharide (LPS)-induced RAW264.7 cells at 20 µM, evidenced by the reduced nitric oxide (NO), tumor necrosis factor α, and interleukin 6 production. Among them, 5 and 6 showed inhibitory effects on NO production comparable with the positive control (BAY11-7083 at 10 µM). Additionally, the LPS-induced mRNA expressions of inducible nitric oxide synthase and cyclooxygenase-2 were also decreased. Interestingly, mRNA expression of nuclear factor erythroid 2-related factor 2 (Nrf2) was downregulated by LPS and recovered by 1-6, suggesting a vital role of Nrf2 in their effect. We further found that pharmacological inhibition of Nrf2 by ML385 largely abrogated the effects of 1-6 on RAW264.7 cells. Therefore, 1-6 may share a common anti-inflammatory mechanism via Nrf2 upregulation and activation.

Antiviral Cyclopropane Acids from Deep-Sea-Derived Fungus Aspergillus sydowii.

Four novel monocyclic cyclopropane acids, namely, sydocyclopropanes A-D (1-4), along with one known congener hamavellone B (5), were isolated from the Aspergillus sydowii MCCC 3A00324 fungus, which was isolated from the deep-sea sediment. The gross structures of novel compounds were established by detailed analyses of the spectroscopic data (HRESIMS and NMR spectra), and their absolute configurations were resolved on the basis of the quantum chemical calculations of ECD and NMR data, in association with DP4+ probability analyses. Sydocyclopropanes A-D, featuring the 1,1,2,3-tetrasubstituted cyclopropane nucleus with different lengthy alkyl side chains, were discovered in nature for the first time. All compounds exhibited antiviral activities against A/WSN/33 (H1N1), with IC50 values ranging from 26.7 to 77.2 µM, of which compound 1 exhibited a moderate inhibitory effect (IC50 = 26.7 µM).

Bisabolane Sesquiterpenes with Anti-Inflammatory Activities from the Endophytic Fungus Penicillium citrinum DF47.

Seven new bisabolane-type sesquiterpenes (1-7), namely penicibisabolanes A-G, together with eight known analogs (8-15) were obtained from the AcOEt extract of the millet fermentation broth of the endophytic fungus Penicillium citrinum DF47, which was isolated from the fresh root of Codonopsis pilosula (Franch.) Nannf. The gross structures of new metabolites were determined on the basis of the spectroscopic data (HR-ESI-MS, 1D and 2D NMR spectra), while their absolute configurations were resolved by comparison of the experimental and calculated ECD spectra, in association with specific rotation data. Compound 1 is a rare seco-trinor-bisabolane sesquiterpene found in nature, while 3 is the first example of phenolic bisabolanes bearing a methoxy group at C-1. All the isolates were evaluated their inhibitory effects against NO production in lipopolysaccharides (LPS) stimulated RAW264.7 cells. Among them, compounds 7 and 13 showed moderately anti-inflammatory effects with the inhibitory rate more than 50 % at the concentration of 20 µM.

Acremolin D, a new acremolin alkaloid from the deep-sea sediment derived Aspergillus sydowii fungus.

Chemical investigation of the deep-sea-derived fungus Aspergillus sydowii MCCC 3A00324 led to the isolation of one new acremolin type alkaloid (acremolin D, 1) and five known alkaloids (2‒6). The planar structure of 1 was established by the extensive analyses of the NMR and HRESIMS data, while its absolute configuration was assigned by the comparison of the experimental and calculated ECD data. Acremolin D (1) represented the second analogue of acremolin found in nature. All compounds were evaluated for their cytotoxic activities against six human cancer cell lines (A549, Hela-S3, MCF-7, HepG2, K562, and SF-268). As a result, compounds 1 and 2 exhibited a certain inhibitory effects against the proliferation of the A549, Hela-S3, HepG2, and K562 cell lines at the concentration of 20 µM.[Formula: see text].

Cis- and Trans-Palmitoleic Acid Isomers Regulate Cholesterol Metabolism in Different Ways.

Hypercholesterolemia is a preventable risk factor for atherosclerosis and cardiovascular disease. However, the mechanisms whereby cis-palmitoleic acid (cPOA) and trans-palmitoleic acid (tPOA) promote cholesterol homeostasis and ameliorate hypercholesterolemia remain elusive. To investigate the effects of cPOA and tPOA on cholesterol metabolism and its mechanisms, we induced hypercholesterolemia in mice using a high-fat diet and then intragastrically administered cPOA or tPOA once daily for 4 weeks. tPOA administration reduced serum cholesterol, low-density lipoprotein, high-density lipoprotein, and hepatic free cholesterol and total bile acids (TBAs). Conversely, cPOA had no effect on these parameters except for TBAs. Histological examination of the liver, however, revealed that cPOA ameliorated hepatic steatosis more effectively than tPOA. tPOA significantly reduced the expression of 3-hydroxy-3-methyl glutaryl coenzyme reductase (HMGCR), LXRα, and intestinal Niemann-Pick C1-Like 1 (NPC1L1) and increased cholesterol 7-alpha hydroxylase (CYP7A1) in the liver, whereas cPOA reduced the expression of HMGCR and CYP7A1 in the liver and had no effect on intestinal NPC1L1. In summary, our results suggest that cPOA and tPOA reduce cholesterol synthesis by decreasing HMGCR levels. Furthermore, tPOA, but not cPOA, inhibited intestinal cholesterol absorption by downregulating NPC1L1. Both high-dose tPOA and cPOA may promote the conversion of cholesterol into bile acids by upregulating CYP7A1. tPOA and cPOA prevent hypercholesterolemia via distinct mechanisms.

Phenolic bisabolane and cuparene sesquiterpenoids with anti-inflammatory activities from the deep-sea-derived Aspergillus sydowii MCCC 3A00324 fungus.

Seventeen undescribed sesquiterpenoids including 14 phenolic bisabolanes, namely asperbisabolanes A-N (1-14), and 3 cuparenes (aspercuparenes A-C, 15-17), together with 10 known bisabolane analogues (18-27) were isolated from the EtOAc extract of fermented cultures of the deep sea sediment-derived fungus Aspergillus sydowii MCCC 3A00324. The new structures were established on the basis of extensive NMR and HRESIMS spectroscopic data analyses, while their absolute configurations were assigned by comparison of the experimental ECD spectra with those of the TDDFT-ECD calculated spectra or reported data in literature. Asperbisabolanes A (1) and B (2) are the first examples of bisabolane sesquiterpenoids featuring a 6/6/6 tricyclic nucleus. Compound 3 possessed a novel seco-bisabolane skeleton with a rare dioxolane ring moiety, while asperbisabolane K (11) represents the first case of bisabolanes bearing a rare methylsulfonyl group. All the isolated compounds (1-27) were evaluated their activities against NO secretion in LPS-activated BV-2 microglia cells. As a result, 6, 12, 16, and 25-27 exhibited the inhibition rate over 45% at a concentration of 10 µM. Moreover, 12 exerted the anti-inflammatory activity by inhibiting the NF-κB-activated pathway in dose-dependent manner.

New Monoterpenoids and Polyketides from the Deep-Sea Sediment-Derived Fungus Aspergillus sydowii MCCC 3A00324.

Chemical study of the secondary metabolites of a deep-sea-derived fungus Aspergillus sydowii MCCC 3A00324 led to the isolation of eleven compounds (1-11), including one novel (1) and one new (2) osmane-related monoterpenoids and two undescribed polyketides (3 and 4). The structures of the metabolites were determined by comprehensive analyses of the NMR and HRESIMS spectra, in association with quantum chemical calculations of the 13C NMR, ECD, and specific rotation data for the configurational assignment. Compound 1 possessed a novel monoterpenoid skeleton, biogenetically probably derived from the osmane-type monoperpenoid after the cyclopentane ring cleavage and oxidation reactions. Additionally, compound 3 was the first example of the α-pyrone derivatives bearing two phenyl units at C-3 and C-5, respectively. The anti-inflammatory activities of 1-11 were tested. As a result, compound 6 showed potent inhibitory nitric oxide production in lipopolysaccharide (LPS)-activated BV-2 microglia cells with an inhibition rate of 94.4% at the concentration of 10 µM. In addition, a plausible biosynthetic pathway for 1 and 2 was also proposed.

Synthesis and Biological Evaluation of Analogues of Butyrolactone I as PTP1B Inhibitors.

In recent years, a large number of pharmacologically active compounds containing a butenolide functional group have been isolated from secondary metabolites of marine microorganisms. Butyrolactone I was found to be produced by Aspergillus terreus isolated from several marine-derived samples. The hypoglycemic activity of butyrolactone I has aroused our great interest. In this study, we synthesized six racemic butenolide derivatives (namely BL-1-BL-6) by modifying the C-4 side chain of butyrolactone I. Among them, BL-3 and BL-5 improved the insulin resistance of HepG2 cells and did not affect the proliferation of RIN-m5f cell line, which indicated the efficacy and safety of BL-3 and BL-5. Furthermore, BL-3, BL-4, BL-5, and BL-6 displayed a significant protein tyrosine phosphatase 1B (PTP1B) inhibitory effect, while the enantiomers of BL-3 displayed different 50% percentage inhibition concentration (IC50) values against PTP1B. The results of molecular docking simulation of the BLs and PTP1B explained the differences of biological consequences observed between the enantiomers of BL-3, which supported BLs as PTP1B inhibitors, and also indicated that the chirality of C-4 might influence the inhibitory effect of the BLs. Our findings provide a novel strategy for the development of butyrolactone derivatives as potential PTP1B inhibitors for the treatment of type 2 diabetes mellitus.

Separation of saturated fatty acids from docosahexaenoic acid-rich algal oil by enzymatic ethanolysis in tandem with molecular distillation.

Algal oil, rich in docosahexaenoic acid (DHA) and an environmentally sustainable source of ω-3 fatty acids, is receiving increasing attention. In the present study, a novel approach combining ethanolysis with a 1,3-specific immobilized lipase (Lipozyme® TL IM) and molecular distillation was investigated to increase the DHA content of algal oil. Algal oil with a 45.94% DHA content was mixed with ethanol, pumped into a column filled with Lipozyme® TL IM, and then circulated for 4 hr at room temperature. The ethanol was then recycled by vacuum distillation. At an evaporator temperature of 150°C, the residue was separated by molecular distillation into a heavy component enriched with DHA glycerides (in the form of triglyceride (TG), diglyceride (DG), and monoglyceride (MG)) and a light component enriched with palmitic acid (PA) and DHA ethyl ester (EE). As a result, 76.55% of the DHA from the algal oil was present in the heavy component, whose DHA content was 70.27%. DHA-MG was collected in the heavy component mostly in the form of 1-MG. Lipozyme® TL IM appeared to specifically target PA rather than DHA at the sn-1(3) position. The Lipozyme® TL IM allowed 90.03% of the initial DHA yield to be retained after seven reaction cycles. Therefore, an eco-friendly and simple method for increasing the DHA content in algal oil has been developed.

Antioxidant Capacity and Hepatoprotective Role of Chitosan-Stabilized Selenium Nanoparticles in Concanavalin A-Induced Liver Injury in Mice.

Selenium nanoparticles (SeNPs) have attracted wide attention for their use in nutritional supplements and nanomedicine applications. However, their potential to protect against autoimmune hepatitis has not been fully investigated, and the role of their antioxidant capacity in hepatoprotection is uncertain. In this study, chitosan-stabilized SeNPs (CS-SeNPs) were prepared by means of rapid ultra-filtration, and then their antioxidant ability and free-radical scavenging capacity were evaluated. The hepatoprotective potential of a spray-dried CS-SeNPs powder against autoimmune liver disease was also studied in the concanavalin A (Con A)-induced liver injury mouse model. CS-SeNPs with size of around 60 nm exhibited acceptable oxygen radical absorbance capacity and were able to scavenge DPPH, superoxide anion, and hydroxyl radicals. The CS-SeNPs powder alleviated Con A-caused hepatocyte necrosis and reduced the elevated levels of serum alanine transaminase, aspartate transaminase, and lactic dehydrogenase in Con A-treated mice. These results suggest that the CS-SeNPs powder protected the mice from Con-A-induced oxidative stress in the liver by retarding lipid oxidation and by boosting the activities of superoxide dismutase, glutathione peroxidase, and catalase, partly because of its ability to improve Se retention. In conclusion, SeNPs present potent hepatoprotective potential against Con A-induced liver damage by enhancing the redox state in the liver; therefore, they deserve further development.

Selenium Nanoparticles-Embedded Chitosan Microspheres and Their Effects Upon Alcohol-Induced Gastric Mucosal Injury in Rats: Rapid Preparation, Oral Delivery, and Gastroprotective Potential of Selenium Nanoparticles.

BACKGROUND: Selenium (Se) is an indispensable trace element required for animals and human beings, whereas Se-deficiency can accelerate the development of acute gastric injury induced by over-consumption of alcohol. Selenium nanoparticles (SeNPs), as a special Se-supplement with favorable properties and unique bioactivities, are expected to play a passive role in gastroprotection. To the best of our knowledge, the gastroprotective potential of SeNPs is unknown and also, a rapid preparation of orally stable SeNPs available for prospective commercial application in the clinic is needed. Thus, SeNPs-embedded chitosan microspheres (SeNPs-CM) were developed to deliver SeNPs, and their gastroprotective potential was evaluated. RESULTS: Herein, a rapid, eco-friendly and economic preparation process, composed of synthesis of SeNPs decorated by chitosan (CS), purification of CS-SeNPs by ultra-filtration (UF) and spray-drying of the purified CS-SeNPs, was introduced to prepare SeNPs-CM. The uniformly distributed SeNPs with a nanosize range of 60 nm were loaded into CS-microspheres, and they could be released from the microspheres in gastric conditions. In addition, SeNPs-CM were safer than selenite in terms of Se dose, with a LD50 of around 8-fold of that of selenite, and it could efficiently enhance the Se retention in Se-deficient Wistar rats. Furthermore, SeNPs-CM pre-treatment might significantly attenuate the ethanol-induced gastric mucosal damage, based on histological evaluation. It might be partly attributed to the systematic antioxidant activities of SeNPs-CM, reflected by the reduction in lipid peroxidation, the augmentation in antioxidant enzymatic activity as well as decreasing aggressive nitric oxides (NO). CONCLUSION: SeNPs-CM could be taken into consideration as a prospective Se-supplement for the oral delivery of SeNPs, with prominent gastroprotective effect against ethanol-induced mucosal injury.

Analgesia Effect of Enteric Sustained-Release Tetrodotoxin Pellets in the Rat.

Tetrodotoxin (TTX) was identified as a latent neurotoxin that has a significant analgesia effect. It was rapidly absorbed and excreted in rat after intramuscular (i.m.) injection. To maintain the effect, frequent injections were required. The enteric sustained-release TTX pellets with sucrose pellets as a drug carrier was prepared by fluidized bed spray irrigation, coated in sequence with Eudragit NE30D as a sustained-release layer, hydroxypropyl methylcellulose (HPMC) as a barrier layer and Eudragit L30D-55 as an enteric coating. TTX in the pellets could be sustained released for 12 h in dissolution test. In vivo, TTX pellets reached Cmax at 5 h, and t1/2 was 14.52 ± 2.37 h after intragastrically (i.g.) administration in rat. In acetic acid induced writhing test in rat, the pellets at the dosages of 20, 40, 60 and 80 µg·kg-1 produced analgesic effect at about 1.5 h to 9 h and the strongest effect was at about 3 h to 6 h. Simultaneously, the LD50 of the enteric sustained-release TTX pellets was 840.13 µg·kg-1, and the ED50 was about 30 µg·kg-1. Thus, the therapeutic index was about 25. The enteric sustained-release TTX pellets with absolute analgesia effect and greatly enhanced safety was prepared.

Selenium-Nanoparticles-Loaded Chitosan/Chitooligosaccharide Microparticles and Their Antioxidant Potential: A Chemical and In Vivo Investigation.

Selenium nanoparticles (SeNPs) have attracted attention due to their favorable properties, unique bioactivities, and potential for use in nutritional supplements and nanomedicine applications. However, the application of SeNPs in the clinic has been greatly hindered by their poor stability, and their potential to protect against alcohol-induced oxidative stress has not been fully investigated. Herein, SeNPs were synthesized in the presence of chitosan (CS) or chitooligosaccharide (COS), and a mixture of SeNPs, CS, and COS was spray-dried to prepare selenium-nanoparticles-loaded chitosan/chitooligosaccharide microparticles (SeNPs-CS/COS-Ms). Their physicochemical properties, including morphology, elemental state, size distribution, surface potential, and characteristic structure, were investigated. The release of SeNPs from the vehicle and the free radical scavenging ability of SeNPs-CS/COS-Ms were also studied. Furthermore, the safety of SeNPs-CS/COS-Ms and their antioxidant activity against alcohol were evaluated in mice. The results indicate that SeNPs-CS/COS-Ms, with a novel structure characterized by their smooth or wrinkled surface, hollow core, and COS body filled with SeNPs-CS nanobeads, were able to release SeNPs and scavenge DPPH and superoxide anion radicals. SeNPs-CS/COS-Ms were found to be much safer than selenite, and they might protect mice from ethanol-induced oxidative stress by reducing lipid and protein oxidation and by boosting glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), and catalase (CAT). In conclusion, SeNPs-CS/COS-Ms offer a new way to develop stable SeNPs with higher efficacy and better biosafety, and the antioxidant potential of SeNPs-CS/COS-Ms against ethanol deserves further development.

Combination Formulation of Tetrodotoxin and Lidocaine as a Potential Therapy for Severe Arrhythmias.

Severe arrhythmias-such as ventricular arrhythmias-can be fatal, but treatment options are limited. The effects of a combined formulation of tetrodotoxin (TTX) and lidocaine (LID) on severe arrhythmias were studied. Patch clamp recording data showed that the combination of LID and TTX had a stronger inhibitory effect on voltage-gated sodium channel 1.5 (Nav1.5) than that of either TTX or LID alone. LID + TTX formulations were prepared with optimal stability containing 1 µg of TTX, 5 mg of LID, 6 mg of mannitol, and 4 mg of dextran-40 and then freeze dried. This formulation significantly delayed the onset and shortened the duration of arrhythmia induced by aconitine in rats. Arrhythmia-originated death was avoided by the combined formulation, with a decrease in the mortality rate from 64% to 0%. The data also suggests that the anti-arrhythmic effect of the combination was greater than that of either TTX or LID alone. This paper offers new approaches to develop effective medications against arrhythmias.

Docosahexaenoic acid inhibits hepatic stellate cell activation to attenuate liver fibrosis in a PPARγ-dependent manner.

Docosahexaenoic acid (DHA) has been found to have a hepatoprotective effect. In this study, we investigated the role of peroxisome proliferator-activated receptor γ (PPARγ) in DHA regulation of liver fibrosis. DHA was found to inhibit hepatic stellate cell (HSC)-LX2 cell viability and downregulate marker proteins of HSC activation. Furthermore, DHA induced cell cycle arrest at G1 phase in HSCs. Antagonism of PPARγ by GW9662 abrogated the effects of DHA on HSCs. Computer-aided molecular docking predicted that DHA bound to PPARγ via hydrogen bonding with residues Ser289, His323, Tyr473, and His499. We overexpressed Ser289 mutant PPARγ in HSC-LX2 cells and investigated fibrotic marker modulation, and found that DHA effects on HSCs were diminished. Thus, bonding with the Ser289 residue might be indispensable for DHA to activate PPARγ to exert its inhibiting effect on activated HSCs. Last, data from a CCl4-treated mouse model confirmed that PPARγ activation was required for DHA to attenuate liver fibrosis.

Comparing the simultaneous determination of cis- and trans-palmitoleic acid in fish oil using HPLC and GC.

BACKGROUND: Cis- and trans-palmitoleic acids (Cis-POA and trans-POA) are isomers of palmitoleic acid, a monounsaturated fatty acid which affects glucose and lipid metabolism, and reduces insulin resistance. Trans-POA is used as a biomarker for indicating the risk of type II diabetes and coronary heart disease, but no methods of analysis or distinguishing between cis-POA and trans-POA have yet been reported. METHOD: An accurate and precise HPLC method was developed to determine cis- and trans-POA simultaneously, and compared with results from a GC method. Cis- and trans-POA were analyzed by HPLC on a reverse-phase BDS-C18 column, equilibrated and eluted with acetonitrile (A) and water (B). In the established and validated GC method used for comparison, potassium hydroxide ester exchange was chosen to derivatize the cis- and trans-POA, before being determined. RESULTS: The calibration curves for cis- and trans-POA were linear over the range 0.05 to 500 µg/mL. The HPLC method exhibited good sensitivity, precision and accuracy. The limits of detection (LOD) for cis- and trans-POA were 0.2 and 0.05 µg/mL, respectively. The method successfully determined cis- and trans-POA in fish oil. For the GC method, the contents of cis-POA quantified were similar to those from the HPLC method, but the contents of trans-POA revealed significant variation between the two methods. CONCLUSIONS: After a comprehensive consideration of the characteristics of the saponification and methyl esterification methods which have been tested and verified, the HPLC method was found to be suitable for determining cis- and trans-POA contents in fish oil. It was also suggested that in natural fish oil, cis-POA may be in the glyceride state, and trans-POA almost completely in the free acid form. In comparison with the GC method, the HPLC method provided a simpler process and faster analyses for identifying and determining cis- and trans-POA. The study has also provided technical support for studying the pharmacological differences and relationship between structure and activity of cis- and trans-POA. This could help physicians to analyze patients' samples more quickly in 10 min and therefore provide a more rapid diagnosis of problems relating to the risk of type II diabetes and coronary heart disease.