Metabolism, Anti-inflammatory And Anti-fungal Activities Of Pogostone And Patchouli Alcohol

Objectives:Pogostemonis Herba is the dried aerial part of Pogostemon cablin (Blanco) Benth.(Labiatae), commonly known as’Guang-Huo-Xiang’in Chinese. It has been traditionally used in Chinese medicine to resolve dampness, whet appetite, arrest vomiting, release the exterior and expel summer-dampness. Pogostemonis Herba is a common Chinese herbal medicine frequently used to treat for common cold, nausea, diarrhea, rhinitis, headaches and fever. To date, Pogostemonis Herba has been used as the main compositions in41kinds of traditional Chinese medicine (TCM) preparations on the market. Moreover, patchouli oil, the essential oil of Pogostemonis Herba, is also used in cosmetics, deodorants and insecticides.Over the last several decades, various studies were conducted to investigate the chemical composition, pharmacological activities, cultivation techniques, genetic characteristics of Pogostemonis Herba. Therefore, the chemical composition, pharmacological activities, cultivation techniques, genetic characteristics of Pogostemonis Herba were almost known. However, many problems are also needed to be further resolved to improve its better development for human health. It is important that, few molecular mechanisms of bioactivities of Pogostemonis Herba compounds are known, which goes against its further clinical application.Pogostone (PO) and Patchouli alcohol (PA) are the major chemical constituents of the essential oil of Pogostemonis Herba. In China, both of them are chemical markers required by law for the quality control of the essential oil of Pogostemonis Herba. However, it remains unclear whether PO and PA have the biological activities of Pogostemonis Herba. Therefore, the aim of this study was to determine the anti-fungal and anti-inflammatory effects of PO and PA to investigate the material basis for efficacy of Pogostemonis Herba. Furthermore, the in vitro and in vivo metabolism of PO and PA were also studied.Methods and Results:1. The anti-inflammatory effect of PO and PAThe in vitro anti-inflammatory effects of PO and PA were investigated in lipopolysaccharide (LPS)-stimulated RAW264.7cells. Results showed that both PO treatment (at the concentrations of1,10or30μM) and PA treatment (at the concentrations of10,20or40μM) could dose-dependently decrease the production of tumor necrosis factor-a (TNF-a), interleukin (IL)-1β, IL-6, nitric oxide (NO) and prostaglandin E2(PGE2) in LPS-stimulated RAW264.7cells. In addition, both PO treatment and PA treatment also reversed the increased mRNA expression of TNF-a, IL-1(3, IL-6, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2(COX-2) caused by LPS in RAW264.7cells. These results indicate that PO and PA are important anti-inflammatory constituents of Pogostemonis Herba, and that its anti-inflammatory effect may be mediated, at least in part, by down-regulation of the mRNA expression of a panel of inflammatory mediators such as TNF-α, IL-1β, IL-6. iNOS and COX-2.The in vivo anti-inflammatory effects of PO and PA were investigated using two common inflammatory animal models i.e., xylene-induced ear edema in mice and carrageenan-induced paw edema in rats. The degree of edema in both inflammatory animals, as well as the protein and mRNA expression of some inflammatory mediators including TNF-a, IL-1β, PGE2and NO in the hind paw of carrageenan-treated rats were measured. Results showed that both PO (20,40,80mg/kg) and PA (10,20,40mg/kg) significantly inhibited the ear edema induced by xylene in mice and the paw edema induced by carrageenan in rats. In addition, treatment with PO (10,20,40mg/kg) or treatment with PA (10,20,40mg/kg) also dose-dependently decreased the production of TNF-a, IL-1β, PGE2and NO in the hind paw of carrageenan-treated rats.The in vivo anti-inflammatory effects of PO and PA were also investigated in LPS-induced endotoxic shock in mice. Results showed that intravenous injection of PO (5,10.20mg/kg) protected mice against endotoxin-mediated mortality, whereas oral administration of PA (20,40,80mg/kg) did not show any protective effect against the endotoxin-mediated mortality. In addition, treatment of PO significantly reduced the serum concentrations of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP), and attenuated LPS-induced liver and lung injury. Furthermore, PO significantly inhibited LPS-induced proinflammatory mediators including TNF-a, IL-1β, IL-6, IFN-y, IL-12, PGE2, and NO in serum. PO potently inhibited the production of the proinflammatory gene expression including TNF-a, IL-1(3, IL-6, IFN-y, IL-12, COX-2and iNOS.To elucidate the underlying mechanism of action associated with anti-inflammatory effect of PO, the intracellular pathways including NF-κB and MAPKs signaling pathways were studied in LPS-stimulated RAW264.7cells. Results showed that pretreatment of PO (1,10,30μM) significantly inhibited the phosphorylation of JNK, p38MAPK, and NF-κB p65in LPS-stimulated RAW264.7cells. Taken together, these results demonstrate that PO has a protective effect against endotoxin-induced shock, it may be mediated, at least in part, by blocking the activation of NF-κB and MAPKs signaling pathways.2. The anti-fungal effect of PO and PAPO and PA were screened for anti-dermatophytes activities by both agar diffusion test and broth dilution test. Results showed that PA did not exhibit any effect on the tested Candida spp. isolates. The MICs for PO against these clinical dermatophytes isolates were between24.38μg/ml and195μg/ml. All dermatophytes strains were resistant to fluconazole (FLC) and itraconazole (MIC>1560μg/ml), and PO was fungicidal against some isolates tested in this study.The efficacies of PO and PA were tested against clinical Candida spp. isolates (differentially sensitive to FLC) and one standard FLC-resistant C. albicans strain (ATCC10231) by broth dilution test. Kill-curve of PO was studied at different concentrations in a clinical strain, and anti-biofilm property was also studied. Results showed that PA did not exhibit any effect on the tested Candida spp. isolates. The MICs for PO against these clinical C. albicans isolates were between3.13μg/ml and50μg/ml. And PO was fungicidal against all isolates tested in this study between50μg/ml and400μg/ml. However, voriconazole (VRC) was fungistatic and the highest concentration (400μg/ml) failed to elicit a fungicidal effect against these Candida spp. isolates. Moreover, the other Candida spp. exhibited from12.5μg/ml to400μg/ml of PO as MICs. By contrast, VRC showed higher activity than PO against these Candida isolates. Furthermore. PO at a concentration of0.5times the MIC could significantly affect the growth of C. albicans. At a sub-MIC concentration (mg/ml) of PO could significantly inhibit the formation of mature (48-h old) biofilm. These results suggest that PO has fungistatic and fungicidal activity against C. albicans. it may be related, at least in part, to its anti-biofilm action. The therapeutic efficacy of oral versus intravaginal PO on experimental vaginitis caused by a fluconazole-resistant Candida albicans isolate were studied, and the effect of PO were compared with VRC. Results showed that oral PO administration (20,40,80mg/kg) and topical PO treatment (1,2,4mg/kg) effectively reduced the fungal load in vagina of vulvovaginal candidiasis mouse models. Furthermore, topical PO administration demonstrated higher activity against the vulvovaginal candidiasis than VRC.3. The metabolism of PO and PAHigh performance liquid chromatography coupled with ion trap mass spectrometry supported with high resolution LTQ-Orbitrap was employed to analyze the metabolism of PO by virtue of the high sensitivity and high selectivity in the measurement. In vitro experiment was carried out using rat liver microsomes while the in vivo study was conducted in rats, which were orally administered with PO (80mg/kg). Through the mass spectrometric characterization of the structural information for the metabolites, three mono-hydroxylated PO, one di-hydroxylated PO and one hydrolyzation metabolite were found in rat liver microsomes. In addition to the aforementioned metabolites, one di-oxygenated metabolite and one mono-oxygenated metabolite were detected in rat plasma. However, no PO and metabolite was found in feces samples in the current work. As a result, a total of seven metabolites of PO were described and hydroxylation was demonstrated to be a major metabolic pathway of PO.High performance liquid chromatography coupled with ion trap mass spectrometry supported with high resolution LTQ-Orbitrap was employed to analyze the metabolism of PA by virtue of the high sensitivity and high selectivity in the measurement. In vitro experiment was carried out using rat liver microsomes while the in vivo study was conducted in rats, which were orally administered with pogostone (80mg/kg). Through the mass spectrometric characterization of the structural information for the metabolite, one carboxylated metabolite was found in urine and feces samples. No metabolite was found in rat liver microsomes.Conclusion:PO and PA are the major chemical constituents of the essential oil of Pogostemonis Herba. However, to the best of our knowledge, the biological activities of PO and PA have been rarely reported. The present study assessed the in vitro and in vivo anti-inflammatory activities of PO and PA. and found that both PO and PA have significant ant i-inflammatory effect. The results demonstrate that PO and PA are important parts of the material basis of Pogostemonis Herba for dampness-induced diseases.Moreover, the study tested the in vitro and in vivo anti-fungal effects of PO and PA, and found that PO possess potent activities against dermatophytes and Candida spp., whereas PA did not exhibit any effect against these funguses. The results suggest that although the content of PO is less than that of PA in the essential oil of Pogostemonis Herba, PO has been proved to be important material basis of Pogostemonis Herba against funguses. PA has long been used as a chemical marker required by law for the quality control of Pogostemonis Herba and the essential oil of Pogostemonis Herba. Since2010, PO has also been used as a chemical marker required by law for the quality control of the essential oil of Pogostemonis Herba, but it have not been used for Pogostemonis Herba yet. This study may provide an evidence for the demand to improve the quality standard for Pogostemonis Herba.Furthermore, the study characterized the metabolism of pogostone in vitro and in vivo using liquid chromatography-mass spectrometry, and found that PO and PA could be rapidly metabolized in rat. These suggest that the underlying mechanism of action of PO and PA may be associated with their metabolites.