广藿香酮及广藿香醇的抗炎、抗真菌活性及药物代谢研究
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摘要
目的：
广藿香Pogostemon cablin (Blanco) Benth为唇形科刺蕊草属植物,以干燥地上部分入药,是临床常用芳香化湿中药,其味辛、性微温,归脾、胃、肺经,具有芳香化浊、和中止呕、发表解暑的功效。临床上用于治疗湿浊中阻、脘痞呕吐、暑湿表证、湿温初起、发热倦怠、胸闷不舒、寒湿闭暑、腹痛吐泻、鼻渊头痛等。广藿香属于我国“十大南药”之一,是41种中成药制剂的重要组成药味。广藿香油,是广藿香的挥发油部位,也作为药用提取物,在市场上占有重要的份额。因此,广藿香是重要的岭南道地药材,有巨大的开发潜力。
过去几十年来,国内外研究者对广藿香进行了广泛的研究,尤其是印度、中国、日本、美国等国家,基本阐明广藿香的化学成分组成、药理活性、栽培技术、生物遗传学等。然而,迄今为止国内外对广藿香的有效成分研究报道甚少,尚未清楚广藿香的有效成分及其作用机制。因此,亟需开展其有效成分物质基础的研究。
广藿香醇及广藿香酮是广藿香油中含量较高的化学成分。长期以来,国家和地方的药材标准将广藿香醇(百秋李醇)的含量作为分析广藿香质量的指标成分,2010年版《中国药典》增加了另一化学成分广藿香酮作为检测指标成分。可见,广藿香醇和广藿香酮是广藿香重要的化学成分。那么广藿香醇和广藿香酮的药理活性如何?它们是否能代表广藿香功效的物质基础呢？解决上述疑惑无疑具有重要的学术价值和重大的应用价值。
因此,本论文选择广藿香醇及广藿香酮,进行抗真菌、抗炎的药理活性研究,为探讨广藿香物质基础提供依据。此外,对广藿香醇及广藿香酮在大鼠体内外代谢产物进行研究,推测其在体内的代谢途径和转化过程,有助于阐明二者在体内的作用机制。本研究旨在初步探索广藿香的药效物质基础,以期为日后进一步研究广藿香药效的其他物质基础提供参考。
方法与结果：
1.广藿香酮和广藿香醇的抗炎活性研究
研究采用LPS诱导的RAW264.7巨噬细胞炎症模型,测定广藿香酮和广藿香醇对LPS诱导巨噬细胞产生的过量炎症介质(TNF-α、IL-1β、IL-6、PGE2、NO)的影响,并检测二者对炎症介质相关基因(TNF-α、IL-1β、IL-6、COX-2、iNOS)表达的影响。结果显示,广藿香酮(1、10、30μM)和广藿香醇(10、20、40μM)对LPS诱导的细胞炎症具有显著抑制作用,且显著抑制LPS诱导的炎症介质相关基因的过度表达。提示它们的作用途径可能是,通过抑制LPS诱导的炎症介质相关基因的过度表达,抑制了炎症介质的过度产生。
采用二甲苯致小鼠耳廓肿胀模型、角叉菜胶致大鼠足肿胀模型,测定广藿香酮和广藿香醇对小鼠耳廓、大鼠足趾肿胀度的影响,以及对大鼠足肿胀组织中炎症介质(IL-1β、TNF-α、PGE2、NO)的影响,以评价广藿香酮和广藿香醇对非感染性炎症的影响。结果显示,广藿香酮(10、20、40mg/kg)和广藿香醇(20、40、80mg/kg)口服给药对二甲苯致小鼠耳廓肿胀和角叉菜胶致大鼠足肿胀具有显著抑制作用,此外,广藿香酮(10、20、40mg/kg)和广藿香醇(10、20、40mg/kg)口服给药可显著抑制大鼠足肿胀组织中炎症介质的过度产生。
采用LPS诱导的内毒素休克小鼠模型,测定广藿香酮和广藿香醇对内毒素休克小鼠死亡率的影响,并测定内毒素休克小鼠生化指标、组织损伤、血清中炎症介质的含量、及组织中炎症介质相关基因表达,评价广藿香酮和广藿香醇对感染性炎症的影响。结果显示,广藿香醇(10、20、40mg/kg)口服给药对内毒素休克小鼠未表现出保护作用；广藿香酮(5、10、20mg/kg)静脉注射给药可提高LPS诱导的内毒素休克小鼠的存活率,下调内毒素休克小鼠血清中的ALP、AST、ALT水平,改善内毒素导致的小鼠肝、肺损伤；广藿香酮可抑制内毒素休克小鼠血清中的炎症介质(TNF-α、IL-1β、 IL-6、IFN-γ、IL-12、PGE2、NO)过量产生,并下调内毒素休克小鼠肝、肺组织中炎症介质相关基因(TNF-α、IL-1β、IL-6、IFN-γ、IL-12、COX-2、iNOS)的表达,提示广藿香酮对内毒素休克小鼠的保护作用,可能通过与其抑制内毒素引起的炎症介质瀑布样级联反应而实现的。
为了进一步研究广藿香酮对LPS诱导的炎症反应的抑制作用机理,检测了广藿香酮对LPS诱导的巨噬细胞内细胞信号通路的影响。结果显示,广藿香酮可抑制LPS诱导的巨噬细胞内NF-κB、JNK/SAPK及p38MAPK信号转导通路的激活。提示广藿香酮对LPS诱导内毒素休克小鼠的保护作用的机制,可能是其阻断了LPS激活细胞的NF-κB和MAPK信号转导通路,进而调控炎症介质相关基因的表达,最终抑制炎症介质的过度产生。
2.广藿香酮和广藿香醇的抗真菌活性研究
采用琼脂稀释法和微量液基稀释法测定广藿香酮和广藿香醇对11株临床皮肤癣菌的影响。结果显示,广藿香酮对各受试皮肤癣菌均有明显抗菌作用,MIC介于24.38～195μg/ml之间,而广藿香醇对受试菌株的MIC>1560μg/ml。在相同条件下所有受试菌株对氟康唑和伊曲康唑均有耐药性(MIC>1560μg/ml),广藿香酮对部分皮肤癣菌有杀菌作用。
采用微量液基稀释法,测定广藿香酮和广藿香醇对临床念珠菌属真菌的影响,并通过测定白色念珠菌的生长曲线、生物被膜活性,初步探讨作用机理。结果显示,广藿香醇对所有受试菌株的MIC>400μg/ml;广藿香酮对受试白色念珠菌的MIC在3.13至50μg／ml之间,广藿香酮对所有受试白色念珠菌株均有杀菌作用,其MFC介于100至400μg／ml之间,而伏立康唑的最高浓度(400μg／m1)仅对其中一株有杀菌作用。对于其他念珠菌属菌种,广藿香酮的MIC介于12.5至400μg／ml之间,均小于伏立康唑；而广藿香酮对其他受试念珠菌种无杀菌作用。广藿香酮可在白色念珠菌的生长初期迅速将其抑制或杀灭,广藿香酮可显著抑制白色念珠菌成熟生物被膜的生长。说明抑制生物膜的生长和形成可能是广藿香酮抗耐药白色念珠菌的作用机制之一
采用雌激素化白色念珠菌小鼠阴道炎模型,测定两种给药方式的广藿香酮(即局部给药和口服给药)对阴道真菌载荷量、阴道粘膜真菌感染的影响,评价广藿香酮体内抗白色念珠菌活性。结果显示,广藿香酮阴道给药(1、2、4mg／kg)和口服给药(20、40、80mg／kg)都能有效减少小鼠阴道真菌载量、缓解雌激素化白色念珠菌小鼠的阴道炎,且受试剂量下广藿香酮阴道给药效果优于口服给药。
3.广藿香酮和广藿香醇的代谢研究
采用肝微粒体体外孵育模型,应用LC-MS/MS法研究广藿香酮在大鼠体外的代谢情况。结果显示,浓度为1.0mg／ml的广藿香酮在大鼠肝微粒体中孵化100min,可检测到广藿香酮的单一羟基化、双羟基化、氧化、水解的代谢产物。采用大鼠口服给药,考察了广藿香酮在大鼠体内的代谢行为。选择健康雄性大鼠,灌胃给予广藿香酮80mg／kg,采集给药后100min时的血液,连续收集24h的尿液、粪便、胆汁,经液-液萃取后用LC-MS/MS法分析。检测到血液、尿液、粪便和胆汁中总共存在5种代谢类型的产物：单一羟基化、双羟基化、氧化、羟基化+氧化、水解产物。
采用肝微粒体体外孵育模型,应用LC-MS/MS法研究广藿香醇在大鼠体外的代谢情况。结果显示,浓度为1.0mg／ml的广藿香醇在大鼠肝微粒体中孵化100main,未检测到广藿香醇代谢产物。采用大鼠口服给药,考察了广藿香醇在大鼠体内的代谢行为。选择健康雄性大鼠,灌胃给予广藿香醇80mg／kg,采集给药后100min时的血液,连续收集24h的尿液、粪便,经液-液萃取后用LC-MS/MS法分析。检测到尿液、粪便中存在1种代谢产物：羧酸化产物。
结论：
广藿香酮和广藿香醇作为广藿香油的主要成分,对二者生物活性的研究颇少。通过体内外抗炎活性试验,首次发现广藿香酮和广藿香醇均有明显的抗炎活性,证实二者均属于广藿香发挥抗炎作用的主要物质基础；广藿香酮作用机制的探索,提示广藿香酮在幽门螺旋杆菌胃炎、炎性肠炎等方面疾病潜在疗效的可能性。通过内外抗真菌试验,证实广藿香酮具有明显的抗皮肤癣菌和抗念珠菌作用,且广藿香酮有用于抗念珠菌外用制剂的潜力。结果发现广藿香酮与广藿香醇对皮肤癣菌的抑菌活性差异较大,提示了“石牌藿香”与“海南藿香”在抗皮肤癣菌方面存在较大差异的可能性。本研究有利于日后广藿香药材在抗皮肤癣菌方面合理应用的研究探索。历版《中国药典》均采用广藿香醇作为分析评价广藿香和广藿香油质量的指标成分,现行版《中国药典》(2010年版)增加了广藿香酮作为检测广藿香油的指标成分,然而广藿香药材仍仅采用广藿香醇作为指标成分。本研究或可为日后提高广藿香药材标准的研究提供理论依据。此外,本文首次采用LC-MS/MS法对广藿香酮和广藿香醇在大鼠体内外的代谢情况进行研究,发现广藿香酮可在大鼠体内迅速代谢为多个一相代谢产物,广藿香醇在大鼠体内可迅速代谢。提示广藿香酮和广藿香醇在体内的药效还可能是由其代谢产物发挥的,研究结果为进一步探讨广藿香酮和广藿香醇抗炎等作用的机制奠定了基础,也为广藿香酮和广藿香醇构效关系的研究提供参考。
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 PA
The 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 PA
PO 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 PA
High 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.
广藿香Pogostemon cablin (Blanco) Benth为唇形科刺蕊草属植物,以干燥地上部分入药,是临床常用芳香化湿中药,其味辛、性微温,归脾、胃、肺经,具有芳香化浊、和中止呕、发表解暑的功效。临床上用于治疗湿浊中阻、脘痞呕吐、暑湿表证、湿温初起、发热倦怠、胸闷不舒、寒湿闭暑、腹痛吐泻、鼻渊头痛等。广藿香属于我国“十大南药”之一,是41种中成药制剂的重要组成药味。广藿香油,是广藿香的挥发油部位,也作为药用提取物,在市场上占有重要的份额。因此,广藿香是重要的岭南道地药材,有巨大的开发潜力。
过去几十年来,国内外研究者对广藿香进行了广泛的研究,尤其是印度、中国、日本、美国等国家,基本阐明广藿香的化学成分组成、药理活性、栽培技术、生物遗传学等。然而,迄今为止国内外对广藿香的有效成分研究报道甚少,尚未清楚广藿香的有效成分及其作用机制。因此,亟需开展其有效成分物质基础的研究。
广藿香醇及广藿香酮是广藿香油中含量较高的化学成分。长期以来,国家和地方的药材标准将广藿香醇(百秋李醇)的含量作为分析广藿香质量的指标成分,2010年版《中国药典》增加了另一化学成分广藿香酮作为检测指标成分。可见,广藿香醇和广藿香酮是广藿香重要的化学成分。那么广藿香醇和广藿香酮的药理活性如何?它们是否能代表广藿香功效的物质基础呢？解决上述疑惑无疑具有重要的学术价值和重大的应用价值。
因此,本论文选择广藿香醇及广藿香酮,进行抗真菌、抗炎的药理活性研究,为探讨广藿香物质基础提供依据。此外,对广藿香醇及广藿香酮在大鼠体内外代谢产物进行研究,推测其在体内的代谢途径和转化过程,有助于阐明二者在体内的作用机制。本研究旨在初步探索广藿香的药效物质基础,以期为日后进一步研究广藿香药效的其他物质基础提供参考。
方法与结果：
1.广藿香酮和广藿香醇的抗炎活性研究
研究采用LPS诱导的RAW264.7巨噬细胞炎症模型,测定广藿香酮和广藿香醇对LPS诱导巨噬细胞产生的过量炎症介质(TNF-α、IL-1β、IL-6、PGE2、NO)的影响,并检测二者对炎症介质相关基因(TNF-α、IL-1β、IL-6、COX-2、iNOS)表达的影响。结果显示,广藿香酮(1、10、30μM)和广藿香醇(10、20、40μM)对LPS诱导的细胞炎症具有显著抑制作用,且显著抑制LPS诱导的炎症介质相关基因的过度表达。提示它们的作用途径可能是,通过抑制LPS诱导的炎症介质相关基因的过度表达,抑制了炎症介质的过度产生。
采用二甲苯致小鼠耳廓肿胀模型、角叉菜胶致大鼠足肿胀模型,测定广藿香酮和广藿香醇对小鼠耳廓、大鼠足趾肿胀度的影响,以及对大鼠足肿胀组织中炎症介质(IL-1β、TNF-α、PGE2、NO)的影响,以评价广藿香酮和广藿香醇对非感染性炎症的影响。结果显示,广藿香酮(10、20、40mg/kg)和广藿香醇(20、40、80mg/kg)口服给药对二甲苯致小鼠耳廓肿胀和角叉菜胶致大鼠足肿胀具有显著抑制作用,此外,广藿香酮(10、20、40mg/kg)和广藿香醇(10、20、40mg/kg)口服给药可显著抑制大鼠足肿胀组织中炎症介质的过度产生。
采用LPS诱导的内毒素休克小鼠模型,测定广藿香酮和广藿香醇对内毒素休克小鼠死亡率的影响,并测定内毒素休克小鼠生化指标、组织损伤、血清中炎症介质的含量、及组织中炎症介质相关基因表达,评价广藿香酮和广藿香醇对感染性炎症的影响。结果显示,广藿香醇(10、20、40mg/kg)口服给药对内毒素休克小鼠未表现出保护作用；广藿香酮(5、10、20mg/kg)静脉注射给药可提高LPS诱导的内毒素休克小鼠的存活率,下调内毒素休克小鼠血清中的ALP、AST、ALT水平,改善内毒素导致的小鼠肝、肺损伤；广藿香酮可抑制内毒素休克小鼠血清中的炎症介质(TNF-α、IL-1β、 IL-6、IFN-γ、IL-12、PGE2、NO)过量产生,并下调内毒素休克小鼠肝、肺组织中炎症介质相关基因(TNF-α、IL-1β、IL-6、IFN-γ、IL-12、COX-2、iNOS)的表达,提示广藿香酮对内毒素休克小鼠的保护作用,可能通过与其抑制内毒素引起的炎症介质瀑布样级联反应而实现的。
为了进一步研究广藿香酮对LPS诱导的炎症反应的抑制作用机理,检测了广藿香酮对LPS诱导的巨噬细胞内细胞信号通路的影响。结果显示,广藿香酮可抑制LPS诱导的巨噬细胞内NF-κB、JNK/SAPK及p38MAPK信号转导通路的激活。提示广藿香酮对LPS诱导内毒素休克小鼠的保护作用的机制,可能是其阻断了LPS激活细胞的NF-κB和MAPK信号转导通路,进而调控炎症介质相关基因的表达,最终抑制炎症介质的过度产生。
2.广藿香酮和广藿香醇的抗真菌活性研究
采用琼脂稀释法和微量液基稀释法测定广藿香酮和广藿香醇对11株临床皮肤癣菌的影响。结果显示,广藿香酮对各受试皮肤癣菌均有明显抗菌作用,MIC介于24.38～195μg/ml之间,而广藿香醇对受试菌株的MIC>1560μg/ml。在相同条件下所有受试菌株对氟康唑和伊曲康唑均有耐药性(MIC>1560μg/ml),广藿香酮对部分皮肤癣菌有杀菌作用。
采用微量液基稀释法,测定广藿香酮和广藿香醇对临床念珠菌属真菌的影响,并通过测定白色念珠菌的生长曲线、生物被膜活性,初步探讨作用机理。结果显示,广藿香醇对所有受试菌株的MIC>400μg/ml;广藿香酮对受试白色念珠菌的MIC在3.13至50μg／ml之间,广藿香酮对所有受试白色念珠菌株均有杀菌作用,其MFC介于100至400μg／ml之间,而伏立康唑的最高浓度(400μg／m1)仅对其中一株有杀菌作用。对于其他念珠菌属菌种,广藿香酮的MIC介于12.5至400μg／ml之间,均小于伏立康唑；而广藿香酮对其他受试念珠菌种无杀菌作用。广藿香酮可在白色念珠菌的生长初期迅速将其抑制或杀灭,广藿香酮可显著抑制白色念珠菌成熟生物被膜的生长。说明抑制生物膜的生长和形成可能是广藿香酮抗耐药白色念珠菌的作用机制之一
采用雌激素化白色念珠菌小鼠阴道炎模型,测定两种给药方式的广藿香酮(即局部给药和口服给药)对阴道真菌载荷量、阴道粘膜真菌感染的影响,评价广藿香酮体内抗白色念珠菌活性。结果显示,广藿香酮阴道给药(1、2、4mg／kg)和口服给药(20、40、80mg／kg)都能有效减少小鼠阴道真菌载量、缓解雌激素化白色念珠菌小鼠的阴道炎,且受试剂量下广藿香酮阴道给药效果优于口服给药。
3.广藿香酮和广藿香醇的代谢研究
采用肝微粒体体外孵育模型,应用LC-MS/MS法研究广藿香酮在大鼠体外的代谢情况。结果显示,浓度为1.0mg／ml的广藿香酮在大鼠肝微粒体中孵化100min,可检测到广藿香酮的单一羟基化、双羟基化、氧化、水解的代谢产物。采用大鼠口服给药,考察了广藿香酮在大鼠体内的代谢行为。选择健康雄性大鼠,灌胃给予广藿香酮80mg／kg,采集给药后100min时的血液,连续收集24h的尿液、粪便、胆汁,经液-液萃取后用LC-MS/MS法分析。检测到血液、尿液、粪便和胆汁中总共存在5种代谢类型的产物：单一羟基化、双羟基化、氧化、羟基化+氧化、水解产物。
采用肝微粒体体外孵育模型,应用LC-MS/MS法研究广藿香醇在大鼠体外的代谢情况。结果显示,浓度为1.0mg／ml的广藿香醇在大鼠肝微粒体中孵化100main,未检测到广藿香醇代谢产物。采用大鼠口服给药,考察了广藿香醇在大鼠体内的代谢行为。选择健康雄性大鼠,灌胃给予广藿香醇80mg／kg,采集给药后100min时的血液,连续收集24h的尿液、粪便,经液-液萃取后用LC-MS/MS法分析。检测到尿液、粪便中存在1种代谢产物：羧酸化产物。
结论：
广藿香酮和广藿香醇作为广藿香油的主要成分,对二者生物活性的研究颇少。通过体内外抗炎活性试验,首次发现广藿香酮和广藿香醇均有明显的抗炎活性,证实二者均属于广藿香发挥抗炎作用的主要物质基础；广藿香酮作用机制的探索,提示广藿香酮在幽门螺旋杆菌胃炎、炎性肠炎等方面疾病潜在疗效的可能性。通过内外抗真菌试验,证实广藿香酮具有明显的抗皮肤癣菌和抗念珠菌作用,且广藿香酮有用于抗念珠菌外用制剂的潜力。结果发现广藿香酮与广藿香醇对皮肤癣菌的抑菌活性差异较大,提示了“石牌藿香”与“海南藿香”在抗皮肤癣菌方面存在较大差异的可能性。本研究有利于日后广藿香药材在抗皮肤癣菌方面合理应用的研究探索。历版《中国药典》均采用广藿香醇作为分析评价广藿香和广藿香油质量的指标成分,现行版《中国药典》(2010年版)增加了广藿香酮作为检测广藿香油的指标成分,然而广藿香药材仍仅采用广藿香醇作为指标成分。本研究或可为日后提高广藿香药材标准的研究提供理论依据。此外,本文首次采用LC-MS/MS法对广藿香酮和广藿香醇在大鼠体内外的代谢情况进行研究,发现广藿香酮可在大鼠体内迅速代谢为多个一相代谢产物,广藿香醇在大鼠体内可迅速代谢。提示广藿香酮和广藿香醇在体内的药效还可能是由其代谢产物发挥的,研究结果为进一步探讨广藿香酮和广藿香醇抗炎等作用的机制奠定了基础,也为广藿香酮和广藿香醇构效关系的研究提供参考。
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 PA
The 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 PA
PO 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 PA
High 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.
引文
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