Subcutaneous adipocytes promote melanoma cell growth by activating the Akt signaling pathway: role of palmitic acid.

Tumorigenesis involves constant communication between tumor cells and neighboring normal cells such as adipocytes. The canonical function of adipocytes is to store triglyceride and release fatty acids for other tissues. This study was aimed to find out if adipocytes promoted melanoma cell growth and to investigate the underlying mechanism. Here we isolated adipocytes from inguinal adipose tissue in mice and co-cultured with melanoma cells. We found that the co-cultured melanoma had higher lipid accumulation compared with mono-cultured melanoma. In addition, fluorescently labeled fatty acid BODIPY® FLC16 signal was detected in melanoma co-cultured with the adipocytes that had been loaded with the fluorescent dye, suggesting that the adipocytes provide fatty acids to melanoma cells. Compared with mono-cultured melanoma, co-cultured melanoma cells had a higher proliferation and phospho-Akt (Ser-473 and Thr-450) expression. Overexpression of Akt mutants in melanoma cells reduced the co-culture-enhanced proliferation. A lipidomic study showed that the co-cultured melanoma had an elevated palmitic acid level. Interestingly, we found that palmitic acid stimulated melanoma cell proliferation, changed the cell cycle distribution, and increased phospho-Akt (Ser-473 and Thr-450) and PI3K but not phospho-PTEN (phosphophosphatase and tensin homolog) expressions. More importantly, the palmitic acid-stimulated proliferation was further enhanced in the Akt-overexpressed melanoma cells and was reduced by LY294002 or knockdown of endogenous Akt or overexpression of Akt mutants. We also found that palmitic acid-pretreated B16F10 cells were grown to a significantly larger tumor in mice compared with control cells. Taken together, we suggest that adipocytes may serve as an exogenous source of palmitic acid that promotes melanoma cell growth by activating Akt.

UHPLC UHD Q-TOF MS/MS analysis of the impact of sulfur fumigation on the chemical profile of Codonopsis Radix (Dangshen).

Over recent decades sulfur fumigation has been becoming abused in processing some freshly harvested Chinese medicinal herbs, although it is questioned whether sulfur fumigation can result in changes in efficacy and safety of the herbs. One of the herbs commonly processed by sulfur fumigation is Codonopsis Radix (Dangshen). A report showed that lobetyolin content in sulfur-fumigated Dangshen was lower than in air-dried Dangshen. Whereas there is no investigation designed to compare the chemical profiles of the sulfur-fumigated Dangshen and the air-dried Dangshen. In the present study, a rapid and versatile ultra-high-performance liquid chromatography coupled with ultra-high resolution quadrupole time-of-flight mass spectrometry (UHPLC UHD Q-TOF MS/MS) method was developed for comprehensive analysis of the chemical profiles of sulfur-fumigated and air-dried Dangshen samples. Principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA) demonstrated that there were significant chemical differences between sulfur-fumigated and air-dried Dangshen samples. Among the changed components, 57 compounds were identified, in which 15 sulfur-containing compounds were detected only in sulfur-fumigated samples. The established methods were successfully applied to discriminate sulfur-fumigated Dangshen among commercial samples. Whether the chemical changes caused by sulfur fumigation affect the clinical efficacy and safety of Dangshen needs to be further investigated.

Lipidomics identification of metabolic biomarkers in chemically induced hypertriglyceridemic mice.

In this study, we aim to identify the potential biomarkers in hTG pathogenesis in schisandrin B-induced hTG mouse model. To investigate whether these identified biomarkers are only specific to schisandrin B-induced hTG mouse model, we also measured these biomarkers in a high fat diet (HFD)-induced hTG mouse model. We employed a LC/MS/MS-based lipidomic approach for the study. Mouse liver and serum metabolites were separated by reversed phase liquid chromatography. Metabolite candidates were identified by matching with marker retention times, isotope distribution patterns, and high-resolution MS/MS fragmentation patterns. Subsequently, target candidates were quantified by quantitative MS. In the schisandrin B-induced hTG mice, we found that the plasma fatty acids, diglyceroids, and phospholipids were significantly increased. Palmitic acid and stearic acid were increased in the plasma; oleic acid, linoleic acid, linolenic acid, arachidonic acid, and docosahexaenoic acid were increased in both the plasma and the liver. Acetyl-CoA, malonyl-CoA, and succinyl-CoA were increased only in the liver. The changes in levels of these identified markers were also observed in HFD-induced hTG mouse model. The consistent results obtained from both hTG models not only suggest novel biomarkers in hTG pathogenesis, but they also provide insight into the underlying mechanism of the schisandrin B-induced hTG.

Anti-inflammatory and analgesic effects of the ethanol extract of Rosa multiflora Thunb. hips.

This study aimed to assess the anti-inflammatory and analgesic effects of Fructus Rosae Multiflorae (FRM, hips of Rosa multiflora Thunb.). FRM was extracted with 75% ethanol and the dried extract (FRME) was administered intragastrically (i.g.) at 100, 200 and 400mg/kg. The anti-inflammatory effect was evaluated in four experimental animal models and analgesic effect in two animal models. Pretreatment with a single dose of FRME produced significant dose-dependent anti-inflammatory effects on carrageenin-induced rat hind paw edema, xylene-induced mouse ear edema and acetic acid-induced mouse vascular permeation. In a 7-day study, daily administration of FRME suppressed cotton pellet-induced rat granuloma formation. Pretreatment with a single dose of FRME also produced dose-dependent anti-nociceptive effects in thermally- and chemically induced mouse pain models. In addition, a single dose of FRME at 2.4g/kg body weight (equivalent to 87.6g of dried hips per kg body weight) produced no observable acute toxicity in mice within seven days. These results demonstrate that FRME possesses anti-inflammatory and analgesic effects and has no obvious acute toxicity, which advanced our understanding of the folk use of FRM in treating various inflammatory disorders.

Schisandrin B regulates lipid metabolism in subcutaneous adipocytes.

Subcutaneous adipocytes in obese subjects have a lower sensitivity to catecholamine-induced lipolysis and a higher sensitivity to insulin anti-lipolytic effects compared to adipocytes in other adipose depots. Therefore, increasing lipolysis in subcutaneous adipocytes coupled with enhanced fatty acid oxidation may be an anti-obesity strategy. Schisandrin B (Sch B) is one of the most abundant active dibenzocyclooctadiene derivatives found in the fruit of Schisandra chinensis which is a commonly prescribed Chinese medicinal herb. We found that Sch B reduced glycerolipid contents in 3T3-L1 adipocytes and subcutaneous adipocytes dissected from DIO mice. Sch B also activated hormone sensitive lipase (HSL) and increased lipolysis in these adipocyte in a protein kinase A-dependent manner. Interestingly, Sch B increased fatty acid oxidation gene expressions in these adipocytes, implying an increase in fatty acid oxidation after treatment. In in vivo model, we found that Sch B increased HSL phosphorylation, reduced glycerolipid levels and increased fatty acid oxidation gene expressions in the subcutaneous adipocytes in the DIO mice. More importantly, Sch B significantly reduced the subcutaneous adipocyte sizes, subcutaneous adipose tissue mass and body weight of the mice. Our study provides scientific evidence to suggest a potential therapeutic function of Sch B or Schisandra chinensis seed containing Sch B in reducing obesity.

Cinnamon induces browning in subcutaneous adipocytes.

Browning is the process of increasing the number of brite cells, which helps to increase energy expenditure and reduce obesity. Consumption of natural and non-toxic herbal extracts that possess the browning effect is an attractive anti-obesity strategy. In this study, we examined the browning effect of cinnamon extract. We found that cinnamon extract (CE) induced typical brown adipocyte multiocular phenotype in 3T3-L1 adipocytes. The treatment also increased brown adipocytes markers and reduced white adipocytes markers in the 3T3-L1 adipocytes. In ex vivo studies, we found that CE increased brown adipocytes markers in the subcutaneous adipocytes isolated from db/db mice and diet-induced obesity (DIO) mice. However, CE did not significantly affect UCP1 expression in the adipocytes isolated from perinephric adipose tissue and epididymal adipose tissue. ß3-adernergic receptor (ß3-AR) antagonist reduced the CE-enhanced UCP1 expression, suggesting an involvement of the ß3-AR activity. Oral administration of CE significantly increased UCP1 expression in the subcutaneous adipose tissue in vivo and reduced the body weight of the DIO mice. Taken together, our data suggest that CE has a browning effect in subcutaneous adipocytes. Our study suggests a natural non-toxic herbal remedy to reduce obesity.

Influence of sulfur fumigation on glycoside profile in Platycodonis Radix (Jiegeng).

BACKGROUND: Over recent decades, sulfur fumigation is becoming abused in processing some freshly harvested herbs used as both medicine and food, although it has been questioned whether sulfur fumigation will change the efficacy and safety of the herbs. One of the herbs commonly processed by sulfur fumigation is Platycodonis Radix (Jiegeng in Chinese). Glycosides are the main bioactive components of Jiegeng. Up to the present, no study has been carried out to evaluate the impact of sulfur fumigation on glycoside profile of Jiegeng. METHODS: A rapid and versatile ultra-high performance liquid chromatography coupled with ultra-high resolution quadrupole time-of-flight mass spectrometry (UHPLC UHD Q-TOF MS/MS) method was developed for comprehensive analysis of the glycoside profiles of sulfur-fumigated and air-dried Jiegeng samples. RESULTS: Twenty-three glycosides were detected in air-dried and sulfur-fumigated Jiegeng samples. After sulfur fumigation, the peak heights of eight glycosides, namely platycogenin A, platycodin D, platycodin D2, platycodin D3, polygalacin D, polygalacin D2, deapio-platycodin D and 3″-O-acetylplatycodin D2, remarkably decreased; while peak heights of five glycosides, namely syringin, lobetyolin, platycoside E, deapio-platycodin D2 and deapio-platycoside E, slightly increased; in addition, peaks of ten glycosides, platycodin A, platycodin C, platycodin V, platycoside C, 16-oxoplatycodin D, 2″-O-acetylpolygalacin D, 2″-O-acetylpolygalacin D2, 3″-O-acetylpolygalacin D, 3″-O-acetylpolygalacin D2, and platycogenic acid B, disappeared. CONCLUSION: Sulfur fumigation caused significant changes of glycoside components of Jiegeng. Further investigations are warranted to explore how these chemical changes occurred and whether these changes would affect the efficacy and safety of Jiegeng.

Metabolomics reveals the mechanisms for the cardiotoxicity of Pinelliae Rhizoma and the toxicity-reducing effect of processing.

Pinelliae Rhizoma (PR) is a commonly used Chinese medicinal herb, but it has been frequently reported about its toxicity. According to the traditional Chinese medicine theory, processing can reduce the toxicity of the herbs. Here, we aim to determine if processing reduces the toxicity of raw PR, and to explore the underlying mechanisms of raw PR-induced toxicities and the toxicity-reducing effect of processing. Biochemical and histopathological approaches were used to evaluate the toxicities of raw and processed PR. Rat serum metabolites were analyzed by LC-TOF-MS. Ingenuity pathway analysis of the metabolomics data highlighted the biological pathways and network functions involved in raw PR-induced toxicities and the toxicity-reducing effect of processing, which were verified by molecular approaches. Results showed that raw PR caused cardiotoxicity, and processing reduced the toxicity. Inhibition of mTOR signaling and activation of the TGF-ß pathway contributed to raw PR-induced cardiotoxicity, and free radical scavenging might be responsible for the toxicity-reducing effect of processing. Our data shed new light on the mechanisms of raw PR-induced cardiotoxicity and the toxicity-reducing effect of processing. This study provides scientific justifications for the traditional processing theory of PR, and should help in optimizing the processing protocol and clinical combinational application of PR.

Assay of free ferulic acid and total ferulic acid for quality assessment of Angelica sinensis.

Activity of Chinese Danggui (DG), the processed root of Angelica sinensis (Oliv.) Diels, is linked to the ferulic acid content but the stability of ferulic acid during extraction for medicinal use is not known. The stabilities of ferulic acid and coniferyl ferulate were evaluated in the extracts of DG using a variety of extraction solvents. These included various combinations and proportions of methanol, water, formic acid, 1 M aqueous hydrochloric acid and 2% sodium hydrogen carbonate (NaHCO3) in water. Coniferyl ferulate was found liable to hydrolyze into ferulic acid in neutral, strongly acidic and basic solvents, where heat and water could facilitate this hydrolysis. However, the hydrolysis was relatively resisted in weakly organic acid. Based on the stability evaluation, two new terms, namely: free ferulic acid and total ferulic acid, were suggested and defined. Free ferulic acid refers to the natural content of ferulic acid in herbs. Total ferulic acid means the sum of free ferulic acid plus the amount of related hydrolyzed components. Meanwhile, the high-performance liquid chromatographic (HPLC) method was developed to assay free ferulic acid and total ferulic acid in DG using methanol-formic acid (95:5) and methanol-2% NaHCO3 in water (95:5) as extraction solvents, respectively. Ten DG samples were investigated on their contents of free and total ferulic acid. The results indicated that the amount variety of free ferulic acid was larger than that of their counterparts, and the ratio of total ferulic acid to free ferulic acid was 4.07 +/- 2.73 (mean +/- SD, n = 10). The chemical assay of DG using total ferulic acid content would be a better choice to assess the herbal quality and was recommended.

Development of high-performance liquid chromatographic fingerprints for distinguishing Chinese Angelica from related umbelliferae herbs.

A high-performance liquid chromatographic (HPLC) fingerprint of Chinese Angelica (CA) was developed basing on the consistent chromatograms of 40 CA samples (Angelica sinensis (Oliv.) Diels). The unique properties of this HPLC fingerprints were validated by analyzing 13 related herbs including 4 Japanese Angelicae Root samples (JA, A. acutiloba Kitagawa and A. acutiloba Kitagawa var. sugiyame Hikino), 6 Szechwan Lovage Rhizome samples (SL, Ligusticum chuanxiong Hort.) and 3 Cnidium Rhizome samples (CR, Cnidium officinale Makino). Both correlation coefficients of similarity in chromatograms and relative peak areas of characteristic compounds were calculated for quantitative expression of the HPLC fingerprints. The amount of senkyunolide A in CA was less than 30-fold of that in SL and CR samples, which was used as a chemical marker to distinguish them. JA was easily distinguished from CA, SL and CR based on either chromatographic patterns or the amount of coniferyl ferulate. No obvious difference between SL and CR chromatograms except the relative amount of some compounds, suggesting that SL and CR might have very close relationship in terms of chemotaxonomy. Ferulic acid and Z-ligustilide were unequivocally determined whilst senkyunolide I, senkyunolide H, coniferyl ferulate, senkyunolide A, butylphthalide, E-ligustilide, E-butylidenephthalide, Z-butylidenephthalide and levistolide A were tentatively identified in chromatograms based on their atmospheric pressure chemical ionization (APCI) MS data and the comparison of their UV spectra with those published in literatures.

Lipidomic-based investigation into the regulatory effect of Schisandrin B on palmitic acid level in non-alcoholic steatotic livers.

Schisandrin B (SchB) is one of the most abundant bioactive dibenzocyclooctadiene derivatives found in the fruit of Schisandra chinensis. Here, we investigated the potential therapeutic effects of SchB on non-alcoholic fatty-liver disease (NAFLD). In lipidomic study, ingenuity pathway analysis highlighted palmitate biosynthesis metabolic pathway in the liver samples of SchB-treated high-fat-diet-fed mice. Further experiments showed that the SchB treatment reduced expression and activity of fatty acid synthase, expressions of hepatic mature sterol regulatory element binding protein-1 and tumor necrosis factor-α, and hepatic level of palmitic acid which is known to promote progression of steatosis to steatohepatitis. Furthermore, the treatment also reduced hepatic fibrosis, activated nuclear factor-erythroid-2-related factor-2 which is known to attenuate the progression of NASH-related fibrosis. Interestingly, in fasting mice, a single high-dose SchB induced transient lipolysis and increased the expressions of adipose triglyceride lipase and phospho-hormone sensitive lipase. The treatment also increased plasma cholesterol levels and 3-hydroxy-3-methylglutaryl-CoA reductase activity, reduced the hepatic low-density-lipoprotein receptor expression in these mice. Our data not only suggest SchB is a potential therapeutic agent for NAFLD, but also provided important information for a safe consumption of SchB because SchB overdosed under fasting condition will have adverse effects on lipid metabolism.

Quantification of ligustilides in the roots of Angelica sinensis and related umbelliferous medicinal plants by high-performance liquid chromatography and liquid chromatography-mass spectrometry.

A reversed phase high-performance liquid chromatographic method was developed for quantifying E-ligustilide (1) and Z-ligustilide (3) in the roots of Angelica sinensis (Oliv.) Diels with confirmation using UV, atmospheric pressure chemical ionisation (APCI) MS and APCI-MS-MS techniques. Based on the UV spectra of compounds 1, E-butylidenephthalide (2), 3 and Z-butylidenephthalide (4), the absorption at 350 nm was chosen as measuring wavelength in which baseline separation of compounds 1 and 3 could be obtained but avoided the interference of compounds 2 and 4. The identity of compounds 1 and 3 in samples were unambiguously determined by the respective quasi-molecular ions ([M+H]+) in APCI-MS. According to the stability data, acetonitrile was chosen for the preparation of standard solutions in order to minimize the isomerization of compound 3. Compounds 1 and 3 were qualitatively and quantitatively analyzed in seven samples of the roots of Angelica sinensis (Oliv.) Diels, Angelica acutiloba Kitagawa, Angelica acutiloba Kitagawa var. sugiyamae Hikino and the rhizome of Ligusticum chuanxiong Hort. Analysis of an extract from a sample root of Angelica gigas Nakai using LC-MS for the first time could not detect the presence of ligustilide in this herb. The overall analytical procedure is rapid and reproducible which is considered suitable for quantitative analysis of large number of samples.

Comparisons of the chemical profiles, cytotoxicities and anti-inflammatory effects of raw and rice wine-processed Herba Siegesbeckiae.

ETHNOPHARMACOLOGICAL RELEVANCE: Although slightly toxic, the Chinese medicinal herb Herba Siegesbeckiae (HS) has long been used as a remedy for traditional Chinese medicine symptoms that resemble inflammatory joint disorders, because it can eliminate the wind-dampness and soothe painful joints. Proper processing can reduce the toxicity and/or enhance the efficacy of raw herbs. In this study, we aim to examine if processing with rice wine reduces the cytotoxicities and/or enhances the anti-inflammatory effects of HS, and to explore the chemical basis behind the potential changes of medicinal properties caused by the processing. MATERIALS AND METHODS: We used cell models to examine the cytotoxicities and anti-inflammatory effects of HS and rice wine-processed HS (WHS). The chemical profiles of HS and WHS were compared using the ultra-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry (UPLC/Q-TOF-MS) analysis. RESULTS: We found that WHS was less toxic than HS in cultured cells as shown in the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Both HS and WHS had anti-inflammatory effects as demonstrated by their abilities to reduce nitric oxide (NO) production as well as protein and mRNA expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. Interestingly, the anti-inflammatory effects of WHS were more potent than that of HS at the concentration of 100 µg/mL. By comparing the chemical profiles, we found that 19 peaks were lower, while 2 other peaks were higher in WHS than in HS. Four compounds including neo-darutoside, darutoside, stigmasterol and 16-O-acetyldarutoside corresponding to 4 individual changed peaks were tentatively identified by matching with empirical molecular formulae and mass fragments. CONCLUSION: Our study showed that processing with rice wine significantly reduced the cytotoxicities and enhanced the anti-inflammatory effects of HS as demonstrated in cell models. We also developed a UPLC/Q-TOF-MS method to clearly differentiate HS from WHS by their different chemical profiles. Further study is warranted to establish the relationship between the alteration of chemical profiles and the changes of medicinal properties caused by processing with rice wine.

A cellular lipidomic study on the Aß-induced neurotoxicity and neuroprotective effects of EGCG by using UPLC/MS-based glycerolipids profiling and multivariate analysis.

The aim of this study was to investigate the cellular lipid metabolism associated with ß-amyloid peptide (Aß)-induced neurotoxicity as well as the neuroprotective effect of (-)-epigallocatechin gallate (EGCG), a major polyphenol in green tea. An ultra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry (UPLC-Q-TOF-MS)-based lipidomic approach was developed to screen and identify changes of the glycerolipids (GL) upon Aß treatment with or without the presence of EGCG in PC12 cells. Principle component analysis (PCA) showed that the Aß-treated group was well separated from the control group, whereas the EGCG group was closer to the control group. The GL levels were significantly elevated in Aß-treated cells compared with the control group, but were restored near to normal levels after EGCG treatment. The elevated phosphatidylcholines (PCs) levels observed in the Aß-treated PC12 cells were quite probably the integrated results of the reduced phospholipase A(2) (PLA(2)) activity and the enhanced activity of lysophospholipid acyltransferases. Moreover, an increased liberation of arachidonic acid (AA) from PCs was observed as another important response of PC12 cells to the Aß aggregates, implying an active inflammatory process occurring in Aß induced neurotoxicity. EGCG treatment can reverse the deregulated metabolism of PCs, which might be one of the biochemical mechanisms contributing to its neuroprotective effect. Collectively, results obtained from the current lipidomic analyses of PC12 cells provided important insight into the biochemical mechanisms underlying Aß-induced neurotoxicity and neuro protective effects of EGCG. This is the first report of the lipidomic study on the neuroprotective effect of EGCG.

Identification of five hepatotoxic pyrrolizidine alkaloids in a commonly used traditional Chinese medicinal herb, Herba Senecionis scandentis (Qianliguang).

Senecio scandens Buch.-Ham is a plant source for a commonly used traditional Chinese medicinal (TCM) herb Qianliguang. A TCM herbal proprietary product containing Qianliguang as the major herb for the treatment of sinusitis has been used in China for several decades, and has also been exported to other regions and countries worldwide. In the present study, the aqueous extract of S. scandens collected in the Shanxi Province of China was determined, for the first time, to contain hepatotoxic and tumorigenic pyrrolizidine alkaloids (PAs) by using high-performance liquid chromatography/mass spectrometric (HPLC/MS) analysis in various scanning modes. A total of nine toxic and two non-toxic PAs were detected in the aqueous extract of S. scandens, of which six PAs, namely neoplatyphylline, senecionine, senecionine N-oxide, seneciphylline, seneciphylline N-oxide and senkirkine, were unequivocally characterized, while other PAs were tentatively assigned as jacobine, jacozine N-oxide (or erucifoline N-oxide), 7-tigloylplatynecine, usaramine and an isomer of yamataimine. The estimated total content of toxic PAs in S. scandens was 10.82 microg/g herb, which was significantly higher than that (> or =1 microg/g herb) recommended by Belgium and Germany not to be used clinically. Among the PAs definitively identified, senecionine, seneciphylline, and senkirkine are known tumorigens capable of inducing liver tumors in experimental animals, while seneciphylline N-oxide and senecionine N-oxide are probably tumorigenic due to their potential conversion into seneciphylline and senecionine via metabolic reduction in the body. Thus, the current finding of the presence of toxic/tumorigenic PAs in S. scandens challenges the safety of using this TCM herb and its proprietary products.

Comparative study on the aristolochic acid I content of Herba Asarifor safe use.

Herba Asari (Xixin, Manchurian Wildginger, Asarum spp.) is a traditional Chinese medicinal herb commonly used as a crude drug and an ingredient in patent medicines. The herb contains aristolochic acid I (AA-I), which has recently caused several incidents of poisoning in Hong Kong. Therefore, the safe use of Asarum is questionable. The present study was undertaken to assess the levels of AA-I using liquid chromatography-mass spectrometry (LC/MS) indifferent medicinal parts of Herba Asari and some proprietary Chinese medicines (PCM) containing it as an ingredient. The AA-I content in the aerial and root portions were compared, in the form of water and methanolic extracts. The results showed that all the aerial portions of Herba Asari generally contain higher levels of AA-I than the roots (in water extract: 0.0870.06 microg/g of root and 0.3270.021 microg/g of aerial), and the methanolic extracts typically contained more AA-I than the water extracts. Moreover, all the three PCM studies showed negligible amounts of AA-I(containing 0.0370.006 microg/g). Therefore, the root portion of Herba Asari was recommended for prescription as a decoction instead of grinding it into powder for oral administration.

Systematic evaluation of organochlorine pesticide residues in Chinese materia medica.

A systematic evaluation on the levels of organochlorine pesticide residues (OCP) was conducted on four selected, authentic Chinese materia medica, namely: Radix Angelicae Sinensis, Radix Notoginseng, Radix Salviae Miltiorrhizae and Radix Ginseng. Altogether ten representative batches of samples were analysed for each herb. Six batches were collected in the major cultivation areas of the Mainland whilst the remaining four batches were procured in the Hong Kong herbal market. All except Radix Angelicae Sinensis have been identified as containing quintozene and hexachlorocyclohexane in various levels. Hexachlorobenzene and lindane were also reported in samples of Radix Ginseng. The banned pesticide, DDT and its derivatives, was also observed in one of the Radix Notoginseng samples. The investigation will be continued for a target list of common used herbs in Hong Kong. All the results will be gathered and analysed for setting up regulatory permissible limits of OCP residues in Chinese materia medica used in Hong Kong.

Determination of patchoulic alcohol in Herba Pogostemonis by GC-MS-MS.

Patchoulic oil, the volatile oil of Pogostemon cablin (Blanco) Benth. and the Chinese crude drug Herba Pogostemonis, is widely used in the cosmetic and oral hygiene industries. Patchoulic alcohol is commonly used as an indicator for the quality assessment of dried P. cablin. However, the complexity of the herbal constituents makes it difficult for using conventional gas chromatography (GC) for analytical purpose. The present study established an accurate, sensitive and reproducible method for the quality assessment of the dried patchouli herb based on patchoulic alcohol content. A gas chromatography-tandem mass spectrometry (GC/MS/MS) method has been successfully developed and demonstrated for the determination of patchoulic alcohol content in the samples of dried P. cablin. The developed method was found to be convenient and facile in particular to tackle the complicated matrix problems always encountered in the herbs which contain high level of essential oils.

Spectrometric studies of cytotoxic protoberberine alkaloids binding to double-stranded DNA.

The noncovalent complexes of five cytotoxic protoberberine alkaloids, that is, berberine, palmatine, jatrorrhizine, coptisine, and berberrubine with several double-stranded oligodeoxynucleotides were systematically investigated by using electrospray ionization mass (ESI-MS) and fluorescence spectrometric methods, with the aim of establishing the structure-activity relationships. ESI-MS spectrometric studies indicated that these five alkaloids showed both 1:1 and 1:2 binding stoichiometries with d(AAGAATTCTT)(2), d(AAGGATCCTT)(2), and d(AAGCATGCTT)(2). Their relative binding affinities toward these three double-stranded DNA were semi-quantitatively evaluated by measuring the ratios of the complex signals ([ds+alkaloid-5H](4-)+[ds+2alkaloid-6H](4-)) to those of the duplexes ([ds-4H](4-)) and also by ESI-MS competitive binding experiments. These experiments established the relative binding affinities of five protoberberine alkaloids in the order of palmatine>jatrorrhizine>coptisine>berberine>berberrubine with d(AAGAATTCTT)(2), palmatinecoptisine>jatrorrhizineberberine>berberrubine with d(AAGGATCCTT)(2) and palmatine>jatrorrhizinecoptisine>berberine>berberrubine with d(AAGCATGCTT)(2). Significantly, these alkaloids except berberrubine bound to d(AAGGATCCTT)(2) and d(AAGCATGCTT)(2) with the affinities comparable to Hoechst 33258, a typical DNA minor groove binder. The relative binding preferences of berberine, palmatine, and coptisine with these three double-stranded DNA were further quantitatively assessed by their association constants obtained from fluorescence titration experiments. The values revealed the order of relative binding affinities as berberine>coptisine>palmatine with d(AAGAATTCTT)(2) and coptisine>berberine>palmatine with d(AAGGATCCTT)(2) and d(AAGCATGCTT)(2). These results were not in full agreement with those obtained from ESI-MS experiments, maybe due to the different measuring solution conditions. The results from ESI-MS and fluorescence titration experiments indicated that the sequence selectivities of these five alkaloids were not significant and remarkable AT- or GC-rich DNA binding preferences were not obtained, in contrast to the report that berberine binds preferentially to AT-rich DNA. To provide further insight into the sequence selectivities, the association constants of berberine with d(AAGATATCTT)(2), 5'-AAGTAATCTT-3'/5'-AAGATTACTT-3', d(AAGGGCCCTT)(2), d(AAGGCGCCTT)(2), and 5'-AAGGCCGCTT-3'/5'-AAGCGGCCTT-3', that is double helical DNA from AT-rich to GC-rich sequences, were further measured by fluorescence titration methods. No significant differences in their association constants were observed, suggesting that berberine showed no remarkable sequence selectivities.

Study on noncovalent complexes of cytotoxic protoberberine alkaloids with double-stranded DNA by using electrospray ionization mass spectrometry.

The noncovalent complexes of four cytotoxic protoberberine alkaloids that is, berberine, palmatine, jatrorrhizine, and coptisine with double-stranded oligodeoxynucleotides d(AAGAATTCTT)(2) were investigated by electrospray ionization mass spectrometry. These four active components from Chinese herbal medicines showed both 1:1 and 1:2 binding stoichiometries, independent on the alkaloid-to-DNA ratios. Binding affinities in the order of palmatine> or =jatrorrhizine>coptisine>berberine with d(AAGAATTCTT)(2) were obtained. Additionally, the preliminary results indicated that berberine had some sequence selectivities.