Ethnopharmacology of five flowers herbal tea, a popular traditional beverage in Hong Kong and South China.

BACKGROUND: It has been a long-standing tradition of using herbal tea for preventive and therapeutic healthcare in Hong Kong and South China and Five Flowers Tea is one of the most popular herbal teas. Based on the principle of traditional Chinese medicine, the pharmacological functions are to clear heat and dispel dampness in the body. Heat and dampness are thought to contribute to a range of health problems, especially during the hot and humid season in South China and Hong Kong. The most prevalent herbs in the formula contain bioactive compounds including flavonoids, alkaloids and terpenoids, which have a wide range of pharmacological properties including anti-inflammation, antivirus, antidiarrhoea, antibacteria, and antioxidation. However, with the composition varies widely, the ethnopharmacological benefits described may not be delivered uniformly. This study is to provide a comprehensive analysis on the composition of the Five Flowers Tea sold in Hong Kong and investigate the rationale behind the selection of herbs used in the formula. This study also provides information on the variation and quality of the Five Flowers Tea in the market. METHODS: Thirty-three Five Flowers Tea samples were collected from various locations in Hong Kong. The size, texture, colour and organoleptic properties were documented. Macroscopic and molecular authentication methods were employed to identify the individual components. RESULTS: Macroscopic identification revealed there were 23 herbs belonging to 18 plant families. The most prevalent herb was Bombax ceiba L., followed by Chrysanthemum morifolium. Ten adulterants and the existence of insect Lasioderma serricorne were confirmed by DNA barcoding techniques. CONCLUSION: This study employed a comprehensive approach to authenticate the herbs in Five Flowers Tea samples collected from various locations in Hong Kong. Macroscopic and molecular methods were used to identify the herbs and adulterants. The findings revealed the varied composition in Five Flowers Tea and the occurrence of adulterants in some samples. This shows that quality assurance of Five Flowers Tea is essential for the effective use of this popular folk medicine.

Simultaneous quantification of six alkaloid components from commercial stemonae radix by solid phase extraction-high-performance liquid chromatography coupled with evaporative light scattering detector.

BACKGROUND: Stemonae radix has been applied in traditional Chinese medicine for centuries. Alkaloids are the main active ingredient in stemonae radix, so their composition and concentration levels are directly linked to clinic effects. OBJECTIVE: The objective was to develop an analytical method with multiple markers for quality survey of commercial stemonae radix. MATERIALS AND METHODS: A method for simultaneous determination of six compounds in commercial stemonae radix was performed using solid-phase extraction and high-performance liquid chromatography coupled with evaporative light scattering detector. The separation was carried out on an Agilent TC-C18 column with 0.1% acetonitrile solution of triethylamine aqueous solution and acetonitrile as the mobile phase under gradient elution within 70 min. The hierarchical clustering analysis (HCA) was successfully used to classify the samples in accordance with their chemical constituents. RESULTS: Linearity (R(2) > 0.9990), intra- and inter-day precision (relative standard deviations <4%), limit of detection (0.011-0.086 µg/mL), limit of quantification (0.033-0.259 µg/mL) of the six alkaloids were determined, and the recoveries were between 96.6% and 103.7%. The method was successfully applied to analysis 36 batches of commercial stemonae radix. All the samples could be classified into five clusters by HCA. CONCLUSION: This article provides an accurate and simple analytical method for quality survey of commercial stemonae radix. Because of the significant chemical variations, careful selection of Stemona sources with obvious antitussive value but devoid of croomine followed by good agricultural practice and good manufacturing practice process is suggested.

Structures and Chemotaxonomic Significance of Stemona Alkaloids from Stemona japonica.

A pair of new alkaloid stereo-isomers, stemocochinin (1) and isostemocochinin (2), was obtained from the roots of Stemona japonica Miq., along with seven known alkaloids, stemonamine (3), isostemonamine (4), maistemonine (5), isomaistemonine (6), croomine (7), stemonine (8), and protostemonine (9). The complete structure and stereochemistry of the pair of isomers were established by extensive analysis of the spectral data. Furthermore, our results indicated that S. japonica is chemically closer to S. sessilifolia than S. tuberosa, which are consistent with our previous DNA study on Stemona species.

Molecular identification and cytotoxicity study of herbal medicinal materials that are confused by Aristolochia herbs.

Herbal materials derived from Aristolochia species contain carcinogenic aristolochic acids (AAs) and have been used as traditional Chinese medicines (TCMs) or adulterants of other TCMs. The purpose of this study is to identify the TCMs Stephaniae Tetrandrae Radix, Akebiae Caulis, Aucklandia Radix and Aristolochiae Fructus by sequencing the matK, rbcL, trnH-psbA and trnL-trnF DNA regions. The cytotoxicities of AAs and these TCMs were also studied in COS-7 and HEK-293 cells. Diagnostic polymorphic sites were identified in all the four DNA loci for the differentiation of genuine herbs from their adulterants/substitute. The 48 h IC50 of AAI were 78 µM (COS-7) and 70 µM (HEK-293) while the IC50 of AAII were higher than 100 µM in both cell lines. Except Aucklandia Radix, cytotoxicity study also showed that AA-containing herbs were more toxic than their corresponding genuine herbs and substitute.

Edible bird's nests--how do the red ones get red?

ETHNOPHARMACOLOGICAL RELEVANCE: Red edible bird's nests are regarded as of higher beneficial value for health and hence fetch a higher price than the white ones. Their red colour remains a myth. AIM OF THE STUDY: To determine if white edible bird's nests can turn red by vapours generated from sodium nitrite in acidic conditions and by vapours from 'bird soil'. MATERIALS AND METHODS: White edible bird's nests were exposed to vapours from sodium nitrite dissolved in 2% HCl or from 'bird soil' in hot and humid conditions. CONCLUSIONS: Vapours from sodium nitrite dissolved in 2% HCl or from 'bird soil' containing guano droppings from swiftlet houses were able to turn white edible bird's nests red. The reddening agent in 'bird soil' was water-soluble and heat-stable. The red colour of edible bird's nests is likely caused by the environmental factors in cave interiors and swiftlet houses.

Antiangiogenic effects and mechanisms of trans-ethyl p-methoxycinnamate from Kaempferia galanga L.

Kaempferia galanga L. (Zingiberaceae) is an aromatic herb and a popular spice used as a condiment in Asian cuisine. The ethanol extract of the dried plant and its successive four subfractions were investigated on zebrafish model by quantitative endogenous alkaline phosphatase assay. Both n-hexane and ethyl acetate fractions had antiangiogenic activity, and two major active components (trans-ethyl p-methoxycinnamate and kaempferol) showed potent antiangiogenic effects on wild-type zebrafish. Because of its much stronger effect and no antiangiogenic activity reported, trans-ethyl p-methoxycinnamate was further investigated for its action mechanism. It dose dependently inhibited vessel formation on both wild- and Tg(fli1a:EGFP)y1-type zebrafish embryos. The semiquantitative reverse transcription polymerase chain reaction assay suggested that trans-ethyl p-methoxycinnamate affects multiple molecular targets related to angiogenesis. In vitro, it specifically inhibited the migration and tube formation of human umbilical vein endothelial cells. In vivo, it could block bFGF-induced vessel formation on Matrigel plug assay.

Identification of Baiying (Herba Solani Lyrati) commodity and its toxic substitute Xungufeng (Herba Aristolochiae Mollissimae) using DNA barcoding and chemical profiling techniques.

Baiying derived from Solanum lyratum Hance is a commonly consumed natural product for ethnomedical treatment of cancer. One of the substitutes present in the market is a carcinogenic aristolochic acids-containing herb Xungufeng derived from Aristolochia mollissima Thunb. The purpose of this study is to establish DNA barcodes, thin layer chromatography (TLC), high performance liquid chromatography (HPLC) and cytotoxicity assay to differentiate Baiying from Xungufeng. A total of 30 DNA sequences from five DNA barcodes (ITS, matK, rbcL, trnH-psbA and trnL-trnF) were generated to differentiate S. lyratum from A. mollissima and authenticate ten samples of Baiying and Xungufeng commodities. Using aristolochic acids as standard markers, TLC and HPLC analyses also successfully authenticated these commodities. In vitro cytotoxicity assay using HEK-293 and Vero cells demonstrated that Xungufeng was significantly more toxic than Baiying. This is the first study applying an integrated molecular, chemical and biological approach to differentiate traditional Chinese medicine from Aristolochia adulterant.

Molecular analyses of the Chinese herb Leigongteng (Tripterygium wilfordii Hook.f.).

Tripterygium wilfordii Hook.f., known as Leigongteng (Thunder God Vine) in traditional Chinese medicine, has attracted much attention for its applications in relieving autoimmune disorders such as rheumatoid arthritis and systemic lupus erythematosus, and for treating cancer. Molecular analyses of the ITS and 5S rDNA sequences indicate that T. hypoglaucum and T. doianum are not distinct from T. wilfordii, while T. regelii should be recognized as a separate species. The results also demonstrate potential value of rDNA sequence data in forensic detection of adulterants derived from Celastrus angulatus in commercial samples of Leigongteng.

Anti-angiogenic effect and mechanism of rhein from Rhizoma Rhei.

PURPOSE: Rhein is a major bioactive component in rhubarb (Dahuang), a famous traditional Chinese medicine derived from the rhizome of Rheum palmatum and related species. It was reported to have antitumor and anti-inflammatory properties. Our previous studies found rhein displaying potent anti-angiogenic activities in a zebrafish embryo model. Its action mechanisms need further elucidation. METHODS: The inhibition effect of vessel formation was checked by microscopic imaging on Tg(fli1a:EGFP)y1 zebrafish embryos. Then the action mechanism of rhein was investigated by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) on wild type zebrafish embryos and further tested on human umbilical vein endothelial cells. RESULTS: At 20µM, rhein could almost completely block intersegmental blood vessels formation at both 48 and 72hpf, and completely inhibit subintestinal vessel plexus formation at 72hpf. Rhein affected multiple molecular targets related to angiogenesis, particularly angpt2 and tie2, and also inhibited endothelial cell migration. CONCLUSION: Rhein could inhibit angiogenesis, which may play a role in antitumor and anti-inflammatory actions.

Anti-angiogenic effects of the fruit of Alpinia oxyphylla.

AIM OF STUDY: The fruit of Alpinia oxyphylla, an herb commonly used in East Asian medicine, is variously used for the treatment of cancer and inflammatory conditions, which may possibly be mediated through anti-angiogenesis. This study aims to check for anti-angiogenic functions in the herb. MATERIALS AND METHODS: The 95% ethanol extract and four subsequent fractions (n-hexane, ethyl acetate, n-butanol and aqueous fractions) of the fruit of A. oxyphylla were tested on zebrafish model by quantitative endogenous alkaline phosphatase assay; then the active fractions were further tested on wild type and Tg(fli1a:EGFP)y1 zebrafish embryos and human umbilical vein endothelial cells and tumor cell lines for the anti-angiogenic effects. RESULTS: The n-hexane and ethyl acetate fractions showed anti-angiogenic potentials in both in vivo and in vitro models. CONCLUSIONS: The use of A. oxyphylla for cancer and inflammation diseases may be partly due to its effects against vessel formation.

Cardiocrinum seeds as a replacement for Aristolochia fruits in treating cough.

AIM OF THE STUDY: The antitussive Chinese herb Madouling derived from Aristolochia species is banned due to aristolochic acid-induced nephropathy. A substitute is found dispensed as Madouling in Taiwan. This study aims to determine the source plant and verify the antitussive properties of the Madouling substitute used in Taiwan. MATERIALS AND METHODS: Forensically informative nucleotide sequencing (FINS) approach based on the trnL-trnF and psbA-trnH regions was applied to facilitate identification of the genuine species and substitute. The antitussive effect of both genuine Madouling and the substitute were evaluated in guinea pigs. RESULTS: FINS approach based on the trnL-trnF and psbA-trnH regions readily identified the sample of Madouling in Taiwan to the seeds of Cardiocrinum giganteum var. yunnanense. Ethanol extracts of the substitute showed significant antitussive properties in guinea pigs. CONCLUSION: Cardiocrinum seeds may have potential as a replacement of Aristolochia fruits.

Antitussive and central respiratory depressant effects of Stemona tuberosa.

AIMS OF THE STUDY: Stemona alkaloids with distinctly different chemical skeletons are recently reported as the active components in the antitussive herb Baibu derived from the root-tubers of Stemona tuberosa. This study aims to determine if alkaloids of this herb contribute equally to the antitussive functions, act on the same sites of cough reflex, and play any role in inducing central respiratory depressant effects. MATERIALS AND METHODS: Antitussive potency of four major alkaloids was evaluated on guinea pigs with citric acid aerosol to induce cough. The action sites of the alkaloids on cough reflex pathway were tested with electrical stimulation of the superior laryngeal nerve in guinea pigs. The central respiratory effects of croomine were also tested on guinea pigs. RESULTS: Croomine, neotuberostemonine and stemoninine showed similar antitussive potency, while tuberostemonine showed much weaker antitussive potency. Neotuberostemonine, tuberostemonine and stemoninine acted on the peripheral cough reflex pathway, while croomine acted on the central part. Croomine also showed obvious central respiratory depressant effects. CONCLUSIONS: The four major Stemona alkaloids in Stemona tuberosa do not contribute equally to antitussive potency in guinea pigs. Neotuberostemonine, tuberostemonine and stemoninine target on peripheral cough reflex pathway. Croomine acts on central sites in the cough reflex pathway and demonstrates central respiratory depressant effects, which can partly account for the adverse reactions reported for the herb.

Development and validation of a rapid capillary zone electrophoresis method for the determination of aconite alkaloids in aconite roots.

INTRODUCTION: Aconites, with aconite alkaloids as the major therapeutic and toxic components, are used for the treatment of analgesic, antirheumatic and neurological symptoms. Quantification of the aconite alkaloids is important for the quality control of aconite-containing drugs. OBJECTIVE: To establish a validated capillary zone electrophoresis (CZE) method for the simultaneous determination of six major alkaloids, namely aconitine, mesaconitine, hypaconitine, benzoylaconine, benzoylmesaconine and benzoylhypaconine, in crude and processed aconite roots. METHODOLOGY: The CZE method was optimised and validated using a stability-indicating method. The optimised running buffer was a mixture of 200 mm Tris, 150 mm perchloric acid and 40% 1,4-dioxane (pH 7.8) with the capillary thermostated at 25 degrees C. RESULTS: Using the optimised method, six aconite alkaloids were well separated. The established method showed good precision, accuracy and recovery. Contents of these alkaloids in crude and processed aconites were determined and it was observed that the levels of individual alkaloids varied between samples. CONCLUSION: The developed CZE method was reliable for the quality control of aconites contained in herbal medicines. The method could also be used as an approach for toxicological studies.

Triptolide functions as a potent angiogenesis inhibitor.

Triptolide is a key anti-inflammatory compound of the Chinese herbal medicine Tripterygium wilfordii Hook. f. (Celastraceae). It also possesses potent antitumor activity. In this study, we show that triptolide is an angiogenesis inhibitor based on various angiogenesis assays. The IC(50) in in vitro assays was 45 nM, which was much lower than the plasma concentrations of triptolide in the rat or human administered with T. wilfordii extracts for treating inflammation. When dosed in vivo, triptolide potently inhibited angiogenesis at 100 nM in Matrigel plug assay. Triptolide at 0.75 mg/kg/day significantly blocked tumor angiogenesis and tumor progression in murine tumorigenesis assay. The underlying mechanism of triptolide correlated with downregulation of proangiogenic Tie2 and VEGFR-2 expression in human umbilical vein endothelial cell by semiquantitative RT-PCR and western blot analysis. Although Tie2 inhibition appeared to be a later event as compared with VEGFR-2, Tie2 overexpression significantly attenuated the inhibitory effect of triptolide on endothelial proliferation and network formation. By contrast, Tie2 knockdown mimicked the inhibitory effect of triptolide on endothelial network formation. Our findings suggest that antitumor action of triptolide is partly via inhibition of tumor angiogenesis by blocking 2 endothelial receptor-mediated signaling pathways, and triptolide can be a promising antiangiogenic agent.

Patent applications for using DNA technologies to authenticate medicinal herbal material.

Herbal medicines are used in many countries for maintaining health and treating diseases. Their efficacy depends on the use of the correct materials, and life-threatening poisoning may occur if toxic adulterants or substitutes are administered instead. Identification of a medicinal material at the DNA level provides an objective and powerful tool for quality control. Extraction of high-quality DNA is the first crucial step in DNA authentication, followed by a battery of DNA techniques including whole genome fingerprinting, DNA sequencing and DNA microarray to establish the identity of the material. New or improved technologies have been developed and valuable data have been collected and compiled for DNA authentication. Some of these technologies and data are patentable. This article provides an overview of some recent patents that cover the extraction of DNA from medicinal materials, the amplification of DNA using improved reaction conditions, the generation of DNA sequences and fingerprints, and the development of high-throughput authentication methods. It also briefly explains why these patents have been granted.

Antiviral constituents against respiratory viruses from Mikania micrantha.

Phytochemical investigation of the dried aerial parts of Mikania micrantha led to the isolation of a new sesquiterpene, 3beta-acetoxy-1,10-epoxy-4-germacrene-12,8;15,6-diolide (1), along with six known constituents: 1,10-epoxy-4-germacrene-12,8;15,6-diolide (2), dihydromikanolide (3), potassium mikanin 3-sulfate (4), mikanin (5), alpinetin (6), and ergosta-7,22-dien-3beta-ol (7). Their structures were elucidated by spectroscopic methods, and the molecular structures and stereochemistry of sesquiterpene lactones 1-3 were revealed by single-crystal X-ray analysis. Compound 2 showed moderate activity against respiratory syncytial virus (IC(50) = 37.4 uM) and parainfluenza type 3 virus (IC(50) = 37.4 uM) with a therapeutic index (TI) of 16.0 for both compounds. Compound 4, the main component of M. micrantha, exhibited inhibitory activity against parainfluenza type 3 virus with IC(50) (19.7 uM) and TI (24.0) values comparable to those of ribavirin, serving as a positive control.

Antiangiogenic activity of Tripterygium wilfordii and its terpenoids.

ETHNOPHARMACOLOGICAL RELEVANCE: Tripterygium wilfordii Hook. f. (Celastraceae) has been traditionally used as folk medicine for centuries in China for the treatment of immune-inflammatory diseases. AIM OF THE STUDY: This study aimed to assess the antiangiogenic activities which support the therapeutic use of Tripterygium wilfordii and its terpenoids for angiogenesis disease such as cancer. MATERIALS AND METHODS: The ethanol extract of Tripterygium wilfordii and subsequent fractions were evaluated on an in vivo antiangiogenic zebrafish embryo model. RESULTS: Three antiangiogenic terpenoids were isolated by bioassay-guided purification, namely, celastrol (4), cangoronine (5) and triptolide (7). Among them, triptolide manifested the most potent antiangiogenic activity against vessel formation by nearly 50% at 1.2 microM. Semi-quantitative RT-PCR analysis revealed that triptolide dose- and time-dependently reduced the mRNA expression of angiopoietin (angpt)2 and tie2 in zebrafish, indicating the involvement of angpt2/tie2 signaling pathway in the antiangiogenic action of triptolide. CONCLUSIONS: The discovery of an alternative pathway further confirms the value of ethnopharmacological investigations into traditional botanicals for leads for potential drug development.

Anti-angiogenic effects of rhubarb and its anthraquinone derivatives.

ETHNOPHARMACOLOGICAL RELEVANCE: Rhubarb root (Dahuang) is often included as an ingredient in traditional Chinese compound prescriptions for the treatment of inflammatory diseases. This application may possibly be mediated through anti-angiogensis and thus would shed light on its potential value in cancer therapy. AIM OF THE STUDY: To elucidate the anti-angiogenic properties of rhubarb root, we tested the inhibitory effects of different fractions and a series of anthraquinone derivatives against vessel formation in zebrafish embryos. MATERIALS AND METHODS: The 95% ethanol extract and four subsequent fractions (n-hexane, ethyl acetate, n-butanol and aqueous fractions) of rhubarb root and five anthraquinone derivatives were investigated on zebrafish model by quantitative endogenous alkaline phosphatase assay and staining assay. RESULTS: Ethyl acetate fraction showed the strongest inhibition of vessel formation by 52%. Three anthraquinones (aloe-emodin, emodin and rhein) displayed potent anti-angiogenic activities. CONCLUSIONS: The angiogenic properties of rhubarb root may partly account for its use in inflammatory diseases. The anthraquinones with acidic or polar, hydrophilic substitution at C-6 or C-3 positions played a substantial role in inhibiting angiogenesis. The value of the zebrafish angiogenic model is further supported.

Authentication of Saussurea lappa, an endangered medicinal material, by ITS DNA and 5S rRNA sequencing.

Wild SAUSSUREA LAPPA in the family Asteraceae is a highly endangered plant. On the other hand, the dried root of cultivated S. LAPPA (Radix Aucklandia, Muxiang) is a popular medicinal material for treating various gastrointestinal diseases. In the market, several medicinal plants including VLADIMIRIA BERARDIOIDEA, V. SOULIEI, V. SOULIEI var. MIRABILIS, INULA HELENIUM and I. RACEMOSA in the family Asteraceae and ARISTOLOCHIA DEBILIS in the family Aristolochiaceae have the trade name of Muxiang. To manage the concerned medicinal material, we investigated if the ITS and 5S rRNA intergenic spacers are effective for discriminating S. LAPPA from its substitutes and adulterants. Sequencing results showed that the similarities of ITS-1, ITS-2 and 5S rRNA intergenic spacers among S. LAPPA and related species were 56.3 - 97.8 %, 58.5 - 97.0 %, and 26.4 - 77.9 %, respectively. The intraspecific variation was much lower. There are also several unique changes in the S. LAPPA sequences that may be used as differentiation markers.

Authentication of Radix Aucklandiae and its substitutes by GC-MS and hierarchical clustering analysis.

Radix Aucklandiae (Muxiang in Chinese), the dried root of Aucklandia lappa, is used as a medicinal material for digestive system disorders in traditional Chinese medicine for centuries. Owing to the similarity of morphologies and trade names, Radix Vladimiriae (Chuan-Muxiang), the roots of Vladimiria souliei and V. souliei var. cinerea, and Radix Inulae (Tu-Muxiang), the roots of Inula helenium and Inula racemosa, as well as the renal toxic aristolochic acid containing Radix Aristolochiae (Qing-Muxiang), the roots of Aristolochia debilis and Aristolochia contorta, are often used confusedly as the substitutes of Radix Aucklandiae. In order to ensure the effective and safe utility of Radix Aucklandiae, a GC-MS method was developed to generate the chemical profiles of essential oils of Radix Aucklandiae and its substitutes. In addition, hierarchical clustering analysis was used to compare the similarities of these chemical profiles. It was found that all the samples of A. lappa have similar chemical profiles and were clustered into one group, while the samples of Radix Vladimiriae, Radix Inulae, and Radix Aristolochiae were clustered into their own independent groups, respectively, suggesting that together with hierarchical clustering analysis, chemical profiles of essential oils generated by GC-MS could objectively discriminate Radix Aucklandiae from its common substitutes.