Clara Bik-San Lau

A review of antimycobacterial natural products

Tuberculosis is a chronic infectious disease caused by several species of mycobacteria. Due to multi‐drug resistant strains of mycobacteria and to a high prevalence of tuberculosis in patients who have acquired human immunodeficiency syndrome (AIDS), the number of patients infected with the disease is increasing worldwide. Thus there is an urgent need for new effective antimycobacterial agents to replace those currently in use. In this instance, the plant kingdom is undoubtedly a valuable source for new anti‐tuberculosis agents. The present review article reports the findings from an extensive literature search of all plants that have been assessed for antimycobacterial/antitubercular activity over the past 20–30 years. An attempt has been made to summarize the information in order to highlight those promising plant species which are worthy of further investigation as leads for drug development. Over 350 plant species from a wide range of families and origins, containing various chemical classes of compounds, have been screened for such activity. A review of the relevant in vitro assays using different species of pathogenic and non‐pathogenic mycobacteria is also included. Copyright

Immunomodulatory and anti-SARS activities of Houttuynia cordata.

Abstract Background Severe acute respiratory syndrome (SARS) is a life-threatening form of pneumonia caused by SARS coronavirus (SARS-CoV). From late 2002 to mid 2003, it infected more than 8000 people worldwide, of which a majority of cases were found in China. Owing to the absence of definitive therapeutic Western medicines, Houttuynia cordata Thunb. (Saururaceae) (HC) was shortlisted by Chinese scientists to tackle SARS problem as it is conventionally used to treat pneumonia. Aim of the study The present study aimed to explore the SARS-preventing mechanisms of HC in the immunological and anti-viral aspects. Results Results showed that HC water extract could stimulate the proliferation of mouse splenic lymphocytes significantly and dose-dependently. By flow cytometry, it was revealed that HC increased the proportion of CD4+ and CD8+ T cells. Moreover, it caused a significant increase in the secretion of IL-2 and IL-10 by mouse splenic lymphocytes. In the anti-viral aspect, HC exhibited significant inhibitory effects on SARS-CoV 3C-like protease (3CLpro) and RNA-dependent RNA polymerase (RdRp). On the other hand, oral acute toxicity test demonstrated that HC was non-toxic to laboratory animals following oral administration at 16g/kg. Conclusion The results of this study provided scientific data to support the efficient and safe use of HC to combat SARS.

Synergistic interaction between Astragali Radix and Rehmanniae Radix in a Chinese herbal formula to promote diabetic wound healing

ETHNOPHARMACOLOGICAL RELEVANCE Astragali Radix (AR) and Rehmanniae Radix (RR) are two traditional Chinese medicines widely used in China for treating diabetes mellitus and its complications, such as diabetic foot ulcer. AIM OF STUDY In our previous study, a herbal formula NF3 comprising AR and RR in the ratio of 2:1 was found effective in enhancing diabetic wound healing in rats through the actions of tissue regeneration, angiogenesis promotion and inflammation inhibition. The aims of the present study were to investigate the herb-herb interaction (or the possible synergistic effect) between AR and RR in NF3 to promote diabetic wound healing and to identify the principal herb in the formula by evaluating the potencies of individual AR and RR in different mechanistic studies. MATERIALS AND METHODS A chemically induced diabetic foot ulcer rat model was used to examine the wound healing effect of NF3 and its individual herbs AR and RR. For mechanistic studies, murine macrophage cell (RAW 264.7) inflammation, human fibroblast (Hs27) proliferation and human endothelial cell (HMEC-1) migration assays were adopted to investigate the anti-inflammatory, granulation formation and angiogenesis-promoting activities of the herbal extracts, respectively. RESULTS In the foot ulcer animal model, neither AR nor RR at clinical relevant dose (0.98g/kg) promoted diabetic wound healing. However, when they were used in combination as NF3, synergistic interaction was demonstrated, of which NF3 could significantly reduce the wound area of rats when compared to water group (p<0.01). For anti-inflammation and granulation formation, AR was more effective than RR in inhibiting lipopolysaccharide (LPS)-induced nitric oxide production from RAW 264.7 cells and promoting Hs27 fibroblast proliferation. In the aspect of angiogenesis promotion, only NF3 promoted cell migration of HMEC-1 cells. CONCLUSIONS AR plays a preeminent role in the anti-inflammatory and fibroblast-proliferating activities of NF3. The inclusion of RR, however, is crucial for NF3 to exert its overall wound-healing as well as the underlying angiogenesis-promoting effects. The results of present study justified the combined usage of AR and RR in the ratio of 2:1 as NF3 to treat diabetic foot ulcer and illustrated that AR is the principal herb in this herbal formula.

The in vivo and in vitro diabetic wound healing effects of a 2-herb formula and its mechanisms of action

ETHNOPHARMACOLOGICAL RELEVANCE The herbs Radix Astragali (RA) and Radix Rehmanniae (RR) have long been used in traditional Chinese Medicine and serve as the principal herbs in treating diabetic foot ulcer. AIM OF STUDY Diabetic complications, such as foot ulcer, impose major public health burdens worldwide. In our previous clinical studies, two Chinese medicine formulae F1 and F2 have achieved over 80% limb salvage. A simplified 2-herb formula (NF3) comprising of RA and RR in the ratio of 2:1 was used for further study. NF3 was examined for the ulcer healing effect in diabetic rats, and its potential mechanisms of action in fibroblast proliferation, angiogenesis and anti-inflammation in vitro. MATERIALS AND METHODS A chemically induced diabetic foot ulcer rat model was used for studying the wound healing effect. In the in vitro mechanistic studies, human fibroblast cells (Hs27), human umbilical vein endothelial cells (HUVEC) and mouse macrophage cells (RAW264.7) were assessed for tissue regeneration, angiogenesis and anti-inflammatory activities, respectively. RESULTS Our in vivo results demonstrated a significant reduction of wound area at day 8 in NF3 (0.98g/kg) group as compared to control (p<0.01). NF3 could significantly stimulate Hs27 proliferation in a dose dependent manner (p<0.05). Besides, NF3 could significantly increase the cell migration and tube formation (p<0.05-0.001) of HUVEC in the angiogenesis study. Furthermore, significant inhibition of nitric oxide production (p<0.01) was found in NF3-treated macrophage cells, suggesting its anti-inflammatory activity. CONCLUSIONS Our study presents for the first time scientific evidence towards the efficacy of the two-herb formula NF3 in enhancing diabetic wound healing through the actions of tissue regeneration, angiogenesis and anti-inflammation.

SYNERGISTIC EFFECTS OF BAICALEIN WITH CIPROFLOXACIN AGAINST NORA OVER-EXPRESSED METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS (MRSA) AND INHIBITION OF MRSA PYRUVATE KINASE

ETHNOPHARMACOLOGICAL RELEVANCE Baicalein, the active constituent derived from Scutellaria baicalensis Georgi., has previously been shown to significantly restore the effectiveness of β-lactam antibiotics and tetracycline against methicillin-resistant Staphylococcus aureus (MRSA). With multiple therapeutic benefits, the antibacterial actions of baicalein may also be involved in overcoming other bacterial resistance mechanisms. The aim of the present study was to further investigate antibacterial activities of baicalein in association with various antibiotics against selected Staphylococcus aureus strains with known specific drug resistance mechanisms. MATERIAL AND METHODS A panel of clinical MRSA strains was used for further confirmation of the antibacterial activities of baicalein. The effect of baicalein on inhibiting the enzymatic activity of a newly discovered MRSA-specific pyruvate kinase (PK), which is essential for Staphylococcus aureus growth and survival was also examined. RESULTS In the checkerboard dilution test and time-kill assay, baicalein at 16 μg/ml could synergistically restore the antibacterial actions of ciprofloxacin against the NorA efflux pump overexpressed SA-1199B, but not with the poor NorA substrate, pefloxacin. Moreover, synergistic effects were observed when baicalein was combined with ciprofloxacin against 12 out of 20 clinical ciprofloxacin resistant strains. For MRSA PK studies, baicalein alone could inhibit the enzymatic activity of MRSA PK in a dose-dependent manner. CONCLUSION Our results demonstrated that baicalein could significantly reverse the ciprofloxacin resistance of MRSA possibly by inhibiting the NorA efflux pump in vitro. The inhibition of MRSA PK by baicalein could lead to a deficiency of ATP which might further contribute to the antibacterial actions of baicalein against MRSA.

Evaluation of in vitro anti-proliferative and immunomodulatory activities of compounds isolated from Curcuma longa

The rhizome of Curcuma longa (CL) has been commonly used in Asia as a potential candidate for the treatment of different diseases, including inflammatory disorders and cancers. The present study evaluated the anti-proliferative activities of the isolated compounds (three curcuminoids and two turmerones) from CL, using human cancer cell lines HepG2, MCF-7 and MDA-MB-231. The immunomodulatory activities of turmerones (alpha and aromatic) isolated from CL were also examined using human peripheral blood mononuclear cells (PBMC). Our results showed that the curcuminoids (curcumin, demethoxycurcumin and bisdemethoxycurcumin) and alpha-turmerone significantly inhibited proliferation of cancer cells in dose-dependent manner. The IC(50) values of these compounds in cancer cells ranged from 11.0 to 41.8 microg/ml. alpha-Turmerone induced MDA-MB-231 cells to undergo apoptosis, which was confirmed by annexin-V and propidium iodide staining, and DNA fragmentation assay. The caspase cascade was activated as shown by a significant decrease of procaspases-3, -8 and -9 in alpha-turmerone treated cells. Both alpha-turmerone and aromatic-turmerone showed stimulatory effects on PBMC proliferation and cytokine production. The anti-proliferative effect of alpha-turmerone and immunomodulatory activities of ar-turmerone was shown for the first time. The findings revealed the potential use of CL crude extract (containing curcuminoids and volatile oil including turmerones) as chemopreventive agent.

Simultaneous quantification of active components in the herbs and products of Si-Wu-Tang by high performance liquid chromatography―mass spectrometry

Si-Wu-Tang (SWT), comprising Paeoniae, Angelicae, Chuanxiong and Rehmanniae, is one of the most popular Traditional Chinese Medicine (TCM) formulae for womans health. Data mining from the available Chinese and English literatures indicated that the major bioactive components of SWT consist of paeoniflorin, paeonol, gallic acid, ferulic acid, Z-ligustilide, ligustrazine, butylphthalide, senkyunolide A and catalpol. Since content determination of the marker compounds is generally considered as an initial step for quality control of TCM product, a high performance liquid chromatography-mass spectrometric method employing both positive and negative electrospray ionization was developed for the simultaneous determination of the nine identified compounds in the raw herbs and products of SWT. The LOQ of the developed assay method for the tested components was 10ng/ml for ligustrazine, 200ng/ml for catalpol, and 100ng/ml for the other seven compounds. The intra-day and inter-day variations of the current assay were within 17.5%. Paeoniflorin, ferulic acid, gallic acid, Z-ligustilide and senkyunolide A were found in all SWT products investigated. Variations in the contents of the studied compounds were observed among batches of raw herbs and SWT products. The currently developed method provides a sensitive and rapid quantification approach that can be useful in the quality control of raw herbs and products of SWT.

Immunostimulatory activities of polysaccharide extract isolated from Curcuma longa

Several curcuminoids and sesquiterpenoids isolated from Curcuma longa (CL) have been shown to have many pharmacological activities. In the present study, the immunomodulatory activities of the polar fractions of CL hot water extracts were investigated using human peripheral blood mononuclear cells (PBMC). Our results showed that the high polarity fraction of the hot water extract exhibited stimulatory effects on PBMC proliferation as shown in [methyl-(3)H]-thymidine incorporation assay. In an attempt to isolate the active components responsible for the activities, further partition with ethyl acetate, n-butanol and ethanol, progressively were performed. The cytokine productions (TGF-beta, TNF-alpha, GM-CSF, IL-1alpha, IL-5, IL-6, IL-8, IL-10, IL-13, etc.) have been modulated by a polysaccharide-enriched fraction as shown in ELISA and cytokine protein array. The proportion of CD14 positive stained PBMC was increased by such fraction. The composition of monosaccharide of the active fraction has been determined by GC-MS and gel permeation chromatography. The immunostimulatory effects of C. longa polysaccharides on PBMC were shown for the first time. The findings revealed the potential use of C. longa crude extract (containing curcuminoids and polysaccharides) as an adjuvant supplement for cancer patients, whose immune activities were suppressed during chemotherapies.

Isolation, Structure Characterization, and Immunomodulating Activity of a Hyperbranched Polysaccharide from the Fruiting Bodies of Ganoderma sinense

A polysaccharide (GSP-6B) with a molecular mass of 1.86 × 10⁶ Da was isolated from the fruiting bodies of Ganoderma sinense . Chemical composition analysis, methylation analysis, infrared spectroscopy, and nuclear magnetic resonance spectroscopy were conducted to elucidate its structure. GSP-6B contains a backbone of (1→6)-linked-β-D-glucopyranosyl residues, bearing branches at the O-3 position of every two sugar residues along the backbone. The side chains contain (1→4)-linked-β-D-glucopyranosyl residues, (1→3)-linked-β-D-glucopyranosyl residues, and nonreducing end β-D-glucopyranosyl residues. An in vitro immunomodulating activity assay revealed that GSP-6B could significantly induce the release of IL-1β and TNF-α in human peripheral blood mononuclear cell (PBMC) and showed no toxicity to either PBMC or a human macrophage cell line THP-1. GSP-6B could also activate dendritic cells (DC) by stimulating the secretion of IL-12 and IL-10 from DC.

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.