The most commonly used spices in Thai traditional medicine: in vitro evaluation of anti-hyperglycemic, antioxidant, polyphenol content, and nitric oxide production inhibitory activities.

Background and purpose: Diabetes mellitus is a persistent hyperglycemic condition. Thai cuisine and medicine incorporate spices: nutmeg, mace, clove buds, cardamom, cinnamon, and coriander. The in vitro impacts of these spices on anti-diabetic, antioxidant, anti-inflammatory, and total phenolic and flavonoid content were assessed. Experimental approach: Alpha-amylase and alpha-glucosidase inhibition assays were conducted. Antioxidant potential was measured through DPPH and ABTS assays. Anti-inflammatory activity was determined by inhibiting nitric oxide generation in RAW 264.7 cells. Total phenolic content was quantified using the Folin Ciocalteu method, while total flavonoid content was estimated via the aluminum chloride colorimetric method. Findings/Results: Ethanolic and aqueous extracts of a blend of spices (Siam cardamom, nutmeg, mace, and clove buds), denoted as 4-GlurE and 4-GlurA, displayed concentration-dependent inhibition of alpha-glucosidase, with IC50 values of 0.373 and 0.435 mg/mL, respectively. 4-GlurE and 4-GlurA exhibited antioxidant activity, by ABTS·+ radical and DPPH scavenging capabilities. 4-GlurE demonstrated anti-inflammatory potential by reducing nitric oxide generation (IC50: 43.95 ± 2.47 µg/mL). 4-GlurE and 4-GlurA possessed total phenolic content (TPC) of 122.47 ± 1.12 and 148.72 ± 0.14 mg GAE/g, respectively. 4-GlurE exhibited a higher total flavonoid content (TFC) compared to the aqueous extract (340.33 ± 4.77 and 94.17 ± 3.36 mg QE/g). Cinnamon and clove aqueous extracts were more potent than acarbose in alpha-glucosidase inhibition with the highest antioxidant activity. Polyphenol levels (TPC and TFC) exhibited strong correlations with antioxidant capacity. Conclusions and implications: Findings are consistent with the traditional use of 4-Glur, with cinnamon, for diabetes prevention and treatment.

Anti-proliferation and induction of mitochondria-mediated apoptosis by Garcinia hanburyi resin in colorectal cancer cells.

Introduction: Several parts of Garcinia hanburyi are used in traditional medicine for many purposes. In this study, Garcinia hanburyi resin (GHR) was explored for possible anti-proliferative effects and the underlying mechanism on colorectal cancer (CRC) cells. Methods: Gambogic acid (GA) content in GHR was analyzed by HPLC method. The cytotoxicities of GA and GHR were assessed in human CRC cell lines (SW480 and Caco-2) and normal colon cells (CCD841 CoN) using a trypan blue exclusion assay, MTS assay, and cell morphology analysis. Cell cycle and apoptosis at its half maximal inhibitory concentration (IC50) were analyzed using flow cytometry. And, the levels of intrinsic apoptosis-related proteins were measured by Western blot analysis. Results: GA was the major compound as 71.26% of the GHR. The cell viability of CRC cells was decreased in a time- and dose-dependent manner after exposure to GHR. The selectivity index indicated that GHR had a high degree of selectivity against CRC cells. The same result was obtained for GA treatment. In addition, GHR markedly induced typical apoptotic morphology of CRC cells, but had no obvious effect on normal colon cells. GHR induced apoptosis with the cell cycle arrest at the G2/M phase. An increase in Bax/Bcl-2 ratio and a decrease in procaspase-3 proteins indicated that GHR promoted apoptosis by disrupting the mitochondrial outer membrane permeability and the subsequent activation of caspase-3. Conclusion: GHR, which contained GA as an active compound, significantly inhibited CRC cell proliferation via the induction of intrinsic apoptosis, while having low toxicity on normal colon cells. Therefore, GHR could be proposed as a potent candidate for the treatment of CRC.

Molecular Changes Following Induction of Hepatocellular Carcinoma by Diethylnitrosamine and Thioacetamide, and Subsequent Treatment with Dioscorea membranacea Extract.

Hepatocellular carcinoma (HCC) is a primary liver cancer commonly found in adults. Previously, we showed the anticancer effects of Thai herbal plant extract, Dioscorea membranacea Pierre (DM), in HCC-bearing rats. In the present study, we further examined the proposed mechanism of DM, including apoptosis and antioxidant activity. Moreover, we used RNA sequencing (RNA-seq) to analyze molecular pathways in the rat model in which HCC was induced by diethylnitrosamine (DEN) and thioacetamide (TAA). The HCC-bearing rats were then treated with 40 mg/kg of DM for 8 weeks, after which experimental and control rats were sacrificed and liver tissues were collected. The RNA-seq data of DEN/TAA-treated rats exhibited upregulation of 16 hallmark pathways, including epithelial mesenchymal transition, inflammatory responses, and angiogenesis (p<0.01). DM extract expanded the Bax protein-positive pericentral zone in the tumor areas and decreased hepatic malondialdehyde levels, implying a decrease in lipid peroxidation in liver. However, DM treatment did not ameliorate the molecular pathways induced in DEN/TAA-treated livers. Our findings indicate that DM extract has antioxidant activity and exerts its pro-apoptotic effect on rat HCCs in vivo at the (post-)translational level.

Exploring in vitro anti-proliferative and anti-inflammatory activities of Prasachandaeng remedy, and its bioactive compounds.

BACKGROUND: Prasachandaeng (PSD) remedy has been empirically used in Thai traditional medicine to treat fever in bile duct and liver and cancer patients through Thai folk doctors. However, there have been no scientific reports on the bioactive compounds and bioactivities related to inflammation-associated carcinogenesis or cytotoxicity against cancer cell lines. In this study, we investigated the chemical content of the remedy, and evaluated its cytotoxic activity against two cancer cell lines in comparison with a non-cancerous cell line and determined tumor necrosis factor-alpha (TNF-α) production in a murine macrophage cell line (RAW 264.7) to evaluate anti-inflammatory activity. A novel HPLC method was used for quality control of its chemical content. METHODS: Pure compounds from the EtOH extract of D. cochinchinensis were isolated using bioassay-guided fractionation and chemical content of the PSD remedy was determined using HPLC. The cytotoxic activity against the hepatocarcinoma cell line (HepG2) and cholangiocarcinoma cell line (KKU-M156), in comparison with non-cancerous cell line (HaCaT), were investigated using antiproliferative assay (SRB). The anti-inflammatory activity measured by TNF-α production in RAW 264.7 was determined using ELISA. RESULTS: All crude extracts and isolated compounds exhibited significant differences from vincristine sulfate (****p < 0.0001) in their cytotoxic activity against HepG2, KKU-M156, and HaCaT. The PSD remedy exhibited cytotoxic activity against HepG2 and KKU-M156 with IC50 values of 10.45 ± 1.98 (SI = 5.3) and 4.53 ± 0.74 (SI = 12.2) µg/mL, respectively. Some constituents from C. sappan, D. cochinchinensis, M. siamensis, and M. fragrans also exhibited cytotoxic activity against HepG2 and KKU-M156, with IC50 values less than 10 µg/mL. The isolated compounds, i.e., Loureirin B (1), 4-Hydroxy-2,4'-dimethoxydihydrochalcone (2), and Eucomol (3) exhibited moderate cytotoxicity against two cancer cell lines. None of the crude extracts and isolated compounds showed cytotoxicity against HaCaT. D. cochinchinensis and PSD remedy exhibited higher anti-inflammatory activity measured as TNF-α production than acetaminophen. CONCLUSION: The findings provide evidence of bioactivity for EtOH extracts of PSD remedy and the isolated compounds of D. Cochinchinensis. The results consistent the use clinical activity and use of PSD remedy as a antipyretic treatment for liver and bile duct cancer patients by Thai traditional practitioners.

Ethnopharmacological nexus between the traditional Thai medicine theory and biologically based cancer treatment.

ETHNOPHARMACOLOGICAL RELEVANCE: The two major theories utilized for diagnosis and treatment in Traditional Thai Medicine (TTM) are the Four Element Theory and the Herbal Flavor Theory. A TTM "Poh-Pu" Remedy has been effectively utilized in Thailand for cancer therapy for centuries. AIMS OF STUDY: To investigate anti-inflammatory activity and liver cancer cytotoxicity of Poh-Pu remedy. To determine relationships between the TTM Herbal Flavor theory and the Four Element theory and total flavonoid content and biological activities of Poh-Pu Remedy plant extracts. MATERIALS AND METHODS: Each plant ingredient was macerated with 95% ethanol. The extracts were investigated for cytotoxic activity against liver cancer using a sulforhodamine B assay, and anti-inflammatory activity was evaluated by inhibition of nitric oxide production. The total flavonoid content was determined by an aluminum chloride colorimetric assay. The relationships between the TTM theories, total flavonoid content, and biological activities were evaluated by correlation and cluster analysis. RESULTS: Mammea siamensis exerted potent cytotoxicity against hepatocellular carcinoma (HepG2) cell lines with an IC50 of 3.15 ± 0.16 µg/mL and low cytotoxicity to the non-cancerous cells (HaCat) with an IC50 33.39 ± 0.40 µg/mL (Selective index (SI) = 10.6). Tiliacora triandra was selectively cytotoxic to cholangiocarcinama (KKU-M156) cells with an IC50 of 12.65 ± 0.92 µg/mL (SI = 6.4). Curcuma comosa was the most potent anti-inflammatory inhibitor of nitric oxide production with an IC50 of 2.75 ± 0.34 µg/mL. Campomanesia aromatica exhibited the highest total flavonoid content of 259.7 ± 3.21 mg quercetin equivalent/g. Pungent plants were most prevalent in the TTM remedy. CONCLUSION: Pungent, fragrant, bitter and nauseating plants utilized in TTM cancer remedy were successfully investigated and identified several lead plants and components with cytotoxic and antiinflammatory activity that require further study. The TTM wind element theory appeared to be aligned with cancer-related activity. Biological activity results of taste from herbs related with The TTM Herbal Flavor theory. The extra-oral locations of flavor receptors are a promising target for biological activity of TTM which require further scrutiny and identified several lead plants and components with cytotoxic and antiinflammatory activities that also require further study.

Therapeutic efficacy of a Dioscorea membranacea extract in a rat model of hepatocellular carcinoma: Histopathological aspects.

Hepatocellular carcinoma (HCC) is most common in adults and has a high mortality rate because of a lack of effective treatment options. We investigated the effect of a medicinal plant as a potential source of drugs against HCC. The rhizomes of Dioscorea membranacea Pierre (DM), Hua-Khao-Yen in Thai, are commonly used as ingredients for alternative treatment of cancer in Thailand. In this study, the anticancer effects of DM extract in HCC-bearing rats were evaluated with respect to gross morphology, histopathology, and leakage of liver enzymes. In untreated HCCs, typical features of liver cancer, including hepatic nodules, thick-cell cords, and pseudoglandular cell arrangements, were observed. In addition, the HCCs showed abnormal reticulin patterns and a high glypican3 expression. In HCC-bearing rats treated with DM the cancer areas and reticulin expression were significantly reduced compared to the untreated group (p < 0.01). Sorafenib, the standard drug to treat HCC, reduced the cancer area further, but increased leakage of liver enzymes and decreased serum albumin concentration, indicating liver toxicity. These findings suggest that DM has an anticancer effect on HCCs in an animal model in vivo with potentially less severe side effects than sorafenib. Therefore, further studies of DM's mechanism of action in HCC should be carried out.

Validation of an HPLC method for quantification of anti-inflammatory markers in an ethanolic extract of Sahastara and its anti-inflammatory activity in vitro.

BACKGROUND AND PURPOSE: Sahastara (SHT) is a traditional Thai medicine for the treatment of musculoskeletal and joint pain. It consists of 21 plant components. A previous study demonstrated the anti-inflammatory activity of SHT on inhibition of nitric oxide production and prostaglandin E2 (PGE2) production, however, inhibitory effects on tumor necrosis factor-alpha (TNF-α) has not been reported. In this study, we evaluated the anti-inflammatory activity of SHT on inhibitory effects on TNF-α and PGE2 production and presented an analytical method for validation of SHT. EXPERIMENTAL APPROACH: Anti-inflammatory activity was evaluated by inhibitory activity on TNF-α and PGE2 production in RAW264.7 cells. The validated procedure was conducted according to ICH guidelines. The validated parameters were specificity/selectivity, linearity, range, the limit of detection (LOD), and limit of quantitation (LOQ). FINDINGS/RESULTS: Ethanolic extract of SHT exerted inhibitory activity on PGE2 production in RAW264.7 cells with IC50 16.97 ± 1.16 µg/mL. Myristica frangrans seed extract showed the highest inhibitory activity on PGE2 production. Piper retrofractum extract showed the highest inhibitory activity on TNF-α production. For the HPLC method, all validated parameters complied with standard requirements. Each analyzed peak showed good selectivity with a baseline resolution greater than 1.51. The linearity of all compounds was > 0.999. The % recovery of all compounds was within 98.0-102.0%. The precision of all compounds was less than 2.0% CV. CONCLUSION AND IMPLICATIONS: Ethanolic extracts of SHT possess anti-inflammatory activity by inhibition of TNF-α and PGE2 production in vitro. This study provides support for the traditional use of SHT. The validated results showed good specificity/selectivity, linearity, precision, and accuracy with appropriate LOD and LOQ. This study is the first report on the validation of the HPLC method of SHT for use as quality control of the SHT extract.

The effects of Benjakul extract and its isolated compounds on cell cycle arrest and apoptosis in human non-small cell lung cancer cell line NCI-H226.

BACKGROUND AND PURPOSE: Benjakul, a traditional Thai formulation for cancer treatment, is composed of five plants. This study aimed to assess the cytotoxicity of Benjakul, its five plants, and its isolated compounds against non-small cell lung cancer (NSCLC) by the sulforhodamine B (SRB) assay. EXPERIMENTAL APPROACH: Analyses of cell cycle and membrane asymmetry changes were performed with different fluorescent dyes and analyzed by flow cytometry in NCI-H226 cells. Activation of caspase-3 was measured using a caspase-3 colorimetric assay kit. The pan-caspase inhibitor Z-VAD-FMK was used in analyses of cell cycle and caspase-3 activity. FINDINGS/RESULTS: Benjakul exhibited cytotoxicity against NSCLC with IC50 between 5.56-5.64 µg/mL. Among its five ingredients, Benjakul displayed the highest selectivity with selectivity index values ranging from 2.93 to 6.88, with the exception of Plumbago indica, indicating its protective effects. Plumbagin and 6- shogaol displayed the highest cytotoxicity and underwent molecular studies in NCI-H226 cells. Flow cytometry analysis revealed that Benjakul and 6-shogaol dose-dependently induced G2/M phase arrest, and plumbagin dose-dependently induced S-G2/M phase arrest with the highest percentage in early incubation time (12-24 h). At the highest doses, Benjakul extract, 6-shogaol, and plumbagin time-dependently increased the population of sub-G1 apoptotic cells with the highest percentage in longer incubation time (60-72 h). Similarly, membrane asymmetry changes showed time-dependent increases in the percentage of early and late apoptotic cells. Moreover, the apoptosis-inducing effect of Benjakul, 6-shogaol, and plumbagin at the highest dose, via the caspase cascade was confirmed by time-dependent induction of caspase-3 activity, followed by its complete reduction and abolished sub-G1 peaks upon addition of Z-VAD-FMK. CONCLUSION AND IMPLICATION: Our findings demonstrated for the first time the effects of Benjakul and its compounds on S-G2/M or G2/M phase arrest and caspase-dependent apoptosis in lung cancer cells.

In Vitro Cytotoxic Activity against Breast, Cervical, and Ovarian Cancer Cells and Flavonoid Content of Plant Ingredients Used in a Selected Thai Traditional Cancer Remedy: Correlation and Hierarchical Cluster Analysis.

This study aimed to investigate in vitro cytotoxic activity of selected plant ingredients from a traditional Thai remedy for the treatment of cancer patients against cancer cells occurring in women such as MCF-7 (breast cancer), SKOV3 (ovarian cancer), and HeLa (cervical cancer) cell lines. The plants and the remedy were macerated with 95% ethanol and boiled in water. Cytotoxic activity of the extracts was analyzed by SRB assay. Total flavonoid contents of the extracts were determined and their correlation with cytotoxic activity was evaluated. The hierarchical cluster analysis (HCA) was used to classify the extracts by their cytotoxic characteristics. A total of 66.7% of the plants was active against the tested cancer cell lines. Among the 44 plants in the remedy used for cancer treatment, nine plants that are also used in Thai cuisine exerted significant cytotoxicity against tested cancer cell lines. Eleven plants in the remedy were active against at least one of the tested cancer cell lines. All extracts were grouped into three groups and illustrated as heat map and hierarchical dendrogram. Total flavonoid content showed weak or no correlation with cytotoxic activity. A. dahurica, F. albopurpurea, and T. indica selectively exerted potent cytotoxic activity against MCF-7 with SI value more than 6. A. galanga, P. amarus, L. striatum, H. indicum, and F. vulgare exerted moderate cytotoxicity to all tested cell with low toxicity to normal cells. The correlation and HCA performed in this study provided an alternative way to investigate biological activities of plant ingredients in polyherbal traditional remedies.

Bactericidal Effect and Anti-Inflammatory Activity of Cassia garettiana Heartwood Extract.

Natural products are used as alternative drugs in traditional medicine to treat infection and inflammation and relieve pain. Heartwood of Cassia garettiana Craib has been investigated as an ingredient in Thai traditional medicine for anti-HIV protease, but there is no report on its antibacterial and anti-inflammatory activities. The objectives of this study were to investigate the anti-inflammatory and antibacterial activities, time-kill profile, and main active constituents of an ethanolic extract of C. garettiana heartwood. The study followed the generally accepted experimental design. All tests were investigated in triplicate. The heartwood of C. garettiana was extracted by maceration with 95% EtOH. The antibacterial activity of the extract and its chemical constituents were determined by their MIC values using resazurin as an indicator. Time-kill profile was determined at 0, 2, 4, 6, 8, 10, 12, and 24 hrs and expressed as log CFU/mL. The anti-inflammatory activity of the extract and its chemical components was investigated by their inhibiting effect on IL-6 and TNF-α production by ELISA. The ethanolic extract was analyzed for its chemical constituents by HPLC technique. The ethanolic extract showed both dose- and time-dependent bactericidal effects against Staphylococcus aureus, methicillin-resistance Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Pseudomonas aeruginosa, Salmonella Typhi, Salmonella Typhimurium, Klebsiella pneumoniae, and Shigella dysenteriae with MIC values of 312.5, 312.5, 312.5, 1,250, 2,500, 625, 625, 2,500, and 625 µg/mL, respectively. It showed an inhibiting effect on IL-6 production at concentrations of 12.5 to 100 µg/mL. The main active chemical constituent of C. garettiana was piceatannol that showed antibacterial activity against all test bacteria except P. aeruginosa. C. garettiana showed a broad spectrum of antibacterial activity against both Gram-negative and Gram-positive bacteria. Piceatannol and resveratrol from the plant strongly inhibited IL-6 production. Based on these results, we concluded that the ethanolic extract of C. garettiana showed both an antibacterial activity and inhibition of IL-6. Piceatannol is the active constituent of the extract and showed anti-inflammatory and antibacterial activities against Gram-negative and Gram-positive bacteria.

Anti-Helicobacter pylori, Anti-Inflammatory, Cytotoxic, and Antioxidant Activities of Mace Extracts from Myristica fragrans.

The aril (mace) of Myristica fragrans, known as Dok-Chan, is a spice that has long been used for treating stomach discomfort, peptic ulcer, and nausea. It is an ingredient in many remedies in Thai traditional medicine, e.g., Ya-Hom-Thep-Bha-Jit, Ya-Hom-Nao-Wa-Kot, and Ya-That-Bun-Job, which are used to treat dyspepsia and other gastrointestinal tract symptoms. The aqueous and ethanolic extracts of mace were used for all tests. Anti-H. pylori activities were determined by the disc diffusion method and agar dilution. Anti-inflammatory activity was determined by the LPS-induced nitric oxide (NO) inhibition in a RAW264.7 cell line, and cytotoxicity was determined against gastric cancer cell lines (Kato III) using the sulphorhodamine B (SRB) assay. The DPPH radical scavenging and ABTS radical cation decolorization assays were used to determine the antioxidant activities. The result found that the ethanolic extract of mace exhibited antimicrobial activity against H. pylori ATCC 43504 and six clinical strains with MIC values of 125-250 µg/ml. The aqueous extract MICs against H. pylori ATCC reference strain and six clinical strains were 500 µg/ml compared with 0.5 µg/ml for the positive control, clarithromycin. The inhibitory effect of LPS-induced NO release and cytotoxic activity of the ethanolic extract had IC50 values of 82.19 µg/ml and 26.06 µg/ml, respectively, and the EC50 values for the DPPH and ABTS antioxidant assays were 13.41 µg/ml and 12.44 µg/ml, respectively. The mace extract also had anticancer properties. In conclusion, the ethanolic mace extract had anti-H. pylori, anti-inflammatory, antioxidant, and anticancer activities. These data support further preclinical and clinical investigation to see if the mace extract could have a role in treating patients with dyspepsia, peptic ulcers, and possibly gastric cancer.

Anti-angiogenic and anti-proliferative effects of Benja-ummarit extract in rats with hepatocellular carcinoma.

The herbal extract Benja-ummarit (BU) is a traditional Thai medicine with a putative cancer-suppressing effect. However, this effect has only been tested in vitro in human hepatocarcinoma cell lines. The present study determined the efficacy of a BU extract to treat hepatocellular carcinoma (HCC) in rats in vivo and established its anti-angiogenic and anti-proliferative properties. The BU extract was prepared in 95% ethanol and its composition determined using liquid chromatography-mass spectrometry. HCC was induced in Wistar rats by an injection of diethylnitrosamine (DEN), followed 2 weeks later by injections of thioacetamide (TAA) thrice weekly for 4 weeks. Following 2 months, the DEN-TAA-treated rats were divided into 6 groups that were treated orally for another 2 months with: i) No treatment; ii) vehicle; iii) 30 mg/kg sorafenib (SF); iv) 1 mg/kg BU; v) 10 mg/kg BU; or vi) 50 mg/kg BU. Liver samples were collected for gross morphological, histological, reverse transcription-quantitative PCR and western blot analyses, and serum samples were collected for liver function tests. The size and number of the cancer nodules were reduced ~10-fold in BU-treated HCC groups and ~14-fold in the SF-treated group compared with the HCC group. Furthermore, the serum parameters of liver damage were lower in BU-compared with SF-treated rats. These results indicate that while each of these formulations strongly reduce HCC expansion, BU extract results in less liver damage. Vascular endothelial growth factor expression was reduced significantly in the BU-and SF-treated HCC groups compared with the HCC group (P<0.05). BU extract antagonizes HCC growth in vivo potently through inhibiting tumor angiogenesis. BU, therefore, qualifies as a promising medical herb requiring further evaluation as a treatment of HCC.

An ethyl-acetate fraction of Holothuria scabra modulates inflammation in vitro through inhibiting the production of nitric oxide and pro-inflammatory cytokines via NF-κB and JNK pathways.

Sea cucumber, Holothuria scabra, is an echinoderm marine animal that has long been used as a traditional therapeutic in various diseases due to its chemical composition and protein enrichment. Many researchers have extensively studied the efficacy of sea cucumber extracts for many health benefits in recent years. Inflammation is a complex process involved in pro-/anti-inflammatory cytokine products. However, the role of the H. scabra extracts in anti-inflammation and its molecular regulations has not been apparently elucidated yet. In this study, we investigated the anti-inflammatory effect of H. scabra extracts by using lipopolysaccharide (LPS) from E. coli to induce an inflammatory response in RAW264.7 macrophage. It was found that ethyl acetate fraction of H. scabra extracts (EAHS) inhibited pro-inflammatory cytokines synthesis at both the transcriptional and translational levels, notably nitric oxide (NO), inducible nitric oxide synthase (iNOS), interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), and prostaglandin E2 (PGE2). In addition, EAHS was able to downregulate IκB/NF-κB, and JNK expressions. These effects may be influenced by high contents of phenolic compound and triterpene glycosides in EAHS. Therefore, EAHS might have the potential to be developed as a natural anti-inflammatory agent.

Potential in vitro anti-allergic, anti-inflammatory and cytotoxic activities of ethanolic extract of Baliospermum montanum root, its major components and a validated HPLC method.

BACKGROUND: The root of Baliospermum montanum has been used as an ingredient of traditional Thai medicines for the treatments of several diseases including itching eczema, muscle and joint inflammation, and cancer. Few studies have been done on phytochemical components of this root. In this study, we isolated major compounds of the crude ethanolic extract of B. montanum root and developed and validated a high performance liquid chromatographic (HPLC) method for the determination of its major components. We then investigated anti-allergic, anti-inflammatory and cytotoxic activities of the extract. METHODS: The aims of this study were to investigate in vitro activities including inhibitory effect of ß-hexosaminidase released from RBL-2H3 cells, inhibition of nitric oxide (NO) production from RAW 264.7 cells and cytotoxic activity against cancerous liver cell lines (HepG2 and KKU M156) by using sulforhodamine B (SRB) assay. Isolation of major components was conducted by using column chromatographic method. Isolated major compounds were analyzed by using high performance liquid chromatography (HPLC). RESULTS: The crude extract exhibited the highest cytotoxic activity, with IC50 less than 1 µg/mL, while its anti-allergy and anti-inflammation were also potent with IC50 less than 6 µg/mL. Three propiophenones isolated from B. montanum root exhibited moderate cytotoxic activities (IC50 > 20 µg/mL). Two of the propiophenones found were major components that can be detected by HPLC. The developed and validated HPLC method showed good accuracy, precision, and linearity. CONCLUSION: The results of this study suggested that ethanolic extract of of B.montanum root can be a potential source of anti-allergy, anti-inflammation, and anti-cancer compounds. The isolated compounds can serve as markers when B. montanum is used in herbal remedies but not as overall responsive markers. The HPLC method developed may be useful for quality control in the production of the extract and for further formulation developments. However, investigation of several associated biological activities is necessary before the development can proceed further. Minor active compounds should be isolated and a more sensitive analytical method should be developed to detail the key responsive components of the ethanolic extract of B. montanum root.

The anti-allergic and anti-inflammatory effects of Benjakul extract (a Thai traditional medicine), its constituent plants and its some pure constituents using in vitro experiments.

Benjakul (BJK), a Thai traditional medicine preparation, has long been used for balanced health, controlled abnormal of element in the body, carminative, and relief of flatulence. It is composed of five plants: Piper interruptum Opiz., Piper longum L., Piper sarmentosum Roxb., Plumbago indica L., and Zingiber officinale Roscoe. The ethanolic extracts of BJK, its five individual plants, and pure constituents of BJK were investigated for their anti-allergic activity using immunoglobulin E (IgE)-sensitized ß-hexosaminidase in the rat basophilic leukemia-2H3 (RBL-2H3) cells and anti-inflammatory activity using lipopolysaccharide (LPS)-induced nitric oxide (NO) and tumor necrosis factor-alpha (TNF-α) in the murine macrophage (RAW 264.7) cells. The ethanolic extracts of BJK showed anti-allergic activity (IC50=12.69µg/ml) and exhibited potent NO inhibitory effect (IC50=16.60µg/ml), but inactive on TNF-α release. Moreover, 6-shogaol and plumbagin, two pure compounds from BJK, showed higher anti-allergic activity than the ethanolic BJK extract with IC50 values of 0.28 and 4.03µg/ml, respectively. These compounds were significantly higher than chlorpheniramine (CPM), standard drug, with IC50 value of 17.98µg/ml. Determination of the anti-inflammatory activity by measuring the inhibition of NO production presented that plumbagin and 6-shogaol exhibited higher than crude BJK extract with IC50 values of 0.002 and 0.92µg/ml, respectively. In particular, plumbagin also showed higher anti-inflammatory than prednisolone, positive control, with IC50 value of 0.59µg/ml. 6-Shogaol also showed inhibitory effect on TNF-α release (IC50=9.16µg/ml). These preliminary results may provide some scientific support for the use of BJK for the anti-allergic treatment and inflammatory disorders through the inhibition of NO production.

Effects of 5,6-Dihydroxy-2,4-Dimethoxy-9,10-Dihydrophenanthrene on G2/M Cell Cycle Arrest and Apoptosis in Human Lung Carcinoma Cells.

5,6-dihydroxy-2,4-dimethoxy-9,10-dihydrophenanthrene (HMP) is an active compound isolated from the rhizome extracts of Dioscorea membranacea Pierre, a Thai medicinal plant. This study aimed to investigate the growth-inhibitory and apoptosis-inducing effects of HMP in human lung cancer A549 cells. The antiproliferative and cytotoxic effects of HMP were analyzed by a Sulforhodamine B assay. Cell division, cell cycle distribution and membrane asymmetry changes were each performed with different fluorescent dyes and then analyzed by flow cytometry. Real-time PCR and immunoblotting were used to detect cell cycle- and apoptosis-related mRNA levels and proteins, respectively. The nuclear morphology of the cells stained with DAPI and DNA fragmentation were detected by fluorescence microscopy and gel electrophoresis, respectively. The results showed that HMP exerted strong antiproliferative and cytotoxic activities in A549 cells with the highest selectivity index. It halted the cell cycle in [Formula: see text]/M phase via down-regulation of the expression levels of regulatory proteins Cdc25C, Cdk1 and cyclinB1. In addition, HMP induced early apoptotic cells with externalized phosphatidylserine and subsequent apoptotic cells in sub-[Formula: see text] phase. HMP increased caspase-3 activity and levels of the cleaved (active) form of caspase-3 whose actions were supported by the cleavage of its target PARP, nuclear condensation and DNA apoptotic ladder. Moreover, HMP significantly increased the mRNA and protein levels of proapoptotic Bax as well as promoted subsequent caspase-9 activation and BID cleavage, indicating HMP-induced apoptosis via both intrinsic and extrinsic pathways. These data support, for the first time, the potential role of HMP as a cell-cycle arrest and apoptosis-inducing agent for lung cancer treatment.

A novel steroid and cytotoxic constituents from Dioscorea membranacea Pierre against hepatocellular carcinoma and cholangiocarcinoma cells.

ETHNOPHARMACOLOGICAL RELEVANCE: The rhizomes of Dioscorea membrancea Pierre have been used in Thai traditional medicine as an ingredient formula for liver cancer and cholangiocarcinoma treatment. AIM OF THE STUDY: To investigate the cytotoxic activity of ethanolic extract and constituents of D. membrancea to support its traditional use. MATERIALS AND METHODS: The SRB assay was used to determine the cytotoxic activity against hepatocellular carcinoma (HepG2), cholangiocarcinoma (KKU-M156) cells and one normal human keratinocyte immortal cells (HaCaT) with its ethanolic extract and isolated compounds. Bioassay guided isolation was used for isolating cytotoxic compounds. RESULTS: The ethanolic extract of D. membranacea rhizome showed weak cytotoxic against KKU-M156 and HepG2 (IC50 at 72h exposure=30.49±0.82 and 38.97±2.04µg/mL respectively). A new steroid [epipanthogenin B (1)], a known steroid [panthogenin B (2)], two napthofuranoxepins [dioscorealide A (3) and dioscorealide B (4)], phenanthraquinone [dioscoreanone (5)] and two phenanthrene [5,6-dihydroxy-2,4-dimethoxy-9,10-dihydrophenanthrene (6) and 2,5,6-trihydroxy-3,4-dimethoxy, 9, 10-dihydrophenanthrene (7)] were isolated from active chloroform fraction. Compound 4 showed the highest cytotoxicity against HepG2 (IC50 at 72h exposure=2.87±0.21µM) and KKU-M156 (IC50 at 72h exposure=1.67±0.10µM) and less toxicity against normal cell line (HaCaT) (IC50 at 72h exposure>100µM). Compound 5 showed selective cytotoxic activity against KKU-M156 (IC50 at 72h exposure=3.46±0.11µM). Compounds 6 and 7 showed weak cytotoxic activity against HepG2 (IC50 at 72h exposure=24.96±2.32 and 51.31±3.52µM). Compounds (1-3) showed no cytotoxic activity against HepG2 and KKU-M156 cell lines (IC50 at 72 h exposure>100µM). CONCLUSION: Seven compounds were isolated from active chloroform fraction of the ethanolic extract of D. membranacea rhizomes. Only dioscorealide B (4) might be served as a good anticancer agent for liver cancer and cholangiocarcinoma cancer because it can kill cancer cell but not toxic on normal cell. This research support Thai traditional medicine use of D. membranacea for liver cancer and cholangiocarcinoma cancer treatment.

Cytotoxic, Anti-inflammatory and Antioxidant Activities of Heliotropium indicum Extracts.

Background: Heliotropium indicum Linn., or 'Indian heliotrope' is very common in India with a long history of traditional medicinal uses in many countries in the world. In Thailand, the plant has been traditionally use to cure various diseases such as fever, insect bites, stings, diarrhea, skin rashes, menstrual disorder and urticaria. In addition, the plant is commonly used by Thai folk doctors as a component in remedies for treatment of lung cancer. Objective: In the present study, we investigated cytotoxicity against two types of lung cancer cell lines (A549 and NCI-H226), anti-inflammatory effect and antioxidant activity of Heliotropium indicum extracts. Material and Method: The water and ethanolic extracts of Heliotropium indicum were tested. The cytotoxic activity against two types of human lung cancer cell lines (A549 and NCI-H226) was evaluated by sulforhodamine B (SRB) assay. The antiinflammatory effect was investigated on lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. LPS-induced nitric oxide (NO) production was determined by Griess reagent. The antioxidant activity was performed by 1, 1-diphenyl-picrylhydrazyl (DPPH) radical scavenging method. Results: The ethanolic extract showed cytotoxic activity only against NCI-H226 (IC50 = 51.90±2.35 µg/ml) whereas the water extract had no cytotoxic activity against both A549 and NCI-H226 (IC50 >100 µg/ml). For anti-inflammatory effect, the results revealed that the ethanolic extract exhibited the most potent inhibitory activity on nitric oxide production (IC50 = 24.17±2.12 µg/ml), followed by Indomethacin (positive control) with an IC50 value of 34.67±6.23 µg/ml while water extract was apparently inactive (IC50 >100 µg/ml). For antioxidant activity, the ethanolic extract showed high antioxidant activity (EC50 = 28.91±4.26 µg/ml) but the water extract showed no antioxidant activity (EC50 >100 µg/ml). Conclusion: These results can support using Heliotropium indicum Linn. for component in lung cancer remedy by Thai folk doctors. However, more studies are required.

Cytotoxic Activity Against Cancer Cell Lines from The Ethanolic Extracts and Its VLC Fractions of Bauhinia strychnifolia Leaves.

Background: Cancer is a leading cause of morbidity and mortality worldwide. Bauhinia strychnifolia Craib leaves is used in Thai traditional medicine for detoxification. Its leaves contain total phenolic content and also exhibit high antioxidant activity. However, there has been no report on cytotoxicity testing from its leave extracts. Thus, the present study aims to investigate its cytotoxic activity against cancer cell lines. Objective: To study cytotoxicity from the ethanolic extracts of B. strychnifolia leaves and its vacuum liquid chromatography fraction against cholangiocarcinoma cell line (KKU-M156) and two types of colon adenocarcinoma cell lines (SW480, LS174T). Material and Method: In vitro cytotoxic activity of the ethanolic extracts against three human cancer cell lines were investigated by using sulforhodamine B (SRB) assay. Results: The 95% ethanolic extract of dried leaves showed the higher cytotoxic activity against KKU-M156, SW480, and LS174T than 50% ethanolic extract of dried leaves. The chloroform fraction from the 95% EtOH extract of dried leaves showed the best cytotoxicity against KKU-M156 and SW480 with IC50 value of 5.79±0.47 and 6.9±0.14 µg/ml, respectively. Conclusion: The chloroform fraction from the 95% ethanolic extract of dried leaves was the most effective fraction against bile duct and colon cancer cell lines, thus this extract should be further investigated for active compounds possessing those observed cytotoxic activity.

Cytotoxic and Anti-inflammatory Activities of Medicinal Plants and Women's Health Remedy Found in "Mahachotarat Scripture" of Thai Traditional Medicine.

Background: "Mahachotarat" is a lesson for woman's care from the Thai traditional medicine book, composed of both medicinal plants and herbal remedies that can treat both pain and cervical cancer. Medicinal plants and herbal remedies, which have often been used in the treatment of pain and cancer, were selected to investigate for biological activity related to woman's health. They were Boesenbergia rotunda Linn, Piper nigrum Linn, Zingiber cassumunar Roxb, Zingiber officinale Roscoe, Zingiber zerumbet (L) Smith, Dioscorea birmanica Prain & Burkill including its ingredient; Prosapogenin A of dioscin and Leard-ngam remedy. Objective: The objective was to investigate cytotoxic and anti-inflammatory activities of all sample plants. Material and Method: Medicinal plants and Leard-ngam remedy were extracted similarly to that practiced by Thai traditional practitioners (ethanol and water extraction). Bioassay guide fractionation was used for isolating pure compound. The structure elucidation of pure compound was proven by spectrophotometry technique. These extracts were tested for their cytotoxic activity against Hela cells, cervical cancer cells, by sulforhodamine B assay, inhibition of nitric oxide and prostaglandin E2 production in lipopolysaccharide-stimulated mouse macrophage RAW 264.7 cells. Result: This study showed that P. nigrum, Z. officinale, B. rotunda and Z. cassumunar showed potent inhibitory activity on nitric oxide and PGE2 production. The 95% ethanolic extract of Z. zerumbet had the highest cytotoxic activity on Hela cells with IC50 value of 4.42±0.20 µg/ml. Diosgenin-3-O-α-L-rhamnosyl (1→2)-ß-D-glucopyranoside or Prosapogenin A of dioscin, which was purified from the 95% ethanolic extract of D. birmanica, showed strong cytotoxic activity on Hela cells with IC50 value of 6.07±0.02 µg/ml. Conclusion: Thai medicinal plants and LG remedies which have often been used in the treatment of pain and cancer by Thai traditional practitioners, showed high anti-inflammatory properties on both pathways which represent chronic and acute inflammation. Interestingly, some medicinal plants were used daily in Thai food. In addition, Z. zerumbet and D. birmanica, which has often been used in the treatment of cancer, also showed high cytotoxic activity against cervical cancer cells.