Analytical determination, antioxidant and anti-inflammatory activities of Bhamrung-Lohit a traditional Thai medicine.

Background and purpose: Bhamrung-Lohit (BRL) remedy is a traditional Thai medicine (TTM). There are few reports of biological activity, the activity of its constituent plants, or quantitative analytical methods for the content of phytochemicals. In this study, we investigated antioxidant, anti-inflammatory activity, and total phenolic and flavonoid content and validated a new analytical method for BRL. Experimental approach: Antioxidant activity was evaluated by a 2,2-diphenyl-1-picrylhydrazyl (DPPH and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radical scavenging. The cellular antioxidant activity was evaluated by inhibition of the superoxide anion (O2●-) production from HL-60 cells and anti-inflammatory activity by inhibition of nitric oxide production in RAW264.7 cells. The total phenolic and flavonoid contents were analyzed using the Folin-Ciocalteu method and an aluminum chloride colorimetric assay, respectively. Validated analytical procedures were conducted according to International Conference on Harmonization (ICH) guidelines. Findings/Results: An ethanolic extract of BRL exerted potent DPPH radical scavenging activity and moderate antioxidant and anti-inflammatory activity. Caesalpinia sappan exerted the greatest effect and the highest content of total phenolics and flavonoids. The HPLC method validated parameters that complied with ICH requirements. Each peak showed selectivity with a baseline resolution of 2.0 and precision was less than 2.0% CV. The linearity of all compounds was > 0.999 and the recovery % was within 98.0%-102.0%. The validated results demonstrated specificity/selectivity, linearity, precision, and accuracy with appropriate LOD and LOQ. Conclusion and implication: BRL remedy, a TTM demonstrated antioxidant and anti-inflammatory properties. This study is the first report on the biological activity and the validation of an HPLC method for BRL remedy.

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.

Anti-Inflammatory Investigations of Extracts of Zanthoxylum rhetsa.

Zanthoxylum rhetsa has been consumed in the diet in northern Thailand and also used as a medicament in ancient scripture for arthropathies. Thus, this study aimed to evaluate the activity of various extracts from differential parts of Z. rhetsa via inhibition of inflammatory mediators (NO, TNF-α, and PGE2) in RAW264.7 macrophages. The chemical composition in active extracts was also analyzed by GC/MS. The parts of this plant studied were whole fruits (F), pericarp (P), and seed (O). The methods of extraction included maceration in hexane, 95% ethanol and 50% ethanol, boiling in water, and water distillation. The results demonstrated that the hexane and 95% ethanolic extract from pericarp (PH and P95) and seed essential oil (SO) were the most active extracts. PH and P95 gave the highest inhibition of NO production with IC50 as 11.99 ± 1.66 µg/ml and 15.33 ± 1.05 µg/ml, respectively, and they also showed the highest anti-inflammatory effect on TNF-α with IC50 as 36.08 ± 0.55 µg/ml and 34.90 ± 2.58 µg/ml, respectively. PH and P95 also showed the highest inhibitory effect on PGE2 but less than SO with IC50 as 13.72 ± 0.81 µg/ml, 12.26 ± 0.71 µg/ml, and 8.61 ± 2.23 µg/ml, respectively. 2,3-Pinanediol was the major anti-inflammatory compound analyzed in PH (11.28%) and P95 (19.82%) while terpinen-4-ol constituted a major anti-inflammatory compound in SO at 35.13%. These findings are the first supportive data for ethnomedical use for analgesic and anti-inflammatory activity in acute (SO) and chronic (PH and P95) inflammation.

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.

In Vitro Wound Healing Activities of Three Most Commonly Used Thai Medicinal Plants and Their Three Markers.

Skin ensures that a constant internal environment can be maintained in an ever-changing external environment. When a wound occurs on the skin, the inflammatory and proliferative phases are initiated in response to injury. Thai traditional medicine (TTM), using medicinal plants and ancient knowledge, has been used to treat wounds. Eight Thai medicinal plants, most commonly used to treat wounds, were evaluated for their in vitro biological activities such as antioxidation by NBT assay, anti-inflammation by production inhibition of NO, promoting fibroblast cell proliferation, and wound closure activities. Plant materials were extracted with 95% ethanol or distilled water and then concentrated and dried. Statistical analysis of data was done using one-way ANOVA at p value of 0.05. The ethanolic extracts of Garcinia mangostana L., Glycyrrhiza glabra L., and Nigella sativa L. could inhibit the production of superoxide anion with the IC50 values of 13.97 ± 0.38, 28.62 ± 1.91, and 71.54 ± 3.22 µg/ml and nitric oxide with the IC50 values of 23.97 ± 0.91, 46.35 ± 0.43, and 78.48 ± 4.46 µg/ml, respectively. These extracts could promote cell proliferation and accelerate wound recovery at the rate of 2.02 ± 0.03, 2.12 ± 0.03, and 2.65 ± 0.05% per hour, respectively. Three established markers from these three plants were selected according to the selection criteria. Alpha-mangostin, glycyrrhizin, and thymoquinone were found to be the active markers for wound closure activities. The ethanolic extracts of G. mangostana, G. glabra, and N. sativa could scavenge superoxide anion and inhibit the production of nitric oxide; therefore these extracts could assist in surpassing the inflammatory phase and protected the cells surrounding the wound area. Most importantly, these extracts also increased the proliferation and accelerated wound closure, indicating that these plant extracts could be promoting wound healing processes and support the use of TTM.

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.

Anti-Allergic Activities of Smilax glabra Rhizome Extracts and Its Isolated Compounds.

BACKGROUND: The rhizomes of Smilax glabra (SG) has long been used in Traditional Chinese and Thai herbal medicine to treat a variety of infectious diseases and immunological disorders. OBJECTIVE: To investigate the in vitro anti-allergic activities of crude extracts andpure isolated flavonoid compounds from SG by determination of inhibitory effect on antigen-induced release of ß-hexosaminidasefrom RBL-2H3 cells. MATERIAL AND METHOD: The in vitro inhibitory effects ofcrude aqueous and organic extracts on ß-hexosaminidase release in RBL-2H3 cells were evaluated as an in vitro indication ofpossible anti-allergic activity. Bioassay-guided fractionation of extracts was used to isolate flavonoid compounds from the ethanolic extracts. RESULTS: The 95% and 50% ethanolic extracts of SG showed remarkably high anti-allergic activity, with IC50 values of 5.74 ± 2.44 and 23.54 ± 4.75 µg/ml, much higher activity than that for Ketotifen (IC50 58.90 µM). The water extract had negligible activity (IC50 > 100 µg/ml). The two isolated flavonols, Engeletin and Astilbin, showed weak anti-allergic activity, IC50 values 97.46 ± 2.04 and >100 µg/ml, respectively. CONCLUSION: The 95% and 50% ethanolic extracts of SG showed strong anti-allergic activity, but two flavonol constituents did not show any significant anti-allergic activity. These findings suggest that a combination of effects of various phytochemicals in crude extracts used in traditional medicine, are responsible for the purported anti-allergic activity of SG herbal preparations. The plethora of constituents in crude extracts, as yet unidentified, are likely to be acting synergistically to account for the strong observed anti-allergic in vitro activity.

Immunomodulatory activity of Dioscorea membranacea Pierre rhizomes and of its main active constituent Dioscorealide B.

BACKGROUND: The rhizomes of Dioscorea membranacea Pierre, also called Hua-Khao-Yen by Thai name, are used as ingredients in many Thai traditional medicines for the alternative or complementary treatment of cancer and AIDs. Preliminary in vitro studies have indicated that D. membranacea extracts exhibited high cytotoxic activity with several cancer cell lines, but the underlining mechanisms are far from clear. The aims of this study were to investigate the effects of ethanolic and aqueous crude extracts from D. membranacea Pierre, and pure compound from D. membranacea Pierre, Dioscorealide B, on natural killer cells activity and on lymphocyte proliferation. METHODS: Immunomodulatory activity was investigated using PBMCs from healthy donors. NK cells activity was performed by the chromium release assay using PBMCs as effector cells, and K562 cells line labelled with chromium as target cells. Lymphocyte proliferation was determined by 3H-thymidine uptake. The degree of activation was expressed as the stimulation index. RESULTS: The crude ethanolic extracts of D. membranacea Pierre significantly stimulated NK cells activity against K562 cells line at lower concentrations of 10 and 100 ng/ml, but not at higher concentrations. The ethanolic extracts showed no observable effect on lymphocyte proliferation. The crude water extracts significantly increased NK cell activity at concentrations of 10 ng/ml, 100 ng/ml, 1 µg/ml, 10 µg/ml and 100 µg/ml, and also activated lymphocyte proliferation at concentration of 1 ng/ml, 10 ng/ml, 100 ng/ml, 1 µg/ml, 5 µg/ml, 10 µg/ml and 100 µg/ml. However, Dioscorealide B had no significant effect at lower concentrations (0-1 µg/ml and 0-0.1 µg/ml, respectively) on NK cell activity and lymphocyte proliferation. In fact higher concentrations (>10 µg/ml and >0.5 µg/ml) of Dioscorealide B cause a significant decrease in NK cell activity and lymphocyte proliferation. CONCLUSIONS: D. membranacea Pierre stimulated NK cells activity and lymphocyte proliferation, but Dioscorealide B either had no effect, and at higher concentrations decreased NK cell activity and lymphocyte proliferation. Our results suggest that both extracts of D. membranacea Pierre significantly increases immune function, but the underlining mechanism is not clearly understood.

Isolation and characterization of a new cytotoxic dihydrophenanthrene from Dioscorea membranacea rhizomes and its activity against five human cancer cell lines.

ETHNOPHARMACOLOGICAL RELEVANCE: The rhizomes of Dioscorea membranacea Pierre (DM) have been used as ingredients in anticancer herbal formulations in Thai traditional medicine (TTM). Thus, the aim of this study was to investigate the active constituents of DM for cytotoxic activity in order to support its TTM use. MATERIALS AND METHODS: A bioassay-guided isolation procedure was used to separate the cytotoxic constituents from ethanolic extract of Dioscorea membranacea rhizomes by testing against five human cancer cell lines, i.e. large cell lung carcinoma, COR-L23; liver cancer cells, HepG2; prostate cancer cells, PC3; breast cancer cells MCF-7; cervical cancer cells, Hela; and one normal human lung cell line (MRC 5) using the SRB assay. RESULTS: Two known dihydrophenanthrene compounds [2,4 dimethoxy-5,6-dihydroxy-9,10-dihydrophenanthrene (1) and 5-hydroxy-2,4,6-trimethoxy-9,10-dihydrophenanthrene (2)], and a new dihydrophenanthrene compound, 5,6,2 -trihydroxy 3,4-methoxy, 9,10-dihydrophenanthrene (3) were isolated and fully characterized. 1 showed the highest cytotoxic activity against COR-L23, MCF-7 and PC3 cell lines (IC50=14.89, 17.49 and 19.04 µM, respectively), and 2 showed selective cytotoxic activity against PC3 (IC50=23.54 µM). The new compound 3 showed selective cytotoxic activity against only MCF-7 cells (IC50=31.41 µM). Interestingly the crude extract of DM was much less toxic to the normal cell line (MRC-5) (IC50>50 µg/ml) compared to the five cancer cell lines, (IC50 value ranged between 6 and 29 µg/ml). CONCLUSION: The phytochemicals isolated from DM may serve as lead compounds for the design of new anti-cancer agents with better selective cytotoxic indices.

Inhibitory Effect on ß -Hexosaminidase Release from RBL-2H3 Cells of Extracts and Some Pure Constituents of Benchalokawichian, a Thai Herbal Remedy, Used for Allergic Disorders.

Introduction. Benchalokawichian (BCW), a Thai traditional herbal formulation, has long been used as antipyretic and to treat skin disorders. It comprises roots from five herbs: Ficus racemosa, Capparis micracantha, Clerodendrum petasites, Harrisonia perforata, and Tiliacora triandra. This polyherbal remedy has recently been included in the Thailand National List of Essential Medicines (Herbal Products list). Methodology. A Bioassay-guided fractionation technique was used to evaluate antiallergy activities of crude extracts, and those obtained by the multistep column chromatography isolation of pure compounds. Inhibitory effect on the release of ß-hexosaminidase from RBL-2H3 cells was used to determine antiallergic activity. Results. Two pure compounds from BCW formulation showed higher antiallergic activity than crude or semipure extracts. Pectolinarigenin showed the highest antiallergic activity, followed by O-methylalloptaeroxylin, with IC50 values of 6.3 µg/mL and 14.16 µg/mL, respectively. Moreover, the highest activities of pure compounds were significantly higher than chlorpheniramine (16.2 µg/mL). Conclusions. This study provides some support for the use of BCW in reducing itching and treatment of other skin allergic disorders. The two isolated constituents exhibited high antiallergic activity and it is necessary to determine their mechanism of action. Further phytochemical and safety studies of pure compounds are required before development of these as antiallergy commercial remedies.

An in vitro inhibitory effect on RAW 264.7 cells by anti-inflammatory compounds from Smilax corbularia Kunth.

BACKGROUND: Smilax corbularia is a Thai medicinal plant locally known as 'Hua-Khao-Yen Neua', which is used for treating inflammatory conditions. OBJECTIVE: To evaluate the anti-inflammatory effect of S. corbularia extracts and its isolated compounds by determination of inhibitory effects on lipopolysaccharide-stimulated PGE2 release, and TNF-alpha and NO production from RAW 264.7 cells. METHODS: The inhibitory effect of aqueous and ethanolic extracts of this plant were determined on LPS-induced NO production, TNF-alpha and PGE2 release in RAW 264.7 cells, as an in vitro indication of possible anti-inflammatory activity. The compounds from active extract were isolated by bioassay-guided fractionation. RESULTS: Only the ethanolic extract of this plant inhibited TNF-alpha and NO production, with IC50 values of 61.97, and 83.90 microg/ml respectively. Three flavonols, engeletin, astilbin and quercetin were isolated from the ethanolic extract. quercetin possessed the highest inhibitory effect on NO production with IC50 11.2 microg/ml (37.1 microM), whereas engeletin and astilbin had no activity (IC50 >100 microg/ml). All three flavonols possessed potent inhibition of PGE2 release with IC50 values of 14.4, 19.6 and 19.9 microg/ml (33.2, 43.5 and 65.8 microM) respectively. Quercetin also exhibited the highest inhibitory effect on TNF-alpha production (IC50 = 1.25 microg/ml or 4.14 microM), but engeletin and astilbin had no activity. CONCLUSION: This is the first report of isolated compounds from S. corbularia with potential anti-inflammatory effects, and the results support the use of this plant by Thai traditional doctors for treatment of inflammatory diseases.