Anti-proliferative and apoptosis induction activities of extracts from Thai medicinal plant recipes selected from MANOSROI II database

Ethonopharmacological relevances

Traditional medicines have long been used by the Thai people. Several medicinal recipes prepared from a mixture of plants are often used by traditional medicinal practitioners for the treatment of many diseases including cancer. The recipes collected from the Thai medicinal text books were recorded in MANOSROI II database. Anticancer recipes were searched and selected by a computer program using the recipe indication keywords including Ma-reng and San which means cancer in Thai, from the database for anticancer activity investigation.

Aim of study

To investigate anti-cancer activities of the Thai medicinal plant recipes selected from the “MANOSROI II” database.

Materials and methods

Anti-proliferative and apoptotic activities of extracts from 121 recipes selected from 56,137 recipes in the Thai medicinal plant recipe “MANOSROI II” database were investigated in two cancer cell lines including human mouth epidermal carcinoma (KB) and human colon adenocarcinoma (HT-29) cell lines using sulforhodamine B (SRB) assay and acridine orange (AO) and ethidium bromide (EB) staining technique, respectively.

Results and conclusions

In the SRB assay, recipes NE028 and, S003 gave the highest anti-proliferation activity on KB and HT29 with the IC₅₀ values of 2.48 ± 0.24 and 6.92 ± 0.49 μg/ml, respectively. In the AO/EB staining assay, recipes S016 and NE028 exhibited the highest apoptotic induction in KB and HT-29 cell lines, respectively. This study has demonstrated that the three Thai medicinal plant recipes selected from “MANOSROI II” database (NE028, S003 and S016) gave active anti-cancer activities according to the NCI classification which can be further developed for anti-cancer treatment.     

Potent anti-cervical cancer activity: Synergistic effects of Thai medicinal plants in recipe N040 selected from the MANOSROI III database

Ethnopharmacological relevances

One of the prestigious Thai/Lanna folklore wisdoms is the medicinal plant recipes. Thai/Lanna medicinal plant recipe database MANOSROI III has been developed by Prof. Dr. Jiradej Manosroi. It consists of over 200,000 recipes covering all diseases including cancer.

Aim of this study

To investigate the in vitro and in vivo anti-cervical cancer activity and the active constituents of the Thai medicinal plant recipe N040 selected from the MANOSROI III database.

Materials and methods

The extracts of recipe N040 and single medicinal plants in the recipe were prepared by hot water and methanol extraction, respectively. The n-hexane, ethyl acetate (EtOAc), n-butanol (n-BuOH) and water fractions of Caesalpinia sappan, the plant which showed the highest anti-proliferative activity were prepared by liquid–liquid partition extraction. The fraction which showed the highest anti-proliferative activity was further isolated for active constituents. Anti-proliferative activity of recipe N040, methanolic extracts, fractions of Caesalpinia sappan and brazilin, an active constituent on HeLa cell were investigated using sulforhodamine B (SRB) assay. Anti-oxidative activities including free radical scavenging and metal ion-chelating activities, as well as the phenolic and flavonoid contents of these fractions were also determined. The in vivo anti-cancer activity of recipe N040 on HeLa cell xenograft and the subchronic toxicity were performed in nude mice and rats, respectively.

Results and conclusions

N040 showed the potent in vitro anti-proliferative activity on HeLa cell with the IC₅₀ value of 0.11 µg/ml. Phytochemicals detected in the plants were steroids/triterpenoids, tannins, flavonoids, saponins and alkaloids. For the single plant, methanolic extract of Caesalpinia sappan gave the highest anti-proliferative activity with the IC₅₀ of 33.46 µg/ml. EtOAc fraction of Caesalpinia sappan showed the highest anti-proliferative and free radical scavenging activities with the IC₅₀ and SC₅₀ of 17.81 and 21.95 µg/ml which were 1.88 and 0.83 folds of its methanolic extract and ascorbic acid, respectively. Poor metal chelating activity (MC₅₀>500 µg/ml) was observed in methanolic extract and all fractions. The highest phenolic and flavonoid contents were observed in the methanolic extract. Brazilin, the known compound isolated from the EtOAc fraction exhibited potent anti-proliferative activity with the IC₅₀ of 0.28 µg/ml which was higher than its methanolic extract and EtOAc fraction of 119.50 and 63.61 folds, respectively, but only 0.39 fold of the recipe extract N040. The tumor size of the HeLa cell xenograft nude mice treated with the recipe N040 at the dose of 44.50 mg/kg body weight per day was significantly smaller (p<0.05) than that of the control with the relative tumor weight inhibition of 57.23% which was 0.65 fold of cisplatin. In the subchronic toxicity study, N040 given orally at the dose of 1000 mg/kg body weight per day for 90 days showed no alteration in body weight gain, hematology [except the increase mean corpuscular hemoglobin (MCH) in the treated male rats] and clinical blood chemistry (except the increase blood glucose in the treated male rats) both in female and male rats. Only minor lesions of the organs including lung, liver, kidney and small intestine were observed in both sexes. This study has demonstrated the synergistic effect of the plants composed in the recipe which resulted in the potent anti-cancer activity and confirmed the traditionally use of the recipe N040. In addition, this study has also suggested the compound brazilin isolated from Caesalpinia sappan for its high potential to be further investigated as a novel anti-cervical cancer drug.     

Hypoglycemic activity of Thai medicinal plants selected from the Thai/Lanna Medicinal Recipe Database MANOSROI II

Ethnopharmacological relevance

Five medicinal plants including Anogeissus acuminata (Roxb. ex DC.) Gills. & Perr. (Combretaceae), Catunaregam tormentosa (Bl. ex DC.) Tirveng (Rubiaceae), Dioecrescis erythroclada (Kurz) Tirveng. (Rubiaceae), Mimosa pudica Linn. var. hispida Bren. (Fabaceae), and Rauwolfia serpentina (L). Benth. ex Kurz. (Apocyanaceae), which have been traditionally used for the treatment of diabetes mellitus and other diseases for several generations by the Thai-Lanna people in the Northern part of Thailand were investigated for their hypoglycemic activity in normoglycemic and alloxan, induced diabetic mice.

Materials and methods

The aqueous extracts of the selected five medicinal plants were tested for their phytochemicals, free radical scavenging activity and hypoglycemic activity on 18 h fasted normoglycemic and alloxan induced diabetic mice over a period of 4 h comparing with the standard anti-diabetic drugs (insulin and glibenclamide) using two way analysis of variance (ANOVA) as analytical tool. Phytochemical analysis was performed using the standard methods while 2,2-diphenyl-1-picrylhydrazine (DPPH) was used to test for free radical scavenging activities of the medicinal plant extracts.

Results

Phytochemicals detected in the extracts were glycosides, xanthones, tannins, alkaloids and saponins. Anogeissus acuminata showed the highest free radical scavenging activity with the IC₅₀ value of 11.00 μg/mL which was 4 folds of the standard ascorbic acid. Significant reduction in fasting blood glucose (FBG) levels of the normoglycemic mice was observed at 4 and 3 h with the extracts of Mimosa pudica (200 mg/kg bw) and Rauwolfia serpentina (100 mg/kg bw), and percentage decreases of 46.15 and 27.94% which were 0.76 and 1.47; 0.53 and 0.91 folds of insulin and glibenclamide, respectively. In alloxan induced diabetic mice, all extracts showed significant (p < 0.05) hypoglycemic activity, with the maximum FBG reduction of 78.96 at 100 mg/kg bw shown by Anogeissus acuminata at the 4 h. The hypoglycemic activity of Anogeissus acuminata was comparable to insulin (1.1 fold), but more potent than glibenclamide (1.76 folds).

Conclusions

Medicinal plants selected from the Thai/Lanna Medicinal Plant Recipe Database MANOSROI II showed hypoglycemic activity in normoglycemic and alloxan induced diabetic mice. This study confirmed the traditional use of these medicinal plants for the treatment of diabetes mellitus and the thiazolidiendiones mimic hypoglycemic effects of the medicinal plants was suggested.     

In vitro screening for anthelmintic and antitumour activity of ethnomedicinal plants from Thailand

Aim of study

This study screened for anthelmintic and/or antitumour bioactive compounds from Thai indigenous plants and evaluated effectiveness against three different worm species and two cancer cell lines.

Materials and methods

Methylene chloride and methanol extracts of 32 plant species were screened for in vitro anthelmintic activity against three species of worms, the nematode Caenorhabditis elegans, the digeneans Paramphistomum epiclitum and Schistosoma mansoni (cercariae). Cytotoxicity of the extracts was evaluated against two cancer cell lines: human amelanotic melanoma (C32) and human cervical carcinoma (HeLa) by the SRB assay. Anthelmintic and anticancer activities were evaluated by the inhibiting concentration at 50% death (IC₅₀) and the selectivity index (SI) relative to human fibroblasts.

Results and conclusions

None of the extracts were active against Paramphistomum epiclitum. Plumbagin, a pure compound from Plumbago indica, had the strongest activity against Caenorhabditis elegans. The methylene chloride extract of Piper chaba fruits had the strongest activity against schistosome cercariae. Strong cytotoxicity was shown by the methylene chloride extract of Michelia champaca bark and the methanol extract of Curcuma longa rhizome against C32 and HeLa, respectively. These extracts had higher SI (>100) than positive controls in relation to either the worms or the cell lines. The methanol extract of Bouea burmanica had a slightly lower activity towards C32 cells than did Michelia champaca but had a much higher SI (>27,000).

Ethnopharmacological relevance

The plant species screened in this research was recorded by several indigenous medicinal practitioners as antiparasitic, anticancer and/or related activities to the human major organ system.     

Herbal medicines used in the treatment of diabetes mellitus in Arunachal Himalaya, northeast, India

Ethnopharmacological relevance

Medicinal plants have played an important role in treating and preventing a variety of diseases throughout the world. Khampti tribal people living in the far-flung Lohit district of the Eastern Arunachal Himalaya, India still depend on medicinal plants and most of them have a general knowledge of medicinal plants which are used for treating a variety of ailments. This survey was undertaken in Lohit district in order to inventory the medicinal plants used in folk medicine to treat diabetes mellitus.

Materials and methods

Field investigations were conducted in seventeen remote villages of Lohit district starting from April 2002 to May 2004 through interviews among 251 key informants who were selected randomly during our household survey. To elucidate community domains and determine differences in indigenous traditional knowledge of medicinal plants with anti-diabetic efficacy, we repeated our field survey starting from April 2008 to May 2010 with one hundred traditional healers locally called as “Chau ya” in Khampti of Lohit district. “Chau ya” traditional healers who know and use medicinal plants for treating diabetes mellitus were interviewed using a semi-structured questionnaire.

Results

This study reports an ethnobotanical survey of medicinal plants in Lohit district of Arunachal Pradesh reputed for the treatment of diabetes mellitus. Forty-six plant species were identified in the study area to treat diabetes mellitus by the Khamptis “Chau ya” traditional healers. Comparative published literature survey analysis of this study with other ethnobotanical surveys of plants used traditionally in treating diabetes mellitus suggests that eleven plant species make claims of new reports on antidiabetic efficacy. These plant species are Begonia roxburghii, Calamus tenuis, Callicarpa arborea, Cuscuta reflexa, Dillenia indica, Diplazium esculentum, Lectuca gracilis, Millingtonia hortensis, Oxalis griffithii, Saccharum spontaneum, and Solanum viarum. Some of the plants reported in this study have an antidiabetic effect on rodent models but none have sufficient clinical evidence of effectiveness.

Conclusions

The wide variety of medicinal plants that are used to treat diabetes mellitus in this area supports the importance of plants in the primary healthcare system of the rural people of Lohit district of Arunachal Pradesh. The finding of new plant uses in the current study reveals the importance of the documentation of such ethnobotanical knowledge.     

In vitro antigonococcal activity of Coscinium fenestratum stem extract

Ethnopharmacological relevance

Gonorrhea is a sexually transmitted disease (STD), which originates from bacteria, Neisseria gonorrhoeae. It is still one of the major health problems worldwide in both men and women. Many medicinal plants have been recorded in Thai folk medicine for relieving STD but there is no scientific report of these plants for antigonococcal activity.

Aim of the study

This study was conducted to evaluate antigonococcal activity of 22 Thai medicinal plants selected from the plants popularly used in Thai folk medicine for treatment of gonorrhea.

Materials and methods

This study was performed by disc diffusion and agar dilution methods against Neisseria gonorrhoeae. Active compound was investigated by bioautographic assay.

Results

Among the selected plants, Coscinium fenestratum (Gaertn.) Colebr. extract showed the most effective activity against Neisseria gonorrhoeae ATCC 49226 with MIC value of 47.39 μg/ml. Bioautographic assay revealed that berberine was the active compound of Coscinium fenestratum against Neisseria gonorrhoeae. The average MIC values of purified berberine against Neisseria gonorrhoeae ATCC 49226 and 11 clinical isolates were 13.51 and 17.66 μg/ml, respectively while average MIC value of the crude extract of Coscinium fenestratum against all clinical isolates was about 56.39 μg/ml. There was no acute toxicity detected at the dose of 5 g of Coscinium fenestratum crude extract per kilogram.

Conclusions

These results provide theoretical support for ethnopharmacological relevance of antigonococcal activity of Coscinium fenestratum and its active compound.     

Traditionally used Thai medicinal plants: In vitro anti-inflammatory,anticancer and antioxidant activities

Aims of the study

In order to assess traditional Thai claims about the therapeutic potential of medicinal plants and to select plants for future phytochemical research, nine plant species with anti-inflammatory uses were selected from Thai textbooks and assessed for their in vitro anti-inflammatory, antiproliferative and antioxidant activities.

Methods

Nuclear factor-kappaB (NF-κB) inhibitory effects in stably transfected HeLa cells were determined by luciferase assay, and effects on LPS-induced pro-inflammatory mediators prostaglandin E2 (PGE2), interleukin (IL)-6, IL-1β, and tumour necrosis factor (TNF)α in primary monocytes were assessed by ELISA. Cytotoxic activities were examined against HeLa cells, human leukaemia CCRF-CEM cells and the multidrug-resistant CEM/ADR5000 subline using the MTT and XTT tests. However, a redox status has been linked with both inflammation and cancer, antioxidant effects were also assessed using the DPPH, lipid-peroxidation, and Folin-Ciocalteau methods.

Results

Among all the nine species, Gynura pseudochina var. hispida and Oroxylum indicum showed the most promising NF-κB inhibitory effects with the lowest IC₅₀ values (41.96 and 47.45 μg/ml, respectively). Muehlenbeckia platyclada did not inhibit the NF-κB activation but effectively inhibited the release of IL-6, IL-1β and TNF-α with IC₅₀ values ranging between 0.28 and 8.67 μg/ml. Pouzolzia indica was the most cytotoxic against CCRF-CEM cells and the multidrug-resistant CEM/ADR5000 cells (9.75% and 10.48% viability, at 10 μg/ml, respectively). Rhinacanthus nasutus was the most potent cytotoxicity against HeLa cells (IC₅₀ 3.63 μg/ml) and showed specific cytotoxicity against the multidrug-resistant CEM/ADR5000 cells (18.72% viability at 10 μg/ml, p < 0.0001 when compared to its cytotoxicity against CCRF-CEM cells). Moreover, Oroxylum indicum showed a high level of antioxidant activity by inhibiting lipid-peroxidation (IC₅₀ 0.08 μg/ml).

Conclusions

This study provides in vitro evidence for the use of the Thai plants, most importantly Gynura pseudochina var. hispida, Oroxylum indicum and Muehlenbeckia platyclada as Thai anti-inflammatory remedies and these plants are now a priority for further phytochemical research.     

A topoisomerase II poison screen of ethnomedicinal Thai plants using a yeast cell-based assay

Ethnopharmacological relevance

The plants used in this study had previously been identified and used by Thai folk practitioners, who had knowledge of Thai traditional medicine, as alternative treatments for cancer. Investigation into the mechanism of the Topoisomerase II (Top2) poison of these plants may give rise to new drug leads for cancer treatment.

Aim of the study

This study aimed to screen ethnomedicinal plants used in Thai traditional medicine for Top2 poison activity using a yeast cell-based assay and also to validate the traditional uses of these plants by examining the Top2 poison activity.

Materials and methods

Thirty Thai medicinal plants were harvested and identified. Plant methanol extracts were prepared and screened in vitro using a yeast cell-based assay. Mutant yeast strains carrying the top2-1 allele, which encodes a temperature-sensitive topoisomerase, were used to establish the yeast spot test. Strains carrying this mutation grow normally at 25 °C and generally have a wild-type drug sensitivity. These yeast strains are able to grow at 30 °C, but the Top2 activity is greatly reduced at this temperature, which causes the strains to be highly drug resistant to anti-Top2 agents. Cell growth was measured by colony survival after exposure to various concentrations of extracts at different temperatures.

Results

The extracts of six out of thirty ethnomedicinal plants, Curcuma longa, C. zedoaria, Derris scandens, Grangea maderaspatana, Stephania pierrei and S. suberosa, were found to have Top2 poison activity against the yeast cells.

Conclusions

The yeast screening system confirmed the proposed anti-cancer mechanisms of plants used in Thai traditional medicine by traditional doctors.     

In vitro and in vivo anti-inflammatory effect of Rhodomyrtus tomentosa methanol extract

Ethnopharmacological relevance

Rhodomyrtus tomentosa (Aiton) Hassk. is a representative Thai medicinal plant traditionally used in South Asian countries to relieve various inflammatory symptoms. However, no systematic studies on its anti-inflammatory activity and mechanisms have been reported.

Materials and methods

The effect of the methanol extract from the leaves of this plant (Rt-ME) on the production of inflammatory mediators [nitric oxide (NO) and prostaglandin E₂ (PGE₂)] and the molecular mechanism of Rt-ME-mediated inhibition, including target enzymes, were studied with RAW264.7, peritoneal macrophage, and HEK293 cells. Additionally, the in vivo anti-inflammatory activity of this extract was evaluated with mouse gastritis and colitis models.

Results

Rt-ME clearly inhibited the production of NO and PGE₂ in lipopolysaccharide (LPS)-activated RAW264.7 cells and peritoneal macrophages in a dose-dependent manner. According to RT-PCR, immunoblotting and immunoprecipitation analyses and a kinase assay with mRNA, whole cell extract, and nucleus lysates from RAW264.7 cells and mice, it was revealed that Rt-ME was capable of suppressing the activation of both nuclear factor (NF)-κB and activator protein (AP)-1 pathways by directly targeting Syk/Src and IRAK1/IRAK4.

Conclusion

Rt-ME could have anti-inflammatory properties by suppressing Syk/Src/NF-kB and IRAK1/IRAK4/AP-1 pathways and will be further developed as a herbal remedy for preventive and/or curative purposes in various inflammatory diseases.