Cytotoxicity and Toxicological Studies of Artocarpus altilis Extracts, Inducing Apoptosis and Cell Cycle Arrest via CASPASE-3 and CASPASE-8 Pathways against Human Breast MCF-7 Cells.

BACKGROUND: In recent biomedical research, the area of cancer and infectious diseases occupies a leading position in the utilization of medicinal plants as a source of drug discovery. Malaysia has a diversity and a large number of underutilized fruits that are rich in phenolic compounds. Artoarpus altilis is considered an underutilized fruit that is rich in phenolic compounds. Methanol extracts of A. altilis have been previously found to contain a high content of antioxidant phytochemicals. OBJECTIVE: The purpose of the study was to evaluate the cytotoxicity and toxicological effect of methanol fruit extracts against MCF-7 cells. To determine the least concentration that might kill or suppress the growth of the cancer cells was in a concentration-dependent manner. METHODS: The variation in the cytotoxic activity among the extracts was indicated by determining the IC50 of each extract against cells at 72 h. The IC50 of the samples was measured using a trypan blue exclusion assay. The methanol extract of the pulp part showed the least inhibition concentration of 15.40±0.91 µg/mL on MCF-7 cells. In the study, the molecular mechanism of methanol extracts-induced apoptosis and cell cycle arrested in human cancer cells were investigated in a time-dependent-manner by using flow cytometry. The treated cells were stained with nexin to detect early and late apoptosis and with propidium iodide (PI) for cell cycle arreste associated with the DNA fragmentation; various cell arrests occurred at G1/S, S, and G2/M phases. Lastly, the gene expression analysis by RT-qPCR method was carried out by analyzing the expression of the gene of interest for the quantification of mRNA levels. RESULTS: Results after cells were treated with IC50 were revealed by upregulating anti-apoptotic genes/downregulated of pro-apoptotic BCL-2 gene expressions triggered the treated cells into CASPASE- 3, intrinsic and extrinsic pathways. CONCLUSION: These findings suggest that the methanol extracts of three parts of A. altilis fruit have potential anticancer activity against MCF-7 cells mainly the pulp part of the fruit.

Determination toxic effects of Hystrix Brachyura Bezoar extracts using cancer cell lines and embryo zebrafish (Danio rerio) models and identification of active principles through GC-MS analysis.

ETHNOPHARMACOLOGICAL RELEVANCE: Porcupine bezoar (PB) is used as folk medicine for various medical conditions including cancer treatment in Malaysia. However, its toxicity profile has never been thoroughly ascertained to confirm its safe nature as an efficacious traditional medicine in the treatment of cancer as well as other ailments. AIM OF THE STUDY: This study was aimed to reveal three different PBs' aqueous extracts(viz. PB-A, PB-B, PB-C) chemical constituent's profile using GC-MS analysis, anticancer property on A375, HeLa and MCF7 cancer cells, toxicity profile on zebrafish embryo morphology, EC50, LC50 and teratogenicity index. MATERIALS AND METHODS: PBs' extracts characterization was performed through GC-MS analysis, in vitro anticancer effect was carried out on A375, HeLa and MCF7 cancer cell lines and finally and toxicity properties on three different PBs aqueous extracts (viz. PB-A, PB-B, PB-C) were determined using zebrafish embryo model. RESULTS: The GC-MS analysis revealed 10 similar compounds in all PBs' extracts. Dilauryl thiodipropionate was found to be a major compound in all PBs' extracts followed by tetradecanoic acid. An in vitro anticancer study revealed PB extracts exerted median inhibition concentration (IC50) <50 µg/mL, on cancer cells viz. A375, HeLa and MCF7 with no significant toxicity on normal cells viz. NHDF cells. In vivo toxicity of PBs extracts found affecting tail detachment, hatching, craniofacial, brain morphology, soft tissues, edema, spinal, somites, notochord and cardiovascular system (brachycardia, disruption of blood circulation) deformities. The LC50 and EC50 demonstrated PB extracts effect as dose and time dependent with median concentration <150.0 µg/mL. Additionally, teratogenicity index (TI) viz. >1.0 revealed teratogenic property for PB extracts. CONCLUSIONS: The findings revealed that all three PBs aqueous extracts possessed anticancer activity and exhibited significant toxicological effects on zebrafish embryos with high teratogenicity index. Hence, its use as an anticancer agent requires further investigation and medical attentions to determine its safe dose.

Anticancer activity of grassy Hystrix brachyura bezoar and its mechanisms of action: An in vitro and in vivo based study.

Porcupine bezoar (PB) is a calcified undigested material generally found in porcupine's (Hystrix brachyura) gastrointestinal tract. The bezoar is traditionally used in South East Asia and Europe for the treatment of cancer, poisoning, dengue, typhoid, etc. However, limited scientific studies have been performed to verify its anticancer potential to substantiate its traditional claims in the treatment of cancers. Hence, this study was aimed at investigating the in vitro and in vivo anticancer properties of two grassy PB aqueous extract (PB-A and PB-B) using A375 cancer cell line and zebrafish model, respectively. This paper presents the first report on in vitro A375 cell viability assay, apoptosis assay, cell cycle arrest assay, migration assay, invasion assay, qPCR experimental assay and in vivo anti-angiogenesis assay using the grassy PBs. Experimental findings revealed IC50 value are 26.59 ± 1.37 µg/mL and 30.12 ± 3.25 µg/mL for PB-A and PB-B respectively. PBs showed anti-proliferative activity with no significant cytotoxic effect on normal human dermal fibroblast (NHDF). PBs were also found to induce apoptosis via intrinsic pathway and arrest cell cycle at G2/M phase. Additionally, the findings indicated its ability to debilitate migration and invasion of A375 cells. Further evaluation using embryo zebrafish model revealed LC50 = 450.0 ± 2.50 µg/mL and 58.7 ± 5.0 µg/mL for PB-A and PB-B which also exerted anti-angiogenesis effect in zebrafish. Moreover, stearic acid, ursodeoxycholic acid and pregnenolone were identified as possible metabolites that might contribute to the anticancer effect of the both PBs. Overall, this study demonstrated that PB-A and PB-B possess potential in vitro and in vivo anticancer effects which are elicited through selective cytotoxic effect, induction of apoptosis, inhibition of migration and invasion and anti-angiogenesis. This study provides scientific evidence that the porcupine bezoar do possess anti-cancer efficacy and further justifies its traditional utility. However, more experiments with higher vertebrae models are still warranted to validate its traditional claims as an anticancer agent.

Occurrence of Intestinal Parasitic Contamination in Select Consumed Local Raw Vegetables and Fruits in Kuantan, Pahang.

Intestinal parasitic infections are one of the most common causes of human diseases that result in serious health and economic issues in many developing and developed countries. Raw vegetables and fruits play an important role in transmitting parasites to humans. Hence, the aim of this study was to investigate the parasitological contamination of select commonly consumed local leafy vegetables and fruits in Kuantan, Malaysia. One kilogram of locally consumed raw vegetables and fruits were collected randomly from the Kuantan wet market (Pasar Tani) during the monsoon season (November 2014-January 2015) and the dry season (February 2015-April 2015). A standard wet mount procedure and modified Ziehl-Neelsen staining were used for the detection of parasites. In the present study, the examination of vegetables revealed five different parasite species. The vegetable samples collected from Kuantan's wet market were positive for both helminthes and protozoa. However, the fruits samples were negative for parasitic contamination. Pegaga was the most contaminated leafy vegetable in this study, and Strongyloides was the parasite found most frequently. Furthermore, there was a high diversity in the type of parasites observed during the dry season compared to the monsoon season. Therefore, further action should be taken to reduce the occurrence of parasitic contamination in vegetables by implementing the principles of good agricultural practice and improving water treatment efficacy.

Identification of Cryptosporidium from Dairy Cattle in Pahang, Malaysia.

Cryptosporidium, a protozoan parasite, can cause cryptosporidiosis which is a gastrointestinal disease that can infect humans and livestock. Cattle are the most common livestock that can be infected with this protozoan. This study was carried out to determine the prevalence of Cryptosporidium infection in cattle in Kuantan, Pahang, Malaysia and to find out the association between the occurrence of infection and 3 different ages of cattle (calves less than 1 year, yearling, and adult cattle). The samples were processed by using formol-ether concentration technique and stained by modified Ziehl Neelsen. The results showed that 15.9% (24/151) of cattle were positive for Cryptosporidium oocysts. The occurrence of Cryptosporidium in calves less than 1 year was the highest with the percentage of 20.0% (11/55) followed by yearling and adult cattle, with the percentage occurrence of 15.6 % (7/45) and 11.8% (6/51), respectively. There was no significant association between the occurrence and age of cattle and presence of diarrhea. Good management practices and proper hygiene management must be taken in order to reduce the infection. It is highly important to control the infection since infected cattle may serve as potential reservoirs of the infection to other animals and humans, especially animal handlers.

Antioxidant activity and phenolic profile of various morphological parts of underutilised Baccaurea angulata fruit.

Baccaurea angulata is an underutilised tropical fruit of Borneo Island of Malaysia. The effect of solvents was examined on yield, total phenolic (TPC), total flavonoids (TFC), total carotene content (TCC), free radical scavenging activities and lipid peroxidation inhibition activities. The results indicated that the pulp (edible portion) had the highest yield, while methanol extracts were significantly (p < 0.01) found to contain higher TPC, TFC and TCC than phosphate buffered saline (PBS) extracts for all the fruits parts. The methanol extracts also showed remarkable antiradical activity and significant lipid peroxidation inhibition activities, with their IC50 results highly comparable to that of commercial blueberry. The variations in the results among the extracts suggest different interactions, such as negative or antagonistic (interference), additive and synergistic effect interactions. The study indicated that B. angulata like other underutilised tropical fruits contained remarkable primary antioxidants. Thus, the fruit has the potential to be sources of antioxidant components.