Abstract Piper sarmentosum is a herbaceous shrub with numerous pharmacological benefits. However, the presence of two toxic phenylpropanoids (α- and β-asarone) limits the medicinal usage of the plant. In this study, the extraction of three asarone isomers, namely α-, β-, and -asarone was optimised using supercritical carbon dioxide extraction (SC-CO2) combined with Box-Behnken experimental design. Comparison of asarone contents in different conventional solvent extracts of P. sarmentosum leaves prior to and after SC-CO2 extraction was performed. The SC-CO2 method successfully maximised the extraction of α-, β-, and ɣ-asarone at P = 81.16 bar, T = 50.11°C, and t = 80.90 min, yielding 13.91% α-asarone, 3.43% β-asarone, and 14.95% ɣ-asarone. The SC-CO2 residue of the leaves re-extracted with conventional solvents showed a significant decrease of asarone ranging from 45% to 100% (p<0.001) compared to their counterparts without SC-CO2 treatment. α-, β-, and ɣ-asarone were completely removed in the ethanol extract of the residue. These findings suggested that the optimised SC-CO2 extraction parameters may serve as a quick treatment step for the selective removal of asarone from P. sarmentosum to develop safer extracts for the food and nutraceutical industries applications.

ABSTRACT Orthosiphon aristatus (Blume) Miq., Lamiaceae, is a medicinal plant from Southeast Asia. Pharmacological effects of O. aristatus are attributed to the presence of lipophilic flavones. This study aimed to carry out accelerated stability studies on O. aristatus ethanolic extract and its nano liposomes. The extracts were exposed to four different temperatures at 30, 40, 50 and 60 °C for 6 months. The samples were analyzed at 0, 1, 2, 3, 4, 5 and 6 months by high performance liquid chromatography using rosmarinic acid, 3′-hydroxy-5,6,7,4′-tetramethoxyflavone, sinensetin and eupatorin as markers. Different chemical kinetic parameters of the markers were evaluated by Arrhenius equation to predict shelf life (t90) at different storage conditions and at room temperature. Moreover, the stability of O. aristatus ethanolic extract and O. aristatus nano liposomes were analyzes by chemical fingerprinting using FTIR spectroscopy, principal component analysis and hierarchical clustering analysis. The degradation of markers in both O. aristatus ethanolic extract and O. aristatus nano liposomes followed the first order degradation reaction (dependening on their initial concentration). The loss of marker compounds in O. aristatus ethanolic extract, stored at 30, 40, 50 and 60 °C for six months were up to 25, 52, 72 and 89% for all compounds, respectively. However, in O. aristatus nano liposomes 16, 71, 85 and 100% of compounds were lost during 6 months of storage at 30, 40, 50 and 60 °C, respectively. Therefore, the markers in O. aristatus nano liposomes seems to be more stable at a temperature below 30 °C compared to O. aristatus ethanolic extract. However, markers present in O. aristatus ethanolic extract are more stable at a higher temperature (above 30 °C). principal component analysis or hierarchical clustering analysis analyses were applied to the FTIR results in order to demonstrate the discrimination between extracts based on the storage conditions. The results show that the functional group of the components in the extracts and their chemistry relationship is influenced by the temperature setup indicating the extracts are not stable during the storage conditions.

OBJECTIVE: Ficus deltoidea leaves have been used in traditional medicine in Southeast Asia to treat diabetes, inflammation, diarrhea, and infections. The present study was conducted to assess the genotoxicity and acute and subchronic toxicity of a standardized methanol extract of F. deltoidea leaves. METHODS: Sprague Dawley rats were orally treated with five different single doses of the extract and screened for signs of toxicity for two weeks after administration. In the subchronic study, three different doses of the extract were administered for 28 days. Mortality, clinical signs, body weight changes, hematological and biochemical parameters, gross findings, organ weights, and histological parameters were monitored during the study. Genotoxicity was assessed using the Ames test with the TA98 and TA100 Salmonella typhimurium strains. Phytochemical standardization was performed using a colorimeter and high-performance liquid chromatography. Heavy metal detection was performed using an atomic absorption spectrometer. RESULTS: The acute toxicity study showed that the LD50 of the extract was greater than 5000 mg/kg. In the subchronic toxicity study, there were no significant adverse effects on food consumption, body weight, organ weights, mortality, clinical chemistry, hematology, gross pathology, or histopathology. However, a dose-dependent increase in the serum urea level was observed. The Ames test revealed that the extract did not have any potential to induce gene mutations in S. typhimurium, either in the presence or absence of S9 activation. Phytochemical analysis of the extract revealed high contents of phenolics, flavonoids, and tannins. High-performance liquid chromatography analysis revealed high levels of vitexin and isovitexin in the extract, and the levels of heavy metals were below the toxic levels. CONCLUSION: The no-observed adverse effect level of F. deltoidea in rats was determined to be 2500 mg/kg.

Syzygium aromaticum (L.) Merr. & L.M. Perry, Myrtaceae, is an evergreen tree with anticarcinogenic, antimutagenic, aphrodisiac, antimicrobial, antioxidant and antiinflammatory properties. This study aims to investigate the anti-breast cancer effect of extracts from leaves, stem and bark of S. aromaticum and to develop a method for preparation of betulinic acid fraction from the leaves. Betulinic acid, ursolic acid and oleanolic acid contents of the extracts were determined by HPLC. A betulinic acid fraction was prepared by simple crystallization of leaves extract and was characterized by HPLC and mass analysis. Anti-breast cancer effects were studied on MCF-7 and MDA-MB-231 cells. The extracts were found to contain high levels of betulinic acid particularly the leaves extract which contained 17% wt/wt. The betulinic acid fraction contains 75% betulinic acid. Cytotoxicity testing reveals high and selective cytotoxic effect of the stem extract on MCF-7 cells with IC50 33±1.6 µg/mL. Cytotoxic effect of the stem extract was due to activation of apoptotic machinery of cell death. Combination studies of stem extract with tamoxifen reveals antagonistic effect at high concentration of tamoxifen and enhancement effect at low concentration. The selective cytotoxicity of the stem extract of S. aromaticum on MCF-7 is not due to betulinic acid but due to other constituents yet to be discovered.

This study aimed to investigate the antitumorigenicity of xanthones-rich extract from Garcinia mangostana L., Clusiaceae, fruit rinds which was obtained by a simple recrystallization of 75% ethanolic extract. α-Mangostin content of the extract was determined qualitatively by TLC and quantitatively by HPLC, and total xanthones content was quantified by UV spectrophotometry. The extract was evaluated for cytotoxicity, apoptosis and antitumorigenicity on HCT 116 human colorectal carcinoma cells. α-Mangostin was found to be the main constituent of the extract which was 71.2±0.1%, and the total xanthones content was 95±4.8% (wt/wt). The extract showed potent dose dependent cytotoxicity with IC50 value 9.2 μg/mL. Apoptosis studies revealed activation of caspases 3 and 7, DNA fragmentation, chromatin condensation and loss of mitochondrial membrane potential. Studies on cell migration and colony formation indicate reduced cell migration ability and clonogenicity of treated HCT 116 cells at sub-inhibitory concentrations. Taken together, the cytotoxic effect of the xanthones extract is mediated through the mitochondrial pathway of apoptosis. The reduced cell migration and clonogenicity of HCT 116 cells might prevent both primary and metastatic tumor growth in vivo which will be the topic of our future work using the metastatic orthotopic colon cancer model.