THE FLAVONOIDS CONTENT IN LEAVES AND FRUITS OF PAPAYA (Carica papaya L.) VAR. CALIFORNIA AND VAR. GANDUL

Papaya has widely used as traditional medicine. Papaya leaves and fruits contain flavonoids that can be used as anti-bacterial, anti-fungal and anti-cancer. Flavonoid is one of secondary metabolite products. Flavonoids are ubiquitous in photosynthesis cells. Flavonoid content can be affected by enzyme which arranged from protein, meanwhile difference formation of gene can affect protein content. The aim of this research was to measure flavonoid content in leaves and fruits of genetical engineering papaya (var. California) and local papaya (var. Gandul). Leaf and fruit samples were dried to make powder. Samples were reflucted with HCl 4 N and extracted with Eter (three times). NaNO3 5 %, AlCl3 10 % and NaOH 1 M were added into samples then analyzed using spectrophotometer (á 454 nm) with quercetin as standard. Data analyzed using T test (p < 0,05). The result showed in California leaves has higher concentration of flavonoid (0,73% w/ w ± 0,05) than Gandul leaves (0,69% w/w ± 0,08). meanwhile California fruits significantly has lower flavonoid concentration (0,59% w/w ± 0,02) than gandul fruits (0,8% w/w ± 0,03). Difference of flavonoids content in leaf and fruit indicates genetic variation affect flavonoids translocation among organ. According to variety California fruits have lower concentration than it’s leaves, meanwhile in gandul variety fruits have higher concentration than leaves. In conclusion, difference of flavonoid content in California variety and gandul variety indicates different ability in flavonoids distribution among individual.Keywords: flavonoids, Carica papaya L. var. California, Carica papaya L. var. Gandul, spectrophotometr

DETECTION OF ALKALOID, FLAVONOID, AND TERPENOID COMPOUNDS IN BREAD (Artocarpus communis Forst.) LEAVES AND PULPS

Leaves of bread tree (Sukun/ Artocarpus communis Forst.) can be used to cure skin diseases, heart, kidneys, and inflamation. The advantageous of bread might be related with its secondary metabolite compounds. The purpose of this research was to detect and compare the content of alkaloid, flavonoid, and terpenoid compounds in bread leaves and pulps using Thin layer Chromatography (TLC) methods. The coloring reagents were dragendorf for alkaloid, sitroborat for flavanoid and sulfate vanillin for terpenoid. Reference compounds used for alkaloid is quinine, rutine for the flavonoid, and terpeniol for terpenoid. The result indicated that alkaloid were detected in chloroform extracts of leaves with Rf score 0.76 and methanol extracts with Rf 0.8, while the alkaloid in pulps weren’t detected. This showed that the leaves were contain of polar and nonpolar alkaloid. Flavonoid were detected in leaves with Rf value 0.89 and in pulps with Rf value 0.9. This result indicated that flavonoid in the leaves and pulps possibly the same compound. Terpenoid were detected in the methanol and chloroform extract of leaves (4 spots) and pulps (2 spots). Moreover, there were spots which have the same Rf value in chloroform and methanol extracts of the leaves (Rf :0,625 and 0,99). In addition, the pulps extracts (chloroform and methanol) have the same Rf values with reference, that was 0,31. It was proved that the pulps contain terpeniol. It can be concluded that alkaloid was only detected in leaves, while flavonoid and terpenoid present in leaves and pulps of bread tree.Keywords : Artocarpus communis Forst., alkaloid, flavonoid, terpenoid, TLC

Apoptosis induction on human breast cancer T47D cell line by extracts of Ancorina sp. [version 2; peer review: 2 approved]

Background: Breast cancer is the second leading cause of death in women. Alternative medicine with high efficacy is needed for breast cancer treatments, for example induction of apoptosis using natural products. It has been found that many natural apoptosis-inducing compounds are isolated from marine sponge. The objective of this study is to analyze the ability of extracts of the sponge Ancorina sp. to induce apoptosis on human breast cancer T47D cell line and find out its mechanism. Methods: T47D cells were treated with crude extracts of methanol, dichloromethane:methanol (1:1) and dichloromethane Ancorina sp. for 24 h, and doxorubicin was used as a positive control. Methods used for this study were MTT assay to examine cell viability and determine IC50 of the three extracts, while the percentage of apoptosis and caspase-3 were investigated by flow cytometry. Results: IC50 values of methanol, dichloromethane:methanol (1:1), and dichloromethane extract were 84.25, 121.45, and 99.85μg/mL respectively. The percentages of apoptotic cells after treatment with methanol, dichloromethane:methanol (1:1), and dichloromethane extracts were 88.68, 27.54 and 53.63% respectively, whereas the percentage of caspase-3 was 77.87, 12.66 and 12.97%, respectively. Conclusions: These results revealed that all extracts of Ancorina sp. have strong or moderate cytotoxicity and have the ability to induce apoptosis on T47D human breast cancer cell line. However, methanol crude extract has high efficacy to induce apoptosis through caspase-3 activation compared to the other extracts. Hence methanol extract warrants further investigation as a natural medicine for human breast cancer

Black Rice Bran Extracts and Fractions Containing Cyanidin 3-glucoside and Peonidin 3-glucoside Induce Apoptosis in Human Cervical Cancer Cells

Anthocyanin of pigmented rice inhibits the growth of cancer cells. The cytotoxicity and apoptosisinducing properties of local black rice (cv Cempo Ireng) extracts and fractions, which contain anthocyaninincluding cyanidin 3-glucoside and peonidin 3-glucoside, on human cervical cancer cell line (HeLa cells) hasbeen evaluated. The pigmented rice bran was extracted and fractionated using methanol-HCl. The MTT testwas performed on HeLa cell cultures to observe the IC50 value. Preparative TLC was performed to obtain thefractions of black rice bran. Cyanidin 3-glucoside and peonidin 3-glucoside were identified in the pigmentedrice bran extract and fractions using UHPLC. Flowcytometry analysis was performed to measure the percentageof apoptotic cells. Our results suggest that the fractions are more toxic than the methanolic crude extract withIC50 values of 85.95 ± 5.56 μg/mL (the lowest one) and 408.13 ± 51.9 μg/mL, respectively. The concentration ofcyanidin 3-glucoside and peonidin 3-glucoside in the methanolic extract were 1.89 and 0.84 μg/mg, respectively.The apoptosis induction by fractions F2 and F4 (52 and 55%) were significantly higher compared to fractionF3 and F5 (30 and 33%) and doxorubicin (21%). Cyanidin 3-glucoside was detected in F4 (0.14 μg/ml) whilepeonidin 3-glucoside in F2 (0.012 μg/ml), however both were not detected in F3 and F5

Anthocyanin, nutrient contents, and antioxidant activity of black rice bran of Oryza sativa L. ‘Cempo Ireng’ from Sleman, Yogyakarta, Indonesia

The chemical contents and health benefits of black rice bran of some rice cultivars have been investigated. However, there has been little research on the ‘Cempo Ireng’ cultivar from Sleman, Yogyakarta. The aim of this present study was to determine the anthocyanin, antioxidant activity, and macro- and micronutrients contents of black rice bran from this local cultivar. The anthocyanin in the black rice bran was extracted using the maceration method with methanol as a solvent. The extract obtained was separated through a preparative thin layer chromatography (TLC) of silica GF254 and a mobile phase composed of n-butanol, acetic acid, and water. Two fractions were collected and analyzed for the anthocyanin content. The preparative TLC spots were separated for further detection and measurement of cyanidin 3-O-glucoside using HPLC followed by LC-MS. The antioxidant activity of the fractions were measured using the DPPH free radical scavenging method. The results showed that the anthocyanin in fraction 1 was identified as cyanidin 3-O-glucoside (66.1 ± 10.6 µg/g). The IC50 of fractions 1 and 2 were 200.96 and 218.36 µg/mL, respectively. Analysis of the macro- and micronutrients revealed that the black rice bran of ‘Cempo Ireng’ had nutrient contents comparable with other rice cultivars. Therefore, this local black rice bran can be used as a source of antioxidants and macro-- and micronutrients

Active Fractions of Black Rice Bran cv Cempo Ireng Inducing Apoptosis and S-phase Cell Cycle Arrest in T47D Breast Cancer Cells

Indonesian black rice cv Cempo Ireng was evaluated for cancer prevention using a T47D cell line model. Methanolic extract of black rice bran (BRB) showed cytotoxicity with an IC50 of 522.13 µg/ml. This result was in contrast to water extract of BRB, which yielded an IC50 of more than 6000 µg/ml. From the methanolic extract of BRB, its 6 fractions were found from the preparative TLC performance, with Rf values ranging from 0 to 0.97. According to the MTT assay of all fractions, fraction 3 (F3) had the lowest IC50 (60.17 ± 1.72µg/ml), while fraction 2 (F2) had an IC50 of 64.3 ± 0.61µg/ml. However, F2 showed the highest induction of apoptosis, i.e. about 75.39 ± 0.43% compared to the T47D cells without treatment (control) (about 8.39 ± 0.16%) and with doxorubicin treatment (about 41.30 ± 0.08%). Furthermore, fraction 5 (F5) not only induced apoptosis but also increased the S-phase arrest percentage (60.36 ± 2.07%), which was significantly higher than for the other treatments. Therefore, each fraction of BRB showed different cytotoxic responses to T47D cells

Effect of Growth Factor In Callus Induction and Bioactive Compounds In Seed Explant of Kaffir Lime (Citrus hystrix DC.)

Our previous study showed that kaffir lime leaf extracts may have anti-cancer properties. However, production of  bioactive compounds is affected by environmental factors. Here, we present a method to control environmental conditions using in vitro culture techniques such as callus induction. Calluses were induced from seed embryo explants of kaffir lime on MS medium with combinations of 2,4-D and BAP at concentrations 1:0.5; 1:1; and 2:1, respectively. Fourty and 60 days-old calluses were extracted using chloroform and ethyl acetate and analyzed by GC-MS. Results showed all combinations of 2,4-D and BAP were able to induce callogenesis from seed embryo explants of kaffir lime with no significant differences of callus initiation time, biomass, morphology and growth rates. However differences were detected in the bioactive compound profiles. In kaffir lime callus, both fatty acids and secondary metabolites were detected. Specifically, in 40 days-old calluses (exponential growth phase) we detected α-pinene and 1.8–cineole in plants treated with 2,4-D: BAP at concentration 1:0.5 and 2:1. In 60 days-old calluses (stationary phase) we detected a number of compounds in plants treated with 2,4-D:BAP at concentrations of 1:0.5 and 2:1, including caryophyllene, linoleoyl chloride, thiogeraniol, stigmasterol, clianosterol, citronellal, neo-isopulegol, citronellol, geraniol, eugenol, cyclopropane, pristane, elemol and farneso

Growth of Kaffir Lime (Citrus hystrix DC) Cell Line Derived from Seed Explant After Yeast Elicitation Using Pure and Technical Grade Yeast

The addition of elicitors in kaffir lime (Citrus hystrix DC.) culture is one of  strategies for obtaining and increasing the production of secondary metabolites.  Saccharomyces cerevisiae is one of the elicitors that can be used to increase secondary metabolites such as terpenoids. However, in its use, the pure cultures of S. cerevisiae are expensive. Therefore, the first objective of this study was to analyze the ability of technical grade (commercial baker’s yeast) to be used as an elicitor and measure the growth of kaffir lime cell line after being elicited by pure and technical grade (commercial baker’s yeast). The second objective is to determine the best time to subculture kaffir lime cell line after elicitation. We observed the morphology and measured the growth curve of pure and technical grade yeast until the 4th subculture generation. Furthermore, we used both grades of yeast for elicitation. Kaffir lime cell suspension was treated with 10 ppm pure grade or 5 ppm and 10 ppm technical grade yeast for 4 days. After elicitation, kaffir lime cell lines were subcultured and their growth was analyzed. The result showed that the morphology and growth curve of technical grade until 4th subculture generations was similar to the pure grade. On the other hand, after elicitation using pure and technical grade yeast and being subcultured, the growth of the elicitated kaffir lime cell line had the same pattern as the control group, but the cell density of the control group was higher than the elicitated group. The initial stationary phase of kaffir lime cell line was on the 17th day which is the best time to subculture. The subculturing process is important to maintain the viability of the kaffir lime cell line.

Identification of BSA B1 Bacteria and Its Potency of Purified Cellulase to Hydrolyze Chlorella zofingiensis

Cellulase has been widely used as biocatalyst in industries. Production of cellulase from microorganisms has many advantages such as short production time and less expense. Our previous study indicated that one of cellulolytic bacteria from digestive tract of milkfish (Chanos chanos), namely BSA B1, showed the highest cellulase activity. The objective of this study was to determine the phylogenetic of BSA B1 strain using 16S rRNA gene sequence. Furthermore, this study also determine the specific activity of purified cellulase from BSA B1 strain and its potency to hydrolyze Chlorella zofingiensis cellulose. Cellulase was purified using ammonium sulphate precipitation, dialysis, and ion exchange chromatography. The purified cellulase was used to hydrolyze cellulose of C. zofingiensis. The result demonstrated that BSA B1 strain was closely related with Bacillus aerius and Bacillus licheniformis. The specific activity of the crude enzyme was 1.543 U mL-1; after dialysis was 4.384 U mL-1; and after chromatography was 7.543 U mL-1. Purified cellulase exhibited activity in hydrolyzed both CMC and C. zofingiensis. Compared to commercial cellulase, purified cellulase had lower activity in hydrolyzed CMC but higher activity in hydrolyzed C. zofingiensis. Ethanol dehydration could potentially increase the reducing sugar yield in cellulose hydrolysis when used appropriately. Morphology of C. zofingiensis cell has changed after incubation with cellulases and ethanol dehydration indicated degradation of cell wall