Indonesian Journal of Biotechnology
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Genetic evaluation of F2 and F3 interspecific hybrids of mung bean (Vigna radiata L. Wilczek) using retrotransposon‐based insertion polymorphism and sequence‐related amplified polymorphism markers
No full textMung bean (Vigna radiata L. Wilczek) is a self‐pollinating and indispensable pulse crop in Indonesia. While low yield productivity is a major concern, genetic improvement is possible through interspecific hybridization. However, interspecific hybridization is relatively infrequent and produces low recombination exchanges, significantly limiting crop breeding efficiency. Thus, a comprehensive study is needed of the selection and genetic diversity evaluation of progenies in advanced generations derived from interspecific hybridization using a specific molecular marker. This study aims to confirm the heterozygosity in the F2 population and assess the genetic diversity in F3 mung bean populations resulting from interspecific hybridization between the mung bean and common bean. We designed the retrotransposon‐based insertion polymorphism (RBIP) marker by identifying the syntenic regions in the flanking sequences of retrotransposon insertion in common bean and mung bean. The RBIP marker can be applied to distinguish the heterozygote progenies from the homozygote progenies. Six combinations of sequence‐related amplified polymorphism (SRAP) primers were used in the genotyping of F3 mung bean progenies. The SRAP marker showed a high degree of polymorphism of up to 100%, while high genetic variation was observed within the population (71%) of mung bean progenies. The F3.4 population had the greatest number of genotypes and displayed the highest number of effective alleles, private alleles, and percentage of polymorphic loci, suggesting the existence of high genetic diversity within this population. These genetic diversity data are exceptionally critical for future genetic research since it has potentially high yield production. The genomic and marker‐assisted selection studies will support the major goals of the mung bean breeding program
Cytotoxic effects of parijoto (Medinilla speciosa Reinw. Ex. Bl.) methanol extract combined with cisplatin on WiDr colon cancer cells through apoptosis induction
No full textParijoto (Medinilla speciosa Reinw. Ex. Bl.) is a medicinal plant with cytotoxic effects on cancer cells in vitro. As only a limited number of studies have reported the effect of parijoto on colon cancer cells, this study initially aimed to measure the total flavonoid levels and potential cytotoxic effects of parijoto methanol extract (PME) through cell viability assays and expression of the apoptotic protein on WiDr colon cancer cells as a model. PME cytotoxic activity was determined by conducting a cytotoxicity test on WiDr colon cancer cells using the MTT assay. The synergistic cytotoxic effects of the PME and cisplatin were tested to obtain the combination index (CI) value. Apoptosis was analyzed by flow cytometry, and the apoptotic protein expression was observed by immunocytochemical tests. Furthermore, quercetin as a major flavonoid in PME was measured using a UV–Vis spectrophotometer. The results showed that PME had a moderate cytotoxic activity with an IC50 of 198.64±1.6 µg/mL, whereas the IC50 of cisplatin was 2.34±0.7 µg/mL. The PME with cisplatin combination test showed a strong synergistic effect with a CI value of <1 (0.1‐0.4). The combination showed increased apoptosis properties compared to PME treatment alone. In addition, immunocytochemistry showed that PME alone or in combination with cisplatin increased the pro‐apoptosis proteins (p53 and caspase‐9) and suppressed Bcl‐2 expression. Moreover, the cell viability value increased as the PME concentration decreased. The administration of PME led to changes in cell morphology, lower cell density, and a decreasing number of living cells. Therefore, the combination of PME and cisplatin had a strong synergistic effect in inducing apoptosis
Anti‐proliferative effects of pentagamaboronon‐0‐sorbitol on HER2‐overexpressing breast cancer cells
No full textHER2‐positive breast cancer is an aggressive form of the disease that is associated with poor prognosis and chemo‐resistance. As such, investigation continues into the development of a new HER2‐targeted drug for breast cancer. This study investigated the anti‐proliferative activities of pentagamaboronon‐0‐sorbitol (PGB‐0‐So) in HER2‐overexpressing breast cancer (MCF‐7/HER2) cells. The cytotoxicity of PGB‐0‐So was assessed via MTT assay. Flow cytometry with propidium iodide and annexin‐V‐FITC staining was conducted to investigate the mechanism of PGB‐0‐So in inhibiting the proliferation of MCF‐7/HER2 cells. Finally, FACS analysis with 2′,7′–dichlorofluorescin diacetate staining was performed to examine intracellular ROS production. PGB‐0‐So exerted cytotoxicity towards MCF‐7/HER2 breast cancer cells with an IC50 value of 36 μM. PGB‐0‐So induced S‐phase arrest and apoptosis in MCF‐7/HER2 cells. Moreover, PGB‐0‐So could increase intracellular ROS production in MCF‐7/HER2 cells. PGB‐0‐So exerted anti‐proliferative activity towards MCF‐7/HER2 cells. This compound may be developed as a chemotherapeutic agent against HER2‐overexpressing breast cancer
The efficacy of captopril and 5-fluorouracil combination in the proliferation and collagen deposition of keloid fibroblast
No full textKeloid is a benign fibroproliferative tissue growth that exceeds the initial wound margins. Captopril has been tested in vitro to reduce fibroblast proliferation and collagen deposition; thus, it has potential for use in the treatment of keloids. Meanwhile, 5‐fluorouracil (5‐FU) has already been used in keloid management. This study aimed to determine the efficacy of the combination of captopril and 5‐FU in keloid fibroblast cultures. Keloid tissues were cultured up to passages 4–7. The study consisted of a control group, captopril in various concentrations (10‐2, 10‐3, 10‐4, and 10‐5 mol/L), 5‐FU 1 mg/mL and a combination of captopril at various concentrations with 5‐FU 1 mg/mL. After 144 hours of treatment, fibroblast proliferation and collagen deposition were measured. The study showed a significant decrease in the mean index of fibroblast proliferation and collagen deposition in the group receiving captopril in various concentrations (10‐2, 10‐3, 10‐4, and 10‐5 mol/L) and the 5‐FU group against the control group (p<0.05). In the combined‐dose group, captopril at a concentration of 10‐2 mol/L and 5‐FU showed a significant reduction in fibroblast proliferation and collagen deposition compared to the 5‐FU group and the captopril at the same dose (p<0.05). In conclusion, the combination of captopril 10‐2 mol/L and 5‐FU 1 mg/mL is better at reducing fibroblast proliferation and collagen deposition in keloid fibroblast cultures than captopril or 5‐FU as a single therapeutic agent
Network pharmacology of black cumin (Nigella sativa L.) as a candidate of OMAI in colorectal cancer: in silico study
No full textColorectal cancer is the third most common cancer globally and the second leading cause of cancer‐related deaths. The management of colorectal cancer requires consideration of various factors due to the non‐selectivity of drugs, meaning that highly effective treatment with lower side effects is needed. Black cumin (Nigella sativa L.) contains thymoquinone and various other metabolites with potential as anticancer effects. The involvement of various genes and the difficulty of drug development have led to a ashift in the drug development paradigm towards plant‐based medicine that is both multicomponent and synergistic in supporting the resulting pharmacological effects. Network pharmacology can predict the synergistic effect of a multicomponent approach. This study aimed to predict the network pharmacology of black cumin as a candidate for OMAI (“Obat Modern Asli Indonesia”, Indonesian‐origin modern medicine) in colorectal cancer. This research was an in silico study using various ethnobotanical databases and software. The results show that seven metabolites in black cumin are correlated with ten surface receptor proteins, 30 intracellular proteins, and mechanisms involving six colorectal cancer signaling pathways. This result indicates that Nigella sativa L. has potential in OMAI and can be a reference for the development of cancer treatment, especially for colorectal cancer
The relationship between morpho‐physiological changes and expression of transcription factors in NTT local rice cultivars as a response to drought stress
No full textResponse by plants to drought occurs through a series of mechanisms that involve transcription regulation. This research was conducted to study transcription factors (TF) and physiological changes in the drought response of local rice cultivars from East Nusa Tenggara (Nusa Tenggara Timur, NTT) during drought stress. Using three NTT local rice cultivars (Boawae Seratus Malam (BSM), Gogo Jak (GJ), and Kisol Manggarai (KM)) and the fraction of transpirable soil water (FTSW) method with two treatment levels, FTSW 1 (control) and FTSW 0.2 (severe stress), we analyzed the TF expression of OsDREB1A, OsDREB2A, OsWRKY45, and OsNAC6. Based on the result, the highest level of TF expression occurred in the BSM, followed by the GJ and KM cultivars. Analysis of physiological characteristics showed an association between TF expression levels and physiological response, with the BSM cultivar showing high pigment levels, high proline content, and lower H2O2 levels. A linkage was also found in relation to water conservation, as indicated by the higher relative water content and cell membrane stability index in the BSM cultivar in contrast to lower electronic leakage and malondialdehyde percentage when exposed to drought. Based on the results, it can be concluded that the BSM cultivar can be considered as a drought‐tolerant local cultivar according to morpho‐physiological analysis. In this study, all NTT local rice cultivars showed a subtle upregulation of stress‐responsive transcription factors OsDREB1A, OsDREB2A, OsWRKY45, and OsNAC6 as responses to drought stress
Effects of different parameters on cellulase production by Trichoderma harzianum TF2 using solid‐state fermentation (SSF)
No full textSolid‐state fermentation is one of the easiest and cheapest methods for producing microbial bioactive com‐ pounds. Trichoderma harzianum has long been recognised as one of the potential fungi for this purpose. Trichoderma sp. were isolated from banana rhizosphere using the soil dilution method and later screened for their ability to produce cellulases using filter paper activity (FPase) and the carboxylmethyl cellulase (CMCase) test. Trichoderma sp. were also subjected to one factor change at a time to determine the effects of different parameters on cellulase production. It was observed that T. harzianum TF2 showed the ability to produce higher cellulase activity when wheat bran was used as the substrate. The results showed that 38.5 U/g of cellulase was produced with the use of wheat bran coupled with an incubation temperature of 28 °C and moisture content of 60%. T. harzianum TF2 showed good potential for use as a culture for cellulase production in this study due to its higher cellulase production under solid‐state fermentation, with the possibility of its application to industry
Anti‐diabetic effect of andrographolide from Sambiloto herbs (Andrographis paniculata (Burm.f.) Nees) through the expression of PPARγ and GLUT‐4 in adipocytes
No full textAndrographolide has been shown to have a pharmacological effect as an antidiabetic. Nevertheless, the comprehensive mechanism of action has yet to be determined. Andrographolide is a primary component of the sambiloto herb (Andrographis paniculata (Burm.f.) Nees), in which a simple isolation process can obtain high yields. This study aimed to explain the anti‐diabetic effect of andrographolide compared to pioglitazone (a positive control) on glucose uptake by measuring the expression levels of peroxisome proliferator‐activated receptor gamma (PPARγ) and glucose transporter type 4 (GLUT‐4) genes in 3T3‐LI mouse adipocytes as an in vitro model. The differentiation of mature adipocytes from 3T3‐L1 fibroblasts was induced with 3‐isobutyl‐1‐methylxanthine, dexamethasone, and insulin. Andrographolide was provided through direct isolation from A. paniculata herbs. The gene expression was detected using the reverse transcription‐polymerase chain reaction (RT‐PCR). Pioglitazone and andrographolide significantly increased glucose uptake capability. Andrographolide was able to increase the mRNA levels of PPARγ and GLUT‐4 compared to pioglitazone with the best concentration at 5.6 µM. In conclusion, andrographolide can improve glucose uptake by increasing mRNA levels of PPARγ and GLUT‐4 that encodes protein, which are key factors for glucose homeostasis. Therefore, this finding further establishes the potency of andrographolide from A. paniculata as an antidiabetic
Early development of self‐administered COVID‐19 rapid test based on nucleocapsid detection in saliva sample
No full textMore than 6,000,000 people have died due to the coronavirus (COVID‐19) pandemic. This disease spread quickly due to its highly contagious nature. The SARS‐CoV‐2 virus that causes the disease can be transmitted through saliva droplets secreted by infected people at a distance of less than 1 m. As a result, saliva has been accepted as an alternative specimen for COVID‐19 detection by the Centers for Disease Control and Prevention (CDC). Furthermore, WHO recommended the use of rapid antigen tests based on lateral flow immunoassay when reverse transcription‐polymerase chain reaction (RT‐PCR) is not available. We developed a saliva‐based rapid antigen test by optimizing the antibody concentration and optimum pH for the conjugation of antibody and gold nanoparticles. We found that the best running buffer formulation consisted of 75 mM sodium phosphate buffer, 1% NaCl, 1% Triton X‐100, 0.5% N‐acetyl‐L‐cysteine, and 0.02% sodium azide. The addition of a mucolytic agent in the buffer can reduce the viscosity of saliva, thus improving sensitivity. The rapid test developed detected the lowest concentration of nucleocapsid protein at 0.1 μg/mL. Our study revealed 100% specificity against negative COVID‐19 saliva and no cross‐reaction with avian influenza virus hemagglutinin
Carrot hairy roots (Daucus carota L.) characterisation and optimisation for high β‐carotene extraction
No full textHairy roots are widely known as a biological system for the production of highly diverse biomolecules. β‐carotene – a precursor for vitamin A – is known to be an anti‐oxidant and anti‐gastric cancer and protection agent against cardiovascular disease, heart disease and stroke. β‐carotene has been chemically synthesised and consumed by humans. However, the chemical process often produces a by‐product that may be harmful to human health. Therefore, this study established a protocol to induce hairy roots (HRs) from a Vietnamese carrot variety and produce natural β‐carotene. The Rhizobium rhizogenes ATCC15834 harbouring Ri plasmid and a Vietnamese carrot variety were used as materials for genetic transformation and HR induction studies. The result showed that approximately 50 HR lines were obtained. Culture medium supplemented with 30 mg/L of sucrose that gave the highest biomass of HR was shown in carrot HR line 30, which had a doubling time of 6.5 days. The highest content of β‐carotene extraction, at 128 mg/100g hairy roots, was achieved with a ratio volume (v/v) of 2‐propanol and plant samples of 20:1, followed by two hours’ incubation with 2‐propanol at 60 °C. Our study reveals a highly efficient protocol for Vietnamese carrot hairy root establishment and multiplication. A very efficient protocol for β‐carotene extraction from the hairy root was established to produce natural β‐carotene that achieves the same β‐carotene quantity as that produced by normal roots. This study provides new insight into the production of high‐content and natural β‐carotene for therapeutic application