PENGUJIAN TOKSISITAS IN VITRO EKSTRAK DAN FRAKSI DARI DAUN JAMBU AIR (SYZYGIUM AQUEUM) DAN KULIT BUAH DELIMA (PUNICA GRANATUM) TERHADAP SEL VERO

Daun jambu air (Syzygium aqueum) dan kulit buah delima (Punica granatum) merupakan tanaman yang pada umumnya terdapat di Indonesia. Kandungan flavonoid pada daun jambu air dan kulit buah delima diketahui memiliki efek antioksidan yang baik untuk tubuh sehingga tanaman ini banyak digunakan untuk pengobatan dan pencegahan beberapa penyakit. Secara tradisional daun jambu air dan kulit buah delima biasa digunakan sebagai antimikroba, antidiabetes, dan pengobatan diare. Penelitian ini bertujuan untuk menentukan toksisitas ekstrak dan fraksi dari daun jambu air dan kulit buah delima secara in vitro terhadap sel Vero. Serbuk simplisia daun jambu air diekstraksi bertingkat dengan metode maserasi, sedangkan serbuk simplisia kulit buah delima diekstraksi bertingkat menggunakan metode refluks. Keduanya diekstraksi bertingkat dengan pelarut n-heksana, etil asetat, dan etanol. Ekstrak dipantau menggunakan kromatografi lapis tipis (KLT). Ekstrak etanol dari daun jambu air dan kulit buah delima difraksinasi dengan metode ekstraksi cair-cair (ECC) menggunakan pelarut n-heksan dan etil asetat. Fraksi dipantau menggunakan kromatografi lapis tipis (KLT). Ekstrak dan fraksi diuji toksisitasnya secara in vitro terhadap sel Vero menggunakan reagen alamar blue. Ekstrak dan fraksi daun jambu air dan kulit buah delima pada konsentrasi uji antara 2 µg/ml sampai dengan 1024 µg/ml bersifat tidak toksik terhadap sel Vero. Berdasarkan penelitian, dapat disimpulkan bahwa ekstrak n-heksana, etil asetat, dan etanol tidak toksik terhadap sel Vero. Fraksi n-heksana, etil asetat, dan air dari tidak toksik terhadap sel Vero.  Kata kunci: Alamar blue, ekstrak, fraksi, Punica granatum, sel Vero, Syzygium aqueum. Syzygium aqueum and Punica granatum are plants commonly found in Indonesia. Flavonoids from Syzygium aqueum leaves and Punica granatum peels are known to have antioxidant effect for the human body. Because of that these plants widely used for treatment several diseases. Traditionally, Syzygium aqueum leaves and Punica granatum peels are commonly used as antibiotic, antidiabetic, and treatment for diarrhea. This study aim to determine in vitro toxicity effect of Syzygium aqueum and Punica granatum extracts and fractions using Vero cell lines. Crude drug of Syzygium aqueum was extracted by maceration, while crude drug of Punica granatum was extracted by reflux. Both crude drugs were extracted using continuous extraction method and solvents with increasing polarity which were n-hexane, ethyl acetate, and ethanol. Extracts were monitored by thin layer chromatography (TLC). The ethanol extract from Syzygium aqueum and Punica granatum were fractionated by liquid-liquid extraction using n-hexane, and ethyl acetate as solvents. Fractions were monitored by thin layer chromatography (TLC). Toxicity effect of Syzygium aqueum and Punica granatum extracts and fractions on Vero cells were evaluated using Alamar blue. Extracts and fractions of Syzygium aqueum leaves and Punica granatum had no toxicity effect against Vero cell at concentrations between 2 µg/ml-1024 µg/ml. Based on our findings all of extracts and fractions had no toxicity effect against Vero cell lines.Keyword: Alamar blue, extract, fraction, Punica granatum, Syzygium aqueum, Vero cell

Overproduction, Purification and Refolding of codon-optimized Hepatitis B Virus X Protein Subgenotype B3 in Escherichia coli BL21(DE3)

Hepatitis B virus (HBV) infects human and causes chronic liver infection, leading to liver cirrhosis and hepatocellular carcinoma. HBV X (Hbx) protein is known to interact with tumor suppressor protein p53 and block its translocation into the nucleus. This study outlines the overproduction of Hbx protein from HBV subgenotype B3 in Escherichia coli BL21(DE3), including its purification and refolding. The gene encoding Hbx was first codon-optimized and inserted into pET16b. The recombinant plasmid was then transformed into E. coli BL21(DE3) as an expression host. Optimization of Hbx expression was performed with variation of IPTG concentration and overproduction temperature. The results showed that Hbx protein was optimally induced by 0.075 mM IPTG and overproduction of Hbx at 17, 25, and 37°C exhibited no difference in protein level and location. The optimal refolding of Hbx was obtained using 0.1 M arginine prior to elution from Nickel column using 100 mM imidazole and 0.25 M arginine. Hbx migrates differently in SDS-PAGE reducing and non-reducing, while the melting curve pattern in TSA analysis changed after the refolding step. Essentially, this purified Hbx protein could potentially be used for interaction study with p53 and the inhibitor candidate of the protein

LP-110 development of GSTA1, CYP2C19, and CYP2B6 gene polymorphism detection methods on the response of cyclophosphamide therapy for lupus nephritis patients

Background Cyclophosphamide, an immunosuppressant in patients with lupus nephritis, aims to prevent recurrence and is a steroid-sparing agent. The clinical response (efficacy and toxicity) of lupus nephritis patients receiving cyclophosphamide varied.1 Cyclophosphamide is a pro-drug metabolized by hydroxylation via the CYP2C19 and CYP2B6 enzymes and detoxification via the glutathione-S-transferase (GST) enzyme.2 The most common type of mutation that occurs is single nucleotide polymorphism (SNP). Several SNPs from previous studies associated with cyclophosphamide response, metabolism, and toxicity in patients with lupus nephritis, namely rs3957356 in the GSTA1, rs4244285 in the CYP2C19, rs7254579 and rs4802101 in the CYP2B6.3 Therefore, detecting and screening SNP genotypes in lupus nephritis patients can help analyze cyclophosphamide response variation. This study aims to develop a method for detecting the genotypes of the four target SNPs and several surrounding SNPs using PCR-Sanger sequencing. Methods The gene polymorphism analysis method was optimized before taking patient samples at the hospital. PCR and Sanger sequencing methods are used for gene polymorphism analysis because they produce chromatograms with target amplicon base lengths and several SNPs that can be analyzed simultaneously. Results The analysis results of the four target SNPs of the GSTA1, CYP2C19, and CYP2B6 (rs3957356, rs4244285, rs7254579, and rs4802101) showed one synonymous SNP and three SNPs (regulatory and intergenic). We have developed an SNP detection assay using four fragments from the GSTA1, CYP2C19, and CYP2B6 that can detect 15 SNPs simultaneously (figure 1). In addition, this method succeeded in distinguishing wild-type, heterozygous and homozygous genotypes. Furthermore, this method can be used to analyze GSTA1, CYP2C19, and CYP2B6 gene polymorphisms in lupus nephritis patients receiving cyclophosphamide therapy, especially in a population in West Java, Indonesia. Conclusions PCR-sanger sequencing is a reliable, accurate, and simple method for determining SNP genotypes from blood samples

LP-110 development of GSTA1, CYP2C19, and CYP2B6 gene polymorphism detection methods on the response of cyclophosphamide therapy for lupus nephritis patients

Background Cyclophosphamide, an immunosuppressant in patients with lupus nephritis, aims to prevent recurrence and is a steroid-sparing agent. The clinical response (efficacy and toxicity) of lupus nephritis patients receiving cyclophosphamide varied.1 Cyclophosphamide is a pro-drug metabolized by hydroxylation via the CYP2C19 and CYP2B6 enzymes and detoxification via the glutathione-S-transferase (GST) enzyme.2 The most common type of mutation that occurs is single nucleotide polymorphism (SNP). Several SNPs from previous studies associated with cyclophosphamide response, metabolism, and toxicity in patients with lupus nephritis, namely rs3957356 in the GSTA1, rs4244285 in the CYP2C19, rs7254579 and rs4802101 in the CYP2B6.3 Therefore, detecting and screening SNP genotypes in lupus nephritis patients can help analyze cyclophosphamide response variation. This study aims to develop a method for detecting the genotypes of the four target SNPs and several surrounding SNPs using PCR-Sanger sequencing. Methods The gene polymorphism analysis method was optimized before taking patient samples at the hospital. PCR and Sanger sequencing methods are used for gene polymorphism analysis because they produce chromatograms with target amplicon base lengths and several SNPs that can be analyzed simultaneously. Results The analysis results of the four target SNPs of the GSTA1, CYP2C19, and CYP2B6 (rs3957356, rs4244285, rs7254579, and rs4802101) showed one synonymous SNP and three SNPs (regulatory and intergenic). We have developed an SNP detection assay using four fragments from the GSTA1, CYP2C19, and CYP2B6 that can detect 15 SNPs simultaneously (figure 1). In addition, this method succeeded in distinguishing wild-type, heterozygous and homozygous genotypes. Furthermore, this method can be used to analyze GSTA1, CYP2C19, and CYP2B6 gene polymorphisms in lupus nephritis patients receiving cyclophosphamide therapy, especially in a population in West Java, Indonesia. Conclusions PCR-sanger sequencing is a reliable, accurate, and simple method for determining SNP genotypes from blood samples

Genetic polymorphisms associated with cyclophosphamide outcome and risk of toxicity in patients with lupus nephritis

A 6-month cyclophosphamide induction therapy followed by maintenance therapy every three months is the first-line treatment for Class III, IV, and V lupus nephritis. Among the 139 single nucleotide polymorphisms (SNPs) associated with cyclophosphamide, four SNPs, namely rs4244285, rs4802101, rs7254579 and rs3957356, are related to the response and risk of toxicity in patients with lupus nephritis. Although pharmacogenetic studies in patients with lupus nephritis (LN) have not been conducted previously in Indonesia, data on rs4244285 are available for several ethnic groups, including Papuans, Bataks, Balinese, Dayaks, Javanese, Bugis, Chinese, Timorese and Malays, even though direct evidence in LN patients is less detectable. However, this can be followed up prior to cyclophosphamide therapy based on the identification of genetic markers. Therefore, clinical studies in patients with lupus nephritis are deemed necessary to evaluate the potential of these markers

Genetic polymorphisms associated with cyclophosphamide outcome and risk of toxicity in patients with lupus nephritis

A 6-month cyclophosphamide induction therapy followed by maintenance therapy every three months is the first-line treatment for Class III, IV, and V lupus nephritis. Among the 139 single nucleotide polymorphisms (SNPs) associated with cyclophosphamide, four SNPs, namely rs4244285, rs4802101, rs7254579 and rs3957356, are related to the response and risk of toxicity in patients with lupus nephritis. Although pharmacogenetic studies in patients with lupus nephritis (LN) have not been conducted previously in Indonesia, data on rs4244285 are available for several ethnic groups, including Papuans, Bataks, Balinese, Dayaks, Javanese, Bugis, Chinese, Timorese and Malays, even though direct evidence in LN patients is less detectable. However, this can be followed up prior to cyclophosphamide therapy based on the identification of genetic markers. Therefore, clinical studies in patients with lupus nephritis are deemed necessary to evaluate the potential of these markers

Improvement of Plasmid Volumetric Yield by Addition of Glycerol and Phosphate Buffer in Escherichia coli TOP10 Batch Culture

The investigation of mRNA development has gained substantial interest, particularly in the ex vivo and in vivo therapy. mRNA is widely used for the development of gene editing-based therapies and mRNA vaccines. The aim of this study was to optimize the medium and harvest time to increase plasmid DNA production as part of mRNA production. This study modified used a medium modification approach to achieve high density culture of Escherichia coli TOP10 pGEMT-N in batch cultivation method. Various media formulations were assessed, including LB; LB with phosphate buffer (K2HPO4 12.549 g/L and KH2PO4 2.31 g/L); LB with glycerol (50 g/L); LB with glycerol and phosphate buffer; LB with phosphate buffer, glycerol, glucose (15 g/L), and galactose (15 g/L). The effect of additional carbon sources and phosphate buffer on culture density was measured through OD600 and wet cell weight analysis. The highest OD600 and wet cell weight was observed when LB with glycerol and phosphate buffer was used, with OD600 of 4.78±0.14 and wet cell weight of 36.00±0.63 mg/ml. Plasmid DNA was subsequently isolated from these cultures following 5- and 7.5-hour incubation periods. The utilization of LB medium with glycerol and phosphate buffer resulted in a substantial increase in the volumetric concentration of plasmid DNA of 1,516.97±385.00 ng/ml after 5 hours of incubation. In conclusion, a remarkable enhancement in plasmid DNA volumetric yield within 5 hours was achieved by addition of glycerol and phosphate buffer to LB medium, leading to incubation period

Aktivitas Makrofag Meningkat Pada Aorta Tikus Hiperkolesterolemia

Aterosklerosis merupakan, kondisi inflamasi kronik, faktor resiko penyakit kardiovaskular disebabkan oleh tingginya kadar kolesterol. Tujuan penelitian ini mengevaluasi peran mieloperoksidase (MPO) dan makrofag di aorta dan jantung tikus yang diinduksi hiperkolesterolemia. Tikus dikelompokkan menjadi kelompok normal dan hiperkolesterolemia. Induksi hiperkolesterolemia dilakukan dengan pemberian pakan tinggi kolesterol, kolesterol murni, asam kolat dan propiltiourasil (KKT) selama 5 bulan. Kolesterol total diukur sebelum induksi, pertengahan, dan setelah induksi. HDL, trigliserida (TG), LDL, indeks aterogenik (IA), jumlah sel darah merah dan sel darah putih diukur setelah induksi. Deteksi ekspresi mieloperoksidase (MPO) dan CD68 pada aorta dan jantung dilakukan dengan metode dot blot dan ELISA. Induksi hiperkolesterolemia selama 5 bulan menghasilkan kadar kolesterol total (364,10 ± 148,46 mg/dL), HDL (7,90 ± 1,29 mg/dL), LDL (307,47 ± 116,91 mg/dL), dan Indeks Aterogenik (1,04 ± 0,23). Kadar kolesterol yang tinggi meningkatkan jumlah sel darah putih yang bersirkulasi namun tidak mempengaruhi jumlah sel darah merah. Jumlah makrofag yang berada di jaringan aorta dan jantung kelompok hiperkolesterolemia meningkat secara signifikan dibanding dengan kelompok normal. Namun, peningkatan aktivitas makrofag yang diukur dari ekspresi MPO hanya teramati pada aorta hewan hiperkolesterolemia, tidak pada jantung. Simpulan, aktivitas makrofag meningkat hanya pada aorta hewan hiperkolesterolemia diduga berperan dalam pembentukan plak ateroma di aorta. Kata kunci: Aorta, CD68, hiperkolesterolemia, makrofag, mieloperoksidase   Macrophage Activity Increases in Hypercholesterolemia Rat Aorta   Abstract   Atherosclerosis, which is an inflammatory chronic condition, is one of the major risk factors of cardiovascular disease caused by hypercholesterolemia. This study aimed to evaluate role of myeloperoxidase (MPO) and macrophage in aorta and heart of rat hypercholesterolemia. Rats were divided into normal and hypercholesterolemia groups. Hypercholesterolemia was induced by cholesterol feeding and CCT (cholesterol, cholic acid and propiltiourasil) oral administration for 5 months. Total cholesterol was measured before induction (T0), in the middle (T2.5), and after induction (T5). HDL, triglyceride (TG), LDL, aterogenic index (IA), red blood, and white blood cell count was measured after induction (T5). Success of induction was proven by the elevation of cholesterol total value of hypercholesterolemia group compared to normal group. Myeloperoxidase (MPO) and CD68 in aorta and heart hypercholesterolemia rat was detected by dot blot and ELISA method. Hypercholesterolemia group showed significant differences in total cholesterol value (364.10±148.46 mg/dL), HDL value (7.90±1.29 mg/dL ), LDL value (307.47±116.91) and Atherogenic Index (1.04±0.23). High level of cholesterol increases circulating white blood cells but have no effect on  circulating red blood cells. Macrophage in the  hypercholesterolemia group increased significantly compared to the normal group. However, the increase in macrophage activity identified throughMPO expression was only seen in hypercholesterolemic aorta but not  in the heart. It is concluded that macrophage activities increase in the aortic tissue, but  not in the heart tissue of the hypercholesterolemia group, which may contribute to the formation of atheroma plaque in aorta. Key words: Aorta, CD68, hypercholesterolemia, macrophage, myeloperoxidas

The long and stumble way to find potential active compounds from plants for defeating hepatitis B and C: review

Hepatitis is a liver illness caused by virus such as hepatitis A virus, hepatitis B virus and hepatitis C virus. Hepatitis B and C are considerably more usual and induce more cirrhosis and dead worldwide than hepatitis A. Although drugs that are currently often used in the medication of hepatitis B and C, the finding of recent drug from various resources including herbal has been intensively developed. Therefore, the purpose of this review is to consider the possibility of plant’s compounds as anti-HBV and anti-HCV. From the results of a review of several articles, several plant’s compound have shown effectiveness againts HBV and HCV by in silico, in vitro and in vivo studies. In conclusion, several plant’s active compounds are possibility to be developed as anti-hepatitis B and C

Design of Adenovirus 5 Vector with Adenovirus 26 Hexon Hypervariable Region Sequence using In Silico Approach

Adenovirus type 5 (Ad5) is one of the vaccine vectors, including the COVID-19 vaccine. Pre-existing immunity to Ad5 may suppress the immunogenicity and efficacy of adenovirus vectored vaccine. The neutralizing antibodies are directed specifically toward seven hypervariable regions (HVR) of hexon proteins located on the outer surface of the capsid. This study aims to design an Ad5 vector that may circumvent anti-Ad5 immunity by designing a chimera Ad5 vector with the sequence of Ad26 HVR (Ad5HVR26) using in silico approach. Substitution of the Ad5 HVR DNA sequence may affect the alternative splicing process of adenovirus mRNA, which then influence the protein product. The splice site prediction of Ad5HVR26 chimera vector was found at HVR5, 6, and 7. The codon change in the splice site was performed to decrease the possibility of incorrect splicing, while retaining the original amino acid sequence. The HVR substitution in chimera vector Ad5HVR26 may also affect the interaction of hexon in the capsid. The HVR2 and HVR4 hexon proteins individually interact with other hexon proteins and IX protein. Thus, two designs of the Ad5HVR26 chimera vector were created in this research. The first design was the Ad5 chimera vector with complete substitution of HVR hexon by Ad26 sequence, with codon modification on the splice site. The second design was Ad5HVR26 chimera vector without the HVR2 and HVR4 substitution to maintain the hexon protein interaction with the capsid proteins. Production of the designed vectors are needed to prove the reduction of vector neutralization by pre-existing immunity