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Optimization of Human Interferon α2b Soluble Protein Overproduction and Primary Recovery of Its Inclusion Bodies NINGRUM, RATIH ASMANA; RETNONINGRUM, DEBBIE SOFIE; CAHYATI, YEYET; RACHMAWATI, HENI
Microbiology Indonesia Vol 5, No 1 (2011): March 2011
Publisher : Indonesian Society for microbiology

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Abstract

The hIFN2b open reading frame has been constructed and overexpressed in Escherichia coli BL21(DE3). The yields of protein purified using nickel column from inclusion bodies (IB) and total soluble proteins were 3.46 mg and 2.57 mg in 1 L culture, respectively. This research was aimed to obtain optimal condition for high level overproduction of soluble hIFNα2b as well as primary recovery of hIFN2b from IB. We used two different conditions for obtaining soluble protein, i.e. induction temperatures and inducer concentrations, and three different conditions for inclusion bodies, i.e. centrifugation speeds, washing and solubilizing buffers. Induction using 0.5 mM of isopropyl thiogalactopyranoside at 25 °C yielded 8.9 mg hIFN2b in 1 L culture. The best recovery of IB was achieved when 10 000 g was applied for centrifugation, 1% Triton X-100  in 50 mM Tris Cl pH 8.0 as washing buffer, and 8M guanidine HCl in 50 mM Tris Cl pH 8.0 containing 800 mM 2-mercaptoethanol as solubilizing buffer were used. At this optimal condition the yield of hIFN2b from IB was 28.85 mg in 1 L culture. The total recovery of hIFNα2b at optimal condition was 50% from IB and 14% from soluble protein. hIFN2b from IB was refolded by 9 d dialysis in refolding buffer (0.2 mM EDTA, 0.25 mM ditiothreitol, 50 mM Tris and 0.4 M urea pH 8.0).
Dissolution test of various low-dose acetylsalicylic acid preparations marketed in Indonesia Sumirtapura, Yeyet C.; Setiawati, Arini; Pamudji, Jessie S.; Rachmawati, Heni
Medical Journal of Indonesia Vol 18, No 3 (2009): July-September
Publisher : Faculty of Medicine Universitas Indonesia

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Abstract

Aim To compare the dissolution profi les of various enteric-coated low-dose acetylsalicylic acid (ASA) tablets marketed in Indonesia.Methods The dissolution study was carried out according to US Pharmacocopoeiae (USP) /European Pharmacopoeiae, method A, using USP apparatus 1 (basket) 100 rpm, with 2 media: 0.1 N HCl, 120 minutes for acid stage, and phosphate buffer pH 6.8, 90 minutes for buffer stage. The sampling points were 120 minutes for the acid stage, and every 10 minutes until 90 minutes for the buffer stage. The acetylsalicylic acid was assayed using spectrophotometry at 280 nm for the acid stage, and at 265 nm for the buffer stage. The free salicylic acid was determined only at the end of the buffer stage with HPLC method. There were 6 test products (Cardio Aspirin® 100 mg, Aptor® 100 mg, Ascardia® 80 mg, Thrombo Aspilet® 80 mg, Astika® 100 mg and Farmasal® 100 mg), 3 batches for each product, and 6 units for each batch.Results The amount of ASA released from each ASA product tested at the end of acid stage (120 minutes) ranged from 1.79% for Cardio Aspirin® to 6.92% for Thrombo Aspilet®, all conformed to the compendial requirement for enteric-coated product (< 10%). The amount of salicylic acid observed at the end of the dissolution test ranged from 3.47% for Cardio Aspirin® to 10.90% for Astika® and 11.90 % for Thrombo Aspilet®. Thrombo Aspilet® showed sustained-release properties, causing high variability in ASA release, such that one of the 3 batches tested did not fulfill the compendial requirement of more than 75% (the release was only 55.11%). High variability in ASA release between batches was also found with Farmasal® at 10, 20, and 30 minutes in buffer medium. The lowest effective dose of ASA as an antiplatelet drug for longterm use is 75 mg of plain ASA, and this is equivalent to 100 mg of enteric-coated ASA.Conclusions All of the low-dose ASA preparations marketed in Indonesia are enteric-coated products, while Thrombo Aspilet® is not only an enteric-coated but also a sustained-release product. Cardio Aspirin®, followed by Aptor®, has the right dose for low-dose enteric-coated preparation (100 mg), produces consistent ASA release between batches,and the most stable towards deacetylation (antiplatelet inactivation). (Med J Indones 2009;18:159-64)Key words: Dissolution profile, enteric coated, deacetylation
PENINGKATAN STABILITAS KURKUMIN MELALUI PEMBENTUKAN KOMPLEKS KURKUMIN--SIKLODEKSTRIN NANOPARTIKEL DALAM BENTUK GEL Ariani Edityaningrum, Citra; Rachmawati, Heni
Pharmaciana Vol 5, No 1 (2015): Pharmaciana
Publisher : Universitas Ahmad Dahlan

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Abstract

Curcumin is a compound derived from turmeric. This compound is practically insoluble inwater and has poor stability. To improve the benefit of curcumin as a potential active compound in agel preparation, better stability are requested. Encapsulation was performed by freeze drying methodsand all evaluation data confirmed that curcumin included in the -cyclodextrin forming curcumin--cyclodextrin nanoparticle. The formula showed particle size of 156.8 ± 38.3 nm, polydispersity indexof 0.174 ± 0.026, and zeta potential of -17.3  0.2 mV. The gelling agents used for formulation of gelbase were HPMC, CMC-Na, carbopol 940, water-soluble chitosan, and viscolam. Viscolam showedbest stability of pH and viscosity after storage at 25 and 40 oC for 28 days. The inclusion complex andcurcumin were incorporated into gel. Both of the formulas showed good stability in pH and viscosityafter storage at 25 and 40oC for 28 days, and the inclusion complex gel showed improvement in thechemical stability which is approximately 2.12-fold (p<0.01) and 1.41-fold (p<0.05), after storage at25 and 40 oC, respectively.
Perancangan Sistem Informasi Manajemen Parkir Dengan Pendekatan Algoritma Hill Climbing di Pusat Perbelanjaan (Studi Kasus: Mal Ska-Pekanbaru) Subehi, Rakhman Gusti; Sari, Dini Nurmala; Rachmawati, Heni
Semantik Vol 3, No 1 (2013): Semantik 2013
Publisher : Semantik

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Abstract

Mal SKA (Sentral Komersil Arengka) merupakan salah satu pusat perbelanjaan modern yang ramai akan datangnya pengunjung khususnya disaat akhir pekan atau sedang berlangsungnya event yang diselanggarakan. Mendapatkan parkir pada waktu tertentu merupakan hal yang sulit dilakukan, apalagi disaat penuh dan ramai. Untuk menemukan tempat parkir yang kosong, mobil harus berjalan pelan dikarenakan pengaturan aktivitas dan ruang parkir yang ada lebih kecil dibandingkan jumlah kendaraan yang datang. Hal itu menyebabkan terjadinya kemacetan di dalam area parkir dan mengakibatkan kurang puasnya pengunjung terhadap pelayanan parkiran. Salah satu metode untuk mengoptimasi lama waktu tunggu pengguna adalah algoritma Hill Climbing. Algoritma Hill Climbing adalah suatu metode untuk mencari dan menentukan rute yang paling singkat dengan memperkecil jumlah tempat yang disinggahi. Maka dibangun aplikasi yang dapatmemberikan informasi lahan parkir yang kosong untuk pengunjung menggunakan algoritma Hill Climbing. Selain itu aplikasi ini dapat melakukan pemesanan tempat parkir bagi Member (Pemilik Properti). Untuk Guest (Tamu) dan Member dapat mengakses melalui Mobile Application berbasis Android. Sedangkan manajemen parkir menggunakan Website Application berbasis PHP. Berdasarkan penelitian yang telah dilakukan aplikasi ini dapat memecahkan kemacetan dalam parkir karena pengguna akan mendapatkan lahan parkir dengan posisi yang diinformasikan.
Tablet of captopril with a cross-linked system of alginate Asyarie, Sukmadjaja; Rachmawati, Heni; Sinambela, Pricillia
INDONESIAN JOURNAL OF PHARMACY Vol 18 No 1, 2007
Publisher : Faculty of Pharmacy Universitas Gadjah Mada, Yogyakarta, Skip Utara, 55281, Indonesia

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Abstract

Captopril has a short biological half life (1-3h) and has been used for long-term treatment of hypertension. The properties of captopril such as freely water-soluble and instability in intestinal environment lead to the difficulty of developing captopril as a sustained-release preparation. . In this study, sustained-release of captopril was prepared with a matrix system using sodium alginat as a polymeric forming-matrix. The ratio of sodium alginat and captopril was 1:2. Matrix system was obtained by forming alginate cross-linked with a various concentration of calcium acetate. Xanthan gum was used to help cross-linked reaction. Tablet was prepared by wet granulation and the dissolution test was performed in HCl 0.01 N, paddle method, 100 rpm, for 12 h. Although release profile of captopril from all formula developed were different, the release of captopril sustained up 12 h. Formula containing 30 % of xanthan gum and 40 % of sodium alginate showed the best release profile of captopril and the hardness of tablets do not influence to the release of captopril. Tablet of captopril with a crosslinked system of alginate sustained the release of captopril until 12 h .Key words: captopril, sustained-release, sodium alginat, calcium acetat.
DEVELOPMENT AND CHARACTERIZATION OF POLYCLONAL ANTIBODY OF RECOMBINANT HUMAN INTERFERON Α2B IN NEW ZEALAND WHITE RABBIT Rachmawati, Heni
INDONESIAN JOURNAL OF PHARMACY Vol 25 No 3, 2014
Publisher : Faculty of Pharmacy Universitas Gadjah Mada, Yogyakarta, Skip Utara, 55281, Indonesia

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Abstract

We have developed recombinant wild type and mutant human interferon α2b (rhIFNα2b) from synthetic gene in Escherichia coli. To identify the successful product of the proteins, immunology-based assay was suggested due to specificity for characterization. This work was aimed to develop and characterize rhIFNα2b polyclonal antibody generated in White New Zealand rabbits. The rhIFNα2b was overproduced in Escherichia coli BL21 containing rhIFNα2b synthetic gene in pET32b.The protein was obtained as inclusion bodies, refolded, purified using nickel affinity chromatography, and characterized using polyacrylamide gel electrophoresis. The purified rhIFNα2b protein was injected into rabbits for 21 days. Absorption of E.coli antibody was done using total E. coli protein to remove antibody againts host cell.   The generation of antibody was monitored using dot blot and Western blot methods and quantified using Enzyme Linked Immunsorbant Assay (ELISA). To do so, rhIFNa2b was used as an antigen. The result showed that the rhIFNα2b was produced as a His-tag protein fusion of 33kDa in size. The results of dot blot and Western blot analyses strongly indicated that antibody against rhIFNα2b was generated and specifically recognized rhIFNα2b. ELISA showed that the titer of the polyclonal anti-rhIFNα2b was 1:10.000. In conclusion, polyclonal antibody spesifically against rhIFNα2b protein was successfully detected with high titer after 21 days after rabbit immunization.
Development of fast disintegrating tablet formula of ketoprofen-β-cyclodextrin inclusion complexes Rachmawati, Heni; Marbun, Estherina Juliana; Pamudji, Jessie S.
INDONESIAN JOURNAL OF PHARMACY Vol 22 No 3, 2011
Publisher : Faculty of Pharmacy Universitas Gadjah Mada, Yogyakarta, Skip Utara, 55281, Indonesia

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Abstract

Ketoprofen  is  one  of  non  steroidal  anti  inflammatory  drugs  (NSAID)  used for  rheumatoid  arthritis.  However,  unpleasant  taste of  ketoprofen  leads  to difficulty  in  the  formulation,  in  particular  for  oral  route.  Therefore,  in  present study, a technique to mask the unacceptable taste of ketoprofen was developed. Then,  a  fast  disintegrating  tablet  on  this  inclusion  complex  was  established  for rapid  release  and  faster  analgesic  effect  of  ketoprofen.  Taste  masking  was prepared  by  complex  inclusion  with  β-cyclodextrin.  The  ratio  of  ketoprofen  and β-cyclodextrin  was  varied.  The  fast  disintegrating  tablet  was  formulated  with direct compression using various ratios of mannitoland lactose as tablet diluent, the main factor influencing the successful of fast disintegrating tablet. Evaluation of  final  product  was  performed  according  to  compendial  standard  and  specific requirements  for  fast  disintegrating  tablet.  The  best  ratio  from  ketoprofen  and β-cyclodextrin  was  2:3  with  concentration  of  ketoprofen  in  inclusion  complex was  40.32%.  The  tablet  met  standard  requirement  was resulted  with  the composition  of  ketoprofen-cyclodextrin  equivalent  to  50  mg  of  pure  ketoprofen and mannitol and lactose (ratio 1:1) as tablet diluent. Fast disintegrating tablet of  modified  ketoprofen  in  inclusion  complex  was  fulfilled  standard  specification for ketoprofen tablet with better acceptance.Key words:ketoprofen, inclusion complex, fast disintegratingtablet, beta cyclodextrin.
Formulasi Mikroenkapsulasi Protein dalam Poli(D,L-Laktida) dengan Teknik Penguapan Pelarut Fitriani, Lili; Rachmawati, Heni; Suciati, Tri
Jurnal Sains dan Teknologi Farmasi Vol 15 No 1 (2010)
Publisher : Fakultas Farmasi Universitas Andalas

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Abstract

Poly (D,L-lactide acid) has been used as scaffold and controlled release device for protein such as growth factor in tissue engineering. In this study, PDLLA microparticles were made and papain was used as model protein. Protein was encapsulated in microparticles using water-oil-water (W1/O/W2) and solid-oil-water (S/O/W) emulsification-solvent evaporation. Types of encapsulation methods and ratios of papain-PEG 20000 were observed in this study to provide the highest encapsulation efficiency. The entrapment efficiency made by W1/O/W2 method was 6,38%±0,025,  whilst S/O/W using ratios of papain-PEG 20000 1:1 ; 1:4 ; and 1:5 were 6,24%±0,91 ; 30,15%±1,66 and 60,67%±4,93, respectively. To conclude, S/O/W is the best method to encapsulate protein with highest entrapment efficiency.
Overproduction and Purification of Soluble Recombinant Human Granulocyte Colony Stimulating Factor in Escherichia coli Using Thioredoxin as Fusion Agustiyanti, Dian Fitria; Retnoningrum, Debbie Sofie; Rachmawati, Heni; Fuad, Asrul Muhamad
ANNALES BOGORIENSES Vol 21, No 1 (2017): Annales Bogorienses
Publisher : Research Center for Biotechnology - Indonesian Institute of Sciences (LIPI)

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Abstract

Recombinant human Granulocyte Colony Stimulating Factor (G-CSF) has been produced in a soluble form in Escherichia coli BL21 (DE3) as a fusion protein. The open reading frame of G-CSF was synthetically constructed in previous work and was codon optimized for best expression in E. coli. In this research, the gene was fused to thioredoxin (Trx) at the N-terminal in pET32 vector. The purpose of this research was to optimize the overproduction and purification processes to obtain high yield recombinant protein in soluble form, and to characterize the Trx-G-CSF fusion protein. Overproduction was performed using IPTG induction method for 3 and 6 hours. The protein was purified by Ni-NTA affinity chromatography and separated using gradient concentration of imidazole. The purified protein was then characterized by SDS-PAGE and Western Blot analysis. Further, enterokinase was used to separate G-CSF from the fusion protein. The purified form of G-CSF was subsequently characterized using Western Blot and mass spectrometry using MALDI-TOF. The results showed that the fusion protein was successfully produced in soluble part as much as 48.25% were obtained after 3 hours of induction. The yield of  fusion protein was 67.37%  from total protein (229.65  mg protein/L culture). The Western Blot analysis showed the G-CSF band at around 18.6 kDa. Mass spectrometry with MALDI-TOF/ TOF revealed that 25.86% of amino acid residue was recognized as part of human G-CSF sequence. 
DNA Condensation Study of Fully Synthesized Lipopeptide-Based Transfection Agent for Gene Delivery Vehicle Tarwadi, Tarwadi; Rachmawati, Heni; Kartasasmita, Rahmana E.; Pambudi, Sabar; Arbianto, Alfan Danny; Restiani, Dewi Esti; Asyarie, Sukmadjaja
ANNALES BOGORIENSES Vol 22, No 2 (2018): Annales Bogorienses
Publisher : Research Center for Biotechnology - Indonesian Institute of Sciences (LIPI)

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Abstract

   The main requirement of transfection agent has to condense DNA in nanoparticle size, protect the DNA from nucleases and other degrading enzymes during its transport in cell cytoplasm and nucleus and should not toxic to target cells. In this research, lipopeptide composed of palmitoyl (C-16) and short peptide sequence have been designed fully synthesized and tested to DNA condensation capability and toxicity. The DNA condensation study was performed using EtBr exclusion assay and cytotoxicity determination was carried out by colorimetric MTT assay. It was revealed that lipopeptide-based transfection agent of Pal-CKKHH and Pal-CKKHH-YGRKKRRQRRR-PKKKRKV condensed DNA molecules efficiently. The lipopeptide was less toxic compared to Lipofectamine and Poly-L-Lysine, that shown by 90% of CHO-K1 cells remained viable when they were treated with 4.36 µM Pal-CKKHHYGRKKRRQRRR-PKKKRKV. Meanwhile, there were only ~75% and 80% of CHO-K1 viable cells when it was treated with PLL and Lipofectamine®2000, respectively. Moreover, cell viability of HepG2 was ~ 75% after treated with 2.18 µM of Pal-CKKHH-YGRKKRRQRRR-PKKKRKV and decreased to ~65% when the lipopeptide concentration increased to 8.72 M. In summary, the synthesized lipopeptide condenses DNA molecules efficiently, less toxic than Lipofectamine®2000 and PLL and has possibility to be explored as a non-viral gene delivery vehicle.