Sismindari .
Faculty of Pharmacy Universitas Gadjah Mada Sekip Utara, Yogyakarta - Indonesia

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Detection of nptII Gene and 35SCaMV Promoter in Tomatoes (Solanum lycopersicum L.) Suratman, A.; Ughude, J. O.; ., Sismindari
Journal of Food and Pharmaceutical Sciences Vol 1, No 1 (2013): J.Food Pharm.Sci (January-April)
Publisher : Fakultas Farmasi, Universitas Gadjah Mada

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (395.879 KB) | DOI: 10.14499/jfps

Abstract

The detection of nptII (kanamycin resistance) as a transgenic marker gene and 35SCaMV as promoter in tomatoes has been carried out. DNA from tomatoes samples was isolated using PureLinkTM plant total DNA purification kit. The purity of DNA samples was estimated using UV-Vis spectrophotometry at 260 nm and 280 nm. They gave an absorbance ratio (A260/A280) of 1.74-1.79 which indicated its purities. The quality of the DNA was confirmed by a clear and thick band, as analyzed in 0.8% agarose gel electrophoresis. In order to identify the transgenic tomatoes, a 786-bp fragment of the nptII gene and a 86-bp fragment of 35SCaMV promoter were amplified using polymerase chain reaction (PCR). PCR reaction was prepared at optimum condition, namely annealing temperature at 56°C and 55°C for nptII gene and 35SCaMV promoter, respectively and 300 ng of DNA template. The PCR results were visualized on 2% agarose gel electrophoresis. The results showed that one of three tomatoes (code ST2) contains 35SCaMV promoter and no tomatoes contain nptII gene, indicating that ST2 is transgenic tomato.Key words: tomatoes, PCR, nptII, 35SCaMV
Cytotoxic Activity of Tegari (Dianella nemorosa Lam.) Methanol Extract Against HeLa Cells Karim, Aditya Krishar; ., Sismindari; Asmara, Widya; ., Istriyati
Indonesian Journal of Biotechnology Vol 17, No 1 (2012)
Publisher : Universitas Gadjah Mada

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (177.336 KB)

Abstract

Dianella nemorosa Lam. also known as tegari belonging to the Liliaceae family. This plant has been utilized for Papua traditional medicine as well as anticancer agent. This research examined potential cytotoxic activity of tegari (D. nemorosa) leaves extract against cervical cancer cell line (HeLa). Methanol extract was obtained by extracting the leaves powder using methanol. Extract was then applied into HeLa cell line to find out the cytotoxic activity. MTT [3-(4,5-dimetilthiazol-2-il)2,5-difeniltetrazolium bromida) assay was used to measure the cytotoxic activity. The result indicated that D. nemorosa leaves extract possessed cytotoxic activity in HeLa cell line with IC50 values were 685,69 µg/ml, 506,43 µg/ml and 708 µg/ml at the incubation period of 24, 48 and 72 h respectively. The strongest cytotoxic was showed by methanol extract incubated in 48 h.
In Vitro CYTOTOXIC ASSAY OF CHLOROFORM EXTRACT OF Brucea javanica L .(Meer), Ipomoea batatas Poir., Mussaenda pubescens Ait.F., AND Portulaca oleracea L. TO HeLa CELLS M, Sonlimar; ., Sismindari; ., Mustofa
INDONESIAN JOURNAL OF PHARMACY Vol 13 No 4, 2002
Publisher : Faculty of Pharmacy Universitas Gadjah Mada, Yogyakarta, Skip Utara, 55281, Indonesia

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.14499/indonesianjpharm0iss0pp215-222

Abstract

Some plants, such as Ipomoea batatas Poir., Mussaenda pubescens Ait. F., Portulaca oleracea L., Brucea javanica L. (Meer), have been used traditionally to treat cancer, even though the scientific base of this activity has yet been completely investigated. The study was conducted to evaluate the cytotoxic activity of these plants on HeLa cell lines.The cytotoxicity of chloroform extract from these plants were determined using micro radioactive method by measuring an incorporation (3H)-hipoxanthine on the cells after 24 and 48 hours incubation with each extract, using doxorubicine as a positive control. Extract free cell culture was refered to as 100% growth. IC50 value, showing 50% inhibition of the cell growth, was determined based on the concentration versus percentage inhibition curves.The result showed the extract B. javanica was the most active to hela cells with the IC50 value of 13.69 1.07 and 55.78.4 g/ml after 24 and 48 hours incubation respectively.Key words: Ipomoea batatas Poir., Mussaenda pubescens Ait. F., Portulaca oleracea L., Brucea javanica L. (Meer), Cytotoxic, HeLa cell lines.
Formulation of nanoparticles from short chain chitosan as gene delivery system and transfection against T47D cell line Winarti, Lina; Martien, Ronny; ., Sismindari
INDONESIAN JOURNAL OF PHARMACY Vol 22 No 3, 2011
Publisher : Faculty of Pharmacy Universitas Gadjah Mada, Yogyakarta, Skip Utara, 55281, Indonesia

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (315.858 KB) | DOI: 10.14499/indonesianjpharm0iss0pp204-211

Abstract

Recently numerous prototype DNA-based biopharmaceuticals can be used to  control  disease  progression  by  induction  and  inhibitin  the  overexpression  of genes.  Since  there  are  poor  cellular  uptake  and  rapid  in  vivo  degradation  of DNA-based  therapeutics  therefore  the  use  of  delivery  systems  to  facilitate cellular internalization and preserve their activity is necessary. Cationic polymers commonly used as carriers to delivery gene because of easy to form complexes and  higher  stability  compared  to  that  lipoplexs.  Chitosan,  a  cationic,  are polymer most widely used in gene delivery systems because of the low toxicity, and biocompatible. The aim of this study was to formulate nanoparticles of short chain  chitosan-pEGFP-C1  and  short  chain  chitosan/TPP-pEGFP-C1  by coaservation  complex  method.  Stability  test  of  the  formula  was  performed  by incubating the nanoparticles complex with DNase I and Artificial Intestinal Fluid. Cytotoxicity  and transfection  studies  were  evaluated  against  T47D  cell line.  The diameter  of  Chitosan-pEGFP-C1  and  chitosan/TPP-pEGFP-C1  nanoparticles  were on the range of 56–282.8 nm. The zeta potential wasdetermined to be +14.03 - +16.6  mV.  Stability  studies  showed  that  chitosan-pEGFP-C1  and  chitosan/TPPpEGFP-C1  nanoparticles  were  stable,  undegradable  by  DNase  I  and  artificial intestinal fluid. Cytotoxic Assay of Chitosan-pEGFP-C1 and  chitosan/TPP-pEGFPC1  nanoparticles  (pH  4.0)  showed  that  the  viability  of cell  was  >  90%  for  all formulas.  EGFP-C1  plasmid  gene  delivered  by  chitosan  nanoparticles  can  be expressed  in  T47D  cell  culture.  According  to  these  results  chitosan  and chitosan/TPP  nanoparticles  had  potentially  to  be  used  as  a  non-viral  vector system delivery for gene therapy.Key words:Chitosan, Nanoparticles, Plasmid EGFP-C1, Cell culture T47D 
Effect of protein fraction of Carica papaya L. leaves on the expressions of p53 and Bcl-2 in breast cancer cells line ., Rumiyati; ., Sismindari; ., Ariyani
INDONESIAN JOURNAL OF PHARMACY Vol 17 No 4, 2006
Publisher : Faculty of Pharmacy Universitas Gadjah Mada, Yogyakarta, Skip Utara, 55281, Indonesia

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (1212.046 KB) | DOI: 10.14499/indonesianjpharm0iss0pp170-176

Abstract

Carica papaya L. leave was known containing Ribosome-inactivating proteins (RIPs), which demonstrated to have an in vitro cytotoxic effect on cancer cell lines. This researh examined the effect of protein fraction containing RIPs isolated from Carica papaya L. on the expressions of p53 and Bcl-2, regulator proteins on apoptosis process, in breast cancer cell lines (T47D).RIPs from Carica papaya L. leaves were isolated by amonium sulfat precipitation. This fraction was analyzed by the activity of cleaving supercoiled double stranded DNA, in order to identify the presence of RIP. The active fraction was then tested of the citotoxic activity on breast cancer cell lines followed by analysing the expressions of p53 and bcl2 using immunohistochemistry technique.The results indicated that the protein fraction possessed cytotoxic activity in breast cancer cell line with the IC50 of 2.8 mg/mL. The expression level of p53 was increased by 59.4%, while Bcl-2 protein was decreased by 63%. These results suggested that this protein could induce apoptotic process.Key words : protein fraction, Carica papaya L. , expression of p53 and Bcl-2
Effects of pH, temperature and storage on the stability of MJ-30 protein isolated from Mirabilis jalapa L leaves ., Sudjadi; Ikawati, Zulies; ., Sismindari; Rahayu, Putu Riana Suastari
INDONESIAN JOURNAL OF PHARMACY Vol 15 No 1, 2004
Publisher : Faculty of Pharmacy Universitas Gadjah Mada, Yogyakarta, Skip Utara, 55281, Indonesia

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (358.748 KB) | DOI: 10.14499/indonesianjpharm0iss0pp1-6

Abstract

The total protein of Mirabilis jalapa L leaves had the ability to cleave supercoiled DNA and showed toxicity on HeLa and Raji cell-lines. The 30 kD protein (MJ-30) purified by cationic exchange chromatography possessed activites as RIP e.g DNA supercoiled cleavage and RNA N-glycosidase activity. The aim of this study was to prepare pure MJ-30 and to observe the stability of MJ-30 .MJ-30 of M.jalapa L leaves was purified using the combination of ammonium sulphate fractionation and cationic exchange chromatography with NaCl gradient elution. MJ-30 was subjected for its stability to assays against pH, temperature, and storage period.Stability assay showed that MJ-30 is stabil at pH 5-6, then the activity decreased as the pH increased. This protein was stable at 300 – 550C, and the activity decreased when the temperature increased. Storage at 40C, MJ-30 was stable until 12 days but the activity was decreasing. However, at 300C, MJ-30 was stable for 3 days only. Glycerol addition to the MJ-30 solution has made the activity stable for 18 days at 40C and 300C storage.Keyword : Protein MJ-30, Leaves of Mirabilis jalapa L, stability, pH, temperature, storage
Cytotoxic effects of an acidic Ribosome-inactivating Protein like protein isolated from Mirabilis jalapa L. leaves on cancer cell-lines ., Sudjadi; Witasari, Lucia D.; Sadarum, Modesta T.; Nastity, Nia; ., Sismindari
INDONESIAN JOURNAL OF PHARMACY Vol 18 No 1, 2007
Publisher : Faculty of Pharmacy Universitas Gadjah Mada, Yogyakarta, Skip Utara, 55281, Indonesia

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (269.61 KB) | DOI: 10.14499/indonesianjpharm0iss0pp8-14

Abstract

Most purification approaches of Ribosome-inactivating Proteins (RIPs) used a cation exchange chromatography resulting highly basic proteins, such as MJ-30 isolated from Mirabilis jalapa leaves. The M.jalapa unbounded protein fractions of the column showed cleavage activity on supercoiled double stranded DNA and possesed cytotoxic effects on cancer cell lines. So the purification and characterization of the acidic protein was needed to be done.Total proteins of M.jalapa leaves was collected from precipitating process of the extract using ammonium sulphate untill saturated. After dialysis, the proteins was applied into the weak base anion exchanger Ionenaustaucher Typ II. The bounded proteins was eluted with 0.0 to 0.5 M NaCl gradient in phosphate buffer. Active protein fractions were determined by supercoilded DNA cleavage activity and then cytotoxic activity was testes.Results showed that protein fraction eluted at 0.35 – 0.40 M NaCl possesed cleavage activity on supercoiled DNA. The acidic protein, MJ-C, was toxic on HeLa, Myeloma and T47D cell lines, with the LC50 of 14.3 μg/mL, 7.4 μg/mL and 27.8 μg/mL, respectively. The MJ-C was more potent than the basic protein MJ-30.Key words: acidic RIP, cytotoxic, Mirabilis jalapa
PURIFICATION OF RIBOSOME-INACTIVATING PROTEIN (RIP) OF MIRABILIS JALAPA L. LEAVES BY CM-SEPHAROSE CL-6B AND SEPHACRYL S-300HR COLUMN ., Sudjadi; ., Sismindari; Herawati, Tenti; Prasetyowati, Alberta Tri
INDONESIAN JOURNAL OF PHARMACY Vol 14 No 2, 2003
Publisher : Faculty of Pharmacy Universitas Gadjah Mada, Yogyakarta, Skip Utara, 55281, Indonesia

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (165.937 KB) | DOI: 10.14499/indonesianjpharm0iss0pp316-321

Abstract

Total protein of Mirabilis jalapa leaves has activities to cleave supercoiled DNA to nick circular and linear and to cleave glycosidic bound of adenin4324 of yeast 26S rRNA. The protein was cytotoxic on HeLa and Raji celllines through apoptosis and necrosis mechanisms, respectively. However, the protein, poseess the activities, has yet been resolved. Therefore, protein furification to obtain single protein is necessary. Crude extract of M.jalapa leaves was prepared using 5 mM sodium phosphate buffer pH 7,2 containing 0,14 M sodium chloride. Total protein was obtained by precipitating the extract at 100% saturated ammonium sulfate and then dialyzed against phosphate buffer. The protein was purified by CM-Sepharose CL-6B, a cation exchange column. After loading the protein, the column was washed with 5 mM sodium phosphate buffer pH 6,5. The proteins were then eluted with linear gradient of increasing sodium chloride concentration. The fraction with supercoiled DNA-cutting activity was performed for N-glycosidase activity. The active fraction was a subject for further purification with Sephacryl S-300HR, a gel filtration column. The purity and size protein were confirmed by SDS-polyacrylamide gel electrophoresis with silver nitrate staining. RIP like protein was eluted from CM-Sepharose CL-6B on 0,25 – 0,3 M sodium chloride. The size of protein is around 30 kD.Key words : RIP purification, M.jalapa leaves, CM-Sepharose CL-6B, Sephacryl S-300HR
TOXICITY OF THE AQUEOUS EXTRACTS FROM THE FRUITING BODY OF GANODERMA SP. BY BRINE LETHALITY TEST OF Artemia salina Leach. ., Rumiyati; ., Sismindari; Widyastuti, S.M.
INDONESIAN JOURNAL OF PHARMACY Vol 13 No 1, 2002
Publisher : Faculty of Pharmacy Universitas Gadjah Mada, Yogyakarta, Skip Utara, 55281, Indonesia

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (148.574 KB) | DOI: 10.14499/indonesianjpharm0iss0pp44-49

Abstract

The level of toxicity of aqueous extract from Ganoderma sp. fruiting body isolated from different plant media was determined in this experiment using Brine shrimp lethality test (BST) on Artemia salina Leach. The influence of media on the toxicity level of aqueous extract from Ganoderma sp. fruiting body was determined in this experiment. Ganoderma sp. and G. Lucidum were grown on three flamboyant, (Delonix regia Bojer ex Hook Rafin), sengon (Paraserianthes falcataria (L) Nielsen) and coconut(Cocos nucifera Linn.) plant media. The aqueous extracts were prepared from fruiting body using 0.02 M phosphate buffer pH 7.2. The toxicity level of these aqueous extract were then screened using the BST method. The level of the toxicity was determined by LC50. The results indicated that the aqueous extracts of Ganoderma sp. and G. lucidum isolated from flamboyant, sengon and coconut plant media were influenced by plant media. Ganoderma sp. was isolated from flamboyant plant media possesed the highest toxicity (LC50 480 g aqueous extract/ml), then followed by Ganoderma sp. isolated from sengon plant media (LC50 770 g aqueous extract /ml) and coconut (LC50 1040 g aqueous extract /ml). G. lucidum from coconut plant media possesed the highest toxicity (LC50 660 g aqueous extract /ml), followed by G. lucidum were isolated from sengon plant media (LC50 1100 g aqueous extract /ml) and flamboyant (LC50 1970 g aqueous extract /ml). It was suggested that G.lucidum having highest toxicity might produce compounds having antitumor activity.Key words: Ganoderma sp., aqueous extracts, toxicity .
THE CLEAVING ACTIVITY ASSAY ON SUPERCOILED DNA BY PROTEIN FRACTIONS FROM Morinda citrifolia LEAVES Sulistyani, Nanik; ., Sismindari; ., Sudjadi
INDONESIAN JOURNAL OF PHARMACY Vol 13 No 4, 2002
Publisher : Faculty of Pharmacy Universitas Gadjah Mada, Yogyakarta, Skip Utara, 55281, Indonesia

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (56.316 KB) | DOI: 10.14499/indonesianjpharm0iss0pp174-179

Abstract

Study on supercoiled DNA cleaving activity assay of protein fraction from Morinda citrifolia leaf had been done. The aim of this research is to compare the supercoiled DNA cleaving abilities among the fractions. This is a preliminary screening to find RIP from Morinda citrifolia Fractionation of protein was done by adding Ammonium Sulphate up to 20%, 40%, 60%, 80% and 100% in the crude extract, saturated solution was then centifugated to get the pellet fractions and coded as F-20, F-40, F-60, F-80 and F-100. The mixture of pUC19 plasmid and protein fractions, in the same protein concentration were incubated at room temperature for one hour, the cleaving ability of protein fraction on supercoiled DNA.was investigated. The results indicated that protein fractions, of F-80 had the highest cleaving activity to supercoiled DNA.Key words : Ribosome-Inactivating Protein (RIP), supercoiled DNA , protein fraction, Morinda citrifolia.