Theaim of this study was to develop the valid analytical method which used for the assay of triamcinolone acetonide (TA) in the investigation of TA loaded nanoparticle formulations. High Performance Liquid Chromatography(HPLC) method was applied in  his study by using an Econosil column,C1810 m, 250x 4.6mm (Alltech Associates Inc, PA,USA )as the stationary phase. Themobile phase consisted of a composition of acetonitrile (ACN) and 20mM phosphate buffer solution (pH 4.2) in the proportion of 50:50 v/v. The HPLCassay of TA was validated for selectivity, linearity, precision, ecovery (accuracy),sensitivity and stability of TA during the assay. Results showed that the concentration of TA in the sample scan be determined against the standard in the concentration range of calibration curve. The system precision and level of recovery were considered to be acceptable, and the method was selective and sensitive.Key words:triamcinoloneacetonide,assay,validatio

Boersma, AWM.

Gandjar, Ibnu G.

Mubarika, Sofia

Nooter, K.

Wahyuningnsih, Mae S.H.

Wahyuono, Subagus

English

The 16,17-dehydrodeacetyl-5α-oleandrin was isolated from an active fraction of Nerium indicum Mill leaves (fam. Apocynaceae). This compound was cytotoxic against various cancer cells, and selective on A498 cells (Renal cancer). However, the apoptosis mechanism was still unknown yet. Therefore, the aim of this study was to know the apoptotic mechanism of 16,17-dehydrodeacetyl-5α-oleandrin on A498 cells by FITC labeled annexin V and immunucytochemical assays. The detection of apoptotic mechanism on A498 cells was performed with FITC-conjugated annexin V using Flow Cytometry. The p53 protein expression were detected using immunocytochemical. Treatment with 16,17-hydrodeacetyl-5α-oleandrin (3.88 x 10-4 mM) using FITC-annexin V increased the percentage of the dead cells in the 24th and 48th hours incubation period. The 16,17-dehydrodeacetyl-5α-oleandrin (1,94x10-4 and 3,88x10-4 mM) raised significanly p53 protein expression (p&lt;0,05). The percentage of the p53 protein expression increased throughout the time of samples incubation.Key words: 16,17-dehydrodeacetyl-5α-oleandrin,fluorescein isothiocyanate-annexin V, immunocytochemically, A498 cells

Open Journal Systems

Detection of apoptosis mechanism on renal cancer cell treated by 16,17-dehydrodeacetyl-5α-oleandrin compound isolated from Nerium indicum Mill. Leaves.

The  aims  of  the  study  was  to  determine  of  the  crystal  fraction  from Zingiber  zerumbet essential  oils.  The  components  examination  was  performed by  GC-MS  method  using  the  solvent  n-hexane  and  anti bacterial  activity  was tested  against  Staphylococcus  aureus ATCC  25923  and  Escherichia  coli ATCC 35218  using  agar  diffusion  method  with  coconut  oil  solvent.  The  components that  can  be  identified  are  α-humulene,  caryophyllene  oxide,  β-selinene, and zerumbone. Crystal fraction solution able to inhibit of S. aureusATCC 25923 at  the  concentration  25  %  and  up  to  50  %  has  not  been  able  to  inhibit  E.  coli ATCC 35218.Key words : Zingiber zerumbet, Lempuyang gajah, Crystal fraction, anti-bacteria

Mulyani, Sri

Majalah Farmasi Indonesia, 21(3), 178 – 184, 2010 Majalah Farmasi Indonesia, 21(3), 2010 178 Komponen  dan anti-bakteri dari fraksi kristal minyak Zingiber zerumbet  Component and antibacterial activity of crystal fraction from Zingiber zerumbet essential oil  Sri Mulyani Fakultas Farmasi UGM  Abstrak  Penelitian ini bertujuan untuk mengetahui komponen penyusun dan aktivitas anti-bakteri fraksi kristal minyak Lempuyang Gajah hasil distilasi uap dan air rimpang lempuyang gajah (Zingiber zerumbet Smith). Pemeriksaan komponen dilakukan dengan metode GC-MS menggunakan pelarut n-heksana sedang aktivitas anti-bakteri diujikan terhadap Staphylococcus aureus ATCC 25923 dan Escherichia coli ATCC 35218 menggunakan metode difusi agar dengan pelarut minyak kelapa. Komponen penyusun yang dapat diidentifikasi adalah α-humulena, kariofilena oksida, β-selinena, dan zerumbona. Larutan fraksi kristal dalam minyak kelapa mampu menghambat pertumbuhan S. aureus ATCC 25923 pada kadar 25 % dan sampai kadar 50 % belum mampu menghambat pertumbuhan E. coli ATCC 35218. Kata Kunci : Zingiber zerumbet, lempuyang gajah, fraksi kristal, anti bakteri  Abstract  The aims of the study was to determine of the crystal fraction from Zingiber zerumbet essential oils. The components examination was performed by GC-MS method using the solvent n-hexane and anti bacterial activity was tested against Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 35218 using agar diffusion method with coconut oil solvent. The components that can be identified are α-humulene, caryophyllene oxide, β-selinene,               and zerumbone. Crystal fraction solution able to inhibit of S. aureus ATCC 25923 at the concentration 25 % and up to 50 % has not been able to inhibit E. coli ATCC 35218. Key words : Zingiber zerumbet, Lempuyang gajah, Crystal fraction, anti-bacterial   Pendahuluan Indonesia merupakan negara yang kaya akan keaneka-ragaman hayati, banyak memiliki berbagai jenis tanaman yang berkhasiat obat, di antaranya adalah tanaman-tanaman dari suku Zingiberaceae, marga Zingiber seperti Zingiber officinale, Zingiber offensii, Zingiber litorale, Zingiber aromaticum, Zingiber american, Zingiber odoriferum, Zingiber purpureum, Zingiber spectabilis, zingiber zerumbet (Anonim, 1986). Zingiber zerumbet (lempuyang gajah), secara tradisional digunakan untuk mengobati sakit perut, kejang pada anak, sakit empedu, borok, kulit, diare, dan disentri (Anonim, 1986). Rimpang lempuyang gajah diketahui mengandung humulena (senyawa dari kelompok seskuiterpena), 5-hidroksi zerumbona, oksida-zerumbona (Jang et al., 2005), alkaloid, saponin, flavonoid, dan polifenol, disamping minyak atsiri. Minyak atsirinya tersusun dari senyawa kelompok seskuiterpena dan terpena (Lechat vahirua et al., 1993). Sedang menurut Agusta (2000), minyak atsiri lempuyang gajah tersusun dari α-pinena, kamfena, kariofilena, β-pinena, α-farnesena, sineol, dl-kamfor, isokario-filena, kariofilena-oksida, dan germakron. Beberapa penelitian aktivitas yang telah dilaporkan adalah mengenai efek anthelmintik terhadap cacing Ascaris suum dari ekstrak air dan minyak atsiri (Sukandar et al., 1997). Ekstrak etanol dari rimpang dilaporkan memiliki aktivitas anti alergi, dengan mekanisme melalui Penghambatan enzim α-heksosaminnidase Sri Mulyani Majalah Farmasi Indonesia, 21(3), 2010 179dengan mengacaukan aktivitas sel-sel RBL-243 (Tewtrakul and Subhadhirasakul, 2007). Oleh Sawangjaroen et al., (2006) dilaporkan bahwa ekstrak kloroform, air dan metanol memiliki aktivitas sebagai anti-amuba. Efek anti-bakteri dari minyak atsirinya, dilaporkan mampu meng-hambat dan membunuh S. aureus, pada kadar 0,0625 % v/v, tetapi sampai kadar 25 % tidak mampu menghambat /membunuh pertumbuh-an E. coli (Bustanul, 2007). Pada penyulingan minyak atsiri dari rimpang lempuyang gajah kering, pada saat air sulingan mendingin, terdapat kristal yang terpisah dari fase minyak maupun airnya (Bustanul, 2007). Sehingga penelitian ini bertujuan untuk mengetahui komponen dari fraksi kristal, serta menguji aktivitas anti-bakterinya terhadap S. aureus  dan E. coli. Ahmad et al., 2008 melaporkan bahwa dari distilasi uap rimpang segar Z. zerumbet diperoleh minyak atsiri yang apabila dialiri dengan air dingin akan diperoleh kristal secara spontan. Dari hasil purifikasi kristal dengan heksana diperoleh senyawa zerumbona. Senyawa ini diketahui memiliki aktivitas anti-bakteri terhadap S. choleraesus yang resisten terhadap methicillin pada kadar 0,13 mg/mL, tetapi sampai kadar 13 mg/mL tidak mem-berikan hambatan terhadap S. aureus yang resisten terhadap methicillin. Aktivitas penghambatan ini ditetapkan dengan metode difusi menggunakan kertas samir.  Metodologi Bahan Bahan yang digunakan dalam penelitian ini adalah rimpang lempuyang gajah yang diperoleh dari pasar Beringharjo Yogyakarta, dan diidentifikasi di laboratorium Farmakognosi, Bagian Biologi Farmasi, Fakultas Farmasi UGM.   Alat  Bakteri uji : S. aureus ATCC 25923, E. coli ATCC 35218. Bahan kimia: n-heksana (pa.), Media Nutrien Agar (NA) (Oxoid), minyak kelapa, Strepto-misin 25 µg/mL. Alat : Seperangkat alat destilasi uap dan air, GCMS (Shimadzu QP 2010 S).  Cara penelitian  Rimpang lempuyang gajah yang telah         bersih dirajang, dikeringkan menggunakan sinar         matahari tak langsung, selanjutnya dilakukan destilasi          minyak atsiri dengan metode distilasi uap dan air.               Distilat yang diperoleh selanjutnya dibiarkan mendingin, dan kristal yang terbentuk selanjutnya dipisahkan dari fase air dan minyak. Pemeriksaan komponen dari fraksi kristal dianalisis komponen penyusunnya dengan GC-MS dengan menggunakan pelarut n-heksana, kemudian diuji aktivitas anti-bakterinya terhadap S. aureus ATCC 25923 dan E. coli ATCC 35218 dengan metode difusi teknik sumuran menggunakan pelarut minyak kelapa. Daya anti-bakterinya ditetapkan dari luas daerah hambatan yang ditimbulkan.  Jumlah sampel dalam sumuran  = 20 πL.  Kondisi GC-MS Kolom Rastek RXi-5MS, panjang 30 m, dia-meter 0,25 mm, detektor FID. Suhu injektor 280o C, suhu kolom diprogram 70° (5 menit) – 270° C (15 menit) dengan kenaikan suhu diatur 10o C/menit. Kecepatan gas pembawa 3,0 mL/menit; tekanan 13,7 k.Pa. Kondisi GC-MS : ion source temp  250° C, interface temp 300° C dan solvent cut time 3 menit. Spektra massa yang dihasilkan dianalisis dengan jalan membandingkan kesamaan spektra massa masing-masing komponen yang diidentifikasi dengan spektra massa dari pustaka Wiley and NIST (1966).  Hasil dan Pembahasan Dari satu kilogram rimpang kering dihasilkan 20 gram fraksi kristal. Dari analisis GC diperoleh 9 puncak dan 4 puncak dianalisis spektra massanya dan diidentifikasi sebagai α-humulena (puncak ke 4), kariofilena oksida (puncak ke 5), β-selinena (puncak ke 6), dan ze-rumbona (puncak ke 8). Data waktu retensi, prosen area, dan jenis senyawa yang teridentifikasi tertera pada Tabel I. Kromatogram (TIC dari GC-MS), serta spektra massa dari komponen yang teridentifikasi beserta spektra massa dari pustaka tertera pada Gambar 1, 2, 3, 4, 5, 6, 7, 8, 9. Hasil pemeriksaan aktivitas anti-bakteri terhadap S. aureus ATCC 25923 dan E. coli ATCC 35218 tertera pada Tabel II.   Dari hasil pemeriksaan antibakteri terlihat, bahwa fraksi kristal mempunyai aktivitas menghambat pertumbuhan S. aureus mulai kadar 25 % tetapi sampai dengan             kadar 50 % tidak mampu menghambat pertumbuhan E. coli. Aktivitas ini sama           dengan aktivitas dari minyak atsirinya yang                   hanya mampu menghambat/membunuh bakteri S. aureus, tetapi tidak mampu menghambat/ membunuh E. coli (Bustanul, 2007).  Komponen  dan anti-bakteri dari fraksi……….. Majalah Farmasi Indonesia, 21(3), 2010 180 Menurut Bustanul (2007), minyak atsiri rimpang lempuyang gajah mampu  menghambat/ membunuh bakteri S. aureus pada kadar 0,0625 %, sedang terhadap E. coli sampai dengan kadar 25 % belum mampu meng-hambat/membunuh pertumbuhan E. coli. Ahmad et al., 2008 melaporkan bahwa kristal zerumbon dari Z. zerumbet sampai kadar                13 mg/mL belum mampu menghambat pertumbuhan S. aureus yang resisten terhadap methicillin. Adanya perbedaan kadar yang dibutuhkan untuk menghambat pertumbuhan S. aureus dapat disebabkan karena adanya perbedaan komposisi kimia antara minyak atsiri dan fraksi kristal ataupun juga metode yang di-gunakan untuk menguji aktivitas anti-bakterinya.    Gambar 1. Kromatogram (TIC GC-MS) fraksi kristal Rimpang Lempuyang Gajah.    Gambar 2. Spektra massa komponen fraksi kristal puncak no 4 dari kromatogram Gambar 1 teridentifikasi sebagai α-humulena.  Tabel I. Waktu retensi, prosen area dan komponen fraksi kristal yang terpisah dari minyak dan air hasil distilasi Rimpang Lempuyang Gajah  No. Waktu retensi Prosen Area Komponen SI 1 06,87 00,95 ----  2 08,84 00,28 ----  3 11.40 00,29 ----  4 16.65 03,63 α-Humulena 92 5 18,48 00,51 Kariofilena oksida 93 6 18,68 01,88 β-Selinena 83 7 18,83 01,44 ----  8 20,56 90,62 Zerumbona 93 9 21,71 00,42 ----   Sri Mulyani Majalah Farmasi Indonesia, 21(3), 2010 181Dalam minyak atsiri terdapat sineol dan kamfer yang tidak teridentifikasi dalam fraksi kristal, sedangkan zerumbona, β-selinena, α-humulena, dan kariofilena oksida (yang merupakan seskuiterpena yang ada di fraksi kristal) tidak terkandung pada minyak atsiri dari rimpang lempuyang gajah. Menurut Carson et al., (2002), sineol mempunyai aktivitas anti-bakteri melalui mekanisme menurunkan toleransi S. aureus terhadap NaCl; yang akhirnya menyebabkan S. aureus kehilangan kemampuan untuk hidup membentuk koloni pada media yang me-ngandung NaCl. Sedangkan kamfor yang merupakan monoterpena dengan gugus keton memiliki aktivitas anti-bakteri di bawah fenol, terpenoid aldehida, tetapi di atas terpenoid alkohol dan hidrokarbon (Knobloch et al., 1986). Meskipun beberapa seskuiterpenoid yang teroksigenasi (berupa keton) dapat ditemukan  di dalam fraksi kristal seperti zerumbona              dan kariofilena oksida, tetapi senyawa ini memiliki memiliki bobot molekul besar.   Gambar 3. Spektra massa dari alfa-Humulena (Wiley 7, LIB).    Gambar 4. Spektra massa komponen fraksi kristal puncak no 5 dari kromatogram Gambar 1 teridentifikasi sebagai Kariofilena oksida.    Gambar 5. Spektra massa dari Kariofilena oksida (NIST 62, LIB).     Gambar 6. Spektra massa komponen fraksi kristal puncak no 6 dari kromatogram Gambar 1 teridentifikasi sebagai Selinena.    Komponen  dan anti-bakteri dari fraksi……….. Majalah Farmasi Indonesia, 21(3), 2010 182 Menurut Jawetz et al., (1996), molekul-molekul dengan bobot molekul besar tidak dapat menembus membran dengan cara difusi pasif. Dengan demikian dapat dipahami, bahwa fraksi kristal memiliki aktivitas yang berbeda dengan minyak atsirinya dan dengan zerumbon murni.  Sedang Mohamad Syahrizal 2006 melaporkan bahwa zerumbol hasil pengubahan zerumbon (hasil fraksinasi dengan heksana dari minyak atsiri Zingiber zerumbet) dengan lithium aluminium hidrida pada suhu -5° hingga -10° C mampu menghambat pertumbuhan Bacillus subtilis pada kadar 0,025 ug/uL dan Pseudomonas aeruginosa pada kadar 0,05 ug/uL. Sharifah Sakinah et al., (2007) melaporkan bahwa senyawa zerumbona dari Zingiber zerumbet me-miliki aktivitas antiproliferasi pada sel-sel HepG2 (sel kanker liver). Sedangkan Sharin (2006) melaporkan bahwa fraksi etil asetat dari ekstrak aseton dan ekstrak etanol menunjukkan aktivitas anti-oksidan yang baik. Dengan demikian kemungkinan fraksi kristal yang   Gambar 7. Spektra massa dari Selinena (Wiley 229, LIB).    Gambar 8. Spektra massa komponen fraksi kristal puncak no 8 dari kromatogram Gambar 1 teridentifikasi sebagai Zerumbona.    Gambar 9. Spektra massa dari Zerumbona (Wiley 229, LIB).   Tabel II. Pemeriksaan aktivitas anti-bakteri fraksi kristal yang terpisah dari minyak dan air hasil distilasi Rimpang Lempuyang Gajah       Diameter hambatan (mm)** Kadar fraksi dalam minyak kelapa S. aureus ATCC 25923 E. coli ATCC 35218 12,5 % 25 % 50 % --- 10 ±0,3 11 ±0,5 --- --- --- Streptomisin 25 µg/mL 17 ±1,4 15 ±1,2 Minyak kelapa --- ---  ** termasuk diameter sumuran   Sri Mulyani Majalah Farmasi Indonesia, 21(3), 2010 183terpisah dari minyak dan air hasil distilasi Zingiber zerumbet ini memiliki aktivitas lain yang lebih baik dibanding aktivitas anti-bakterinya, sehingga perlu dilakukan penelitian lebih lanjut.  Kesimpulan Komponen yang dapat diidentifikasi dari fraksi kristal yang terpisah dari air dan minyak hasil destilasi uap dan air rimpang lempuyang gajah adalah α-humulena, kariofilena oksida,           β-silenena, dan zerumbona. Larutan dari fraksi kristal dalam minyak kelapa mampu menghambat pertumbuhan S. aureus ATCC 25923 pada kadar 25 % dan sampai kadar 50 % belum mampu menghambat pertumbuhan E. coli ATCC 35218.   Daftar Pustaka  Agusta A, 2000. Minyak Atsiri Tumbuhan Tropik Indonesia, 89., ITB, Bandung Ahmad, B. A., Siddig, I. A., Adel, SAZ., Manal, M. E., and Syam M. M., 2008, Anticancer and Antimicrobial Activites of Zerumbone from the Rhizomes of Zingiber zerumbet.. International Journal of Pharmacology 4 (4), 301-304 Anonim, 1986, Medicinal Herbs Index in Indonesia., PT Eisei Indonesia ., 348-349 Bustanul, A., 2007, Uji Daya Antibakteri Minyak Atsiri Lempuyang Gajah (Zingiber zerumbet) terhadap Staphylococcus aureus ATCC 25923 dan Escherichia coli ATCC 35218, Skripsi, Fakultas Kedokteran UGM.  Carson, C. F, Mee, B. J., and Riley, T. V, 2002, Mechanism of Action of Melaleuca alternifolia               (Tea Tree) Oil on Staphylococcus aureus Determined by Time Kill, Lysis,                     Leakage, and salt tolerance assays and electron microscopy. http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=127210 Jang, D. S., Min H. Y., Kim M. S., Han, A. R., Wudono, T., Jeohu G. H., Kong, S. S., Lee, S. K., and Seo, E. K, 2005, Humulens Derivative from Zingiber zerumbet with the Inhibitory Effect on Lipopolysacharide-induced Nitric-oxide Production., Chem Pharm Bull (Tokyo) 53 (7), 829-831. http://agris.fao.org/agris-search/search/display.do?f= 1995/ US/US95207.xml;US9443476 Jawets, E, Melnick, J. L, and Adelberg, E. A, 1996, Mikrobiologi Kedokteran., Edisi XX., 234-240, Diterjemahkan oleh Nugroho Edi dan Maulany., EGC. Jakarta. Knobloch, K, Weis, N., and Weigand, H., 1986, Mechanism of Antimicrobial Activity of  Essential Oil, Planta Medica 6, 556. Lechat-Vahirua, I., Francois, P., Menut, C., Lamaty, G., and Bessiere, G. M., 1993, Essential oils from three Polynesian Zingiberaceae, J Riv. Ital. EPPOS, V 4, N spec. Num., p. 740-2. http://www.google.co.id/#hl=id&&sa=X&ei=L591TOX6M4KivQP5lpC2Bg&ved=0CBEQBSgA&q=Le+Chat-essential+oils+from+three+polynesian+ zingiberaceae & spell=1&fp=dc4b9ba5e001c2f8 Syahrizal, M., 2006, Kajian Kimia Zerumbon daripada Zingiber zerumbet Smith, Thesis Fakulti Sains Universiti Teknologi Malaysia. http://eprints.utm.my/5325/ Sawangjaroen, N., Phongpaichit, S., Subhadhirasakul, S., Vissuthi, M., Srisuwan, N., and Thammapalerd, N.,  2006, The Anti-amoebic Activity of Some Medicinal Plants Used by  AIDS Patients in Southern Thailand, Parasitol Res. 98 (6) : 588-92. Epub 2006 Jan 31. http://www.ncbi.nlm.nih.gov/pubmed/16447069 Sakinah, S., Tri Handayani., and Hawariah, A., 2007, Zerumbone Induced apoptosis in liver cancer           cells via modulation of Bax/Bcl-2 ratio, Cancer Cells International, 7:4, doi: 10. 1186/1475-2867.7-4. Pub. 3 April 2007. http://cancerci.com/ content/7 /1/4/a bstract Komponen  dan anti-bakteri dari fraksi……….. Majalah Farmasi Indonesia, 21(3), 2010 184 Sharin, 2006, Pemprofilan dan Pencirian Melalui LC-MS/MS Terhadap Komponen Aktif  Daripada Zinger Ubatan (Curcuma xanthorrhiza dan Zingiber zerumbet), Tesis, Universiti Putra Malaysia.http://psasir.upm.edu.my/374/2/549096_ib_2006_6_abstrak je dh pdf.pdf Sukandar, E. Y.,  Suganda, A. G.,  and Kristiana, A. S. R. D. W., 1997, Efek Antihelmintik            Zingiber zerumbet, terhadap cacing Ascaris suum., Majalah Farmasi Indonesia Edisi 8 No. 1, 12-22.  Tewtrakul, S., and Subhadhirasakul, S., 2007, Anti allergic Activity of Some Selected Plants in the Zingiberaceae Family, J. Ethnopharmacol, 109 (3) : 535-8. Epub 2006 Aug 15. http://www.ncbi.nlm.nih.gov/pubmed?term=%22Tewtrakul%20S%22[Author]    Korespondensi : Sri Mulyani Fakultas Farmasi UGM Skip Utara Yogyakarta 55281   

Indonesian

Component  and  antibacterial  activity  of  crystal  fraction  from Zingiber zerumbetessential oil

Indonesian Journal of Pharmacy

The aim of this study was to develop the valid analytical method which used for the assay of triamcinolone acetonide (TA) in the investigation of TA-loaded nanoparticle formulations. High Performance Liquid Chromatography (HPLC) method was applied in this study by using an Econosil column, C18 10 'm, 250 x 4.6 mm (Alltech Associates Inc, PA, USA) as the stationary phase. The mobile phase consisted of a composition of acetonitrile (ACN) and 20mM phosphate buffer solution (pH 4.2) in the proportion of 50:50 v/v. The HPLC assay of TA was validated for selectivity, linearity, precision, recovery (accuracy), sensitivity and stability of TA during the assay. Results showed that the concentration of TA in the samples can be determined against the standard in the concentration range of calibration curve. The system precision and level of recovery were considered to be acceptable, and the method was selective and sensitive.Key words: triamcinolone acetonide, assay, validatio

Nastiti, Christofori Maria Ratna Rini

Assay method validation of triamcinolone acetonide (TA) to support the investigation of TA-loaded nanoparticles

Rini Nastiti, Cr. Maria Ratna

Majalah Farmasi Indonesia, 20(4), 178 – 184, 2009 Majalah Farmasi Indonesia, 20(4), 2009 178 Assay method validation of triamcinolone acetonide (TA) to support the investigation of TA-loaded nanoparticles   Validasi metode penetapan kadar triamsinolon asetonida (TA) untuk mendukung investigasi nanopartikel TA  Christofori Maria Ratna Rini Nastiti Faculty of Pharmacy, Sanata Dharma University, Yogyakarta  Abstract  The aim of this study was to develop the valid analytical method which used for the assay of triamcinolone acetonide (TA) in the investigation of TA-loaded nanoparticle formulations. High Performance Liquid Chromatography (HPLC) method was applied in this study by using an Econosil column, C18 10 µm, 250 x 4.6 mm (Alltech Associates Inc, PA, USA) as the stationary phase. The mobile phase consisted of a composition of acetonitrile (ACN) and 20mM phosphate buffer solution (pH 4.2) in the proportion of 50:50 v/v. The HPLC assay of TA was validated for selectivity, linearity, precision, recovery (accuracy), sensitivity and stability of TA during the assay. Results showed that the concentration of TA in the samples can be determined against the standard in the concentration range of calibration curve. The system precision and level of recovery were considered to be acceptable, and the method was selective and sensitive. Key words: triamcinolone acetonide, assay, validation  Abstrak  Tujuan penelitian ini adalah untuk mengembangkan suatu metode penetapan kadar triamsinolon asetonida (TA) terkait dengan riset nanopartikel TA. Metode HPLC diaplikasikan dengan kolom Econosil sebagai fase diam. Sebagai fase gerak, diigunakan komposisi asetonitril: 2 nmM buffer fosfat               (pH 4.2) dengan perbandingan 50:50. Validasi metode penetapan kadar                 TA ditetapkan dalam hal selektivitas metode, linearitas respon, ketelitian, ketepatan, sensitivitas dan stabilitas TA selama penetapan kadar. Hasil menunjukkan bahwa kadar TA dapat ditetapkan dalam rentang kurva baku yang dikehendaki. Ketelitian dan ketepatan metode dapat diterima, dan metode selektif serta sensitif dalam menetapkan kadar TA pada sampel. Kata kunci: triamsinolon asetonida, penetapan kadar, validasi  Introduction  Recently, Triamcinolone acetonide (TA, MW: 434.5) with the structure as shown in Figure 1, has been extensively investigated in the treatment of a variety of ocular diseases associated with inflammation and neovascularisation (Jonas et al., 2005; Rechtman    et al., 2003; Ciulla     et al., 2003). The angiostatic effect of TA in treating neovascularisation (Obata et al., 2007), may provide potential for this agent to be developed as an antiangiogenesis agent, which could be useful in the treatment of a range of cancers. TA is stable in its solid state, but it is unstable in aqueous-alkaline and ethanolic solutions.  TA solution is more stable at pH values less than 5.5 and minimal degradation occurred at pH 3.4 (Gupta, 1982). As TA does not ionise, no information on the pKa of TA is available.  Christofori Maria Ratna Rini Nastiti Majalah Farmasi Indonesia, 20(4), 2009 179 By loading TA into nanoparticles (NPs), the nature of TA can be modified, leading to the improved delivery of the drug to the target site, which is facilitated by                 a controlled release. Besides, the NPs                    can protect TA from the unfriendly environment (Kreuter, 1994). Although TA loaded NPs have been initially investigated by Krause (1985), further research is needed using other biodegradable polymers and potentially superior methods, to improve the quality of NPs. To accommodate the further TA-loaded NPs  investigation, a valid analytical method of determining TA needs to be developed.  Several publications have reported the novel HPLC assay for TA (Gupta, 1982; Derendorf et al., 1986; Matysova et al., 2003). A method established by Gupta (1982) was adopted in this research with some modification since it is stability-indicating with relatively short retention time.  Material and Methods Materials Micronised TA, batch number: 2196NMO0100421, chemical grade (Farmabios, Italy); Acetonitrile, HPLC grade (LabScan, Thailand); Orthophosphoric acid,  Mw 98.00, BDH ‘AnalaR’, analytical grade, Min 85 % (BDH, Germany); Phosphate Buffer Solution pH 7.4 preserved by Sodium azide (PBS-NaN3) in house production; empty poly-DL-lactide NPs (PDLLA-NPs), empty poly(lactic-co-glycolic acid) NPs (PLGA-NPs) and empty methoxy-polyethyleneglycol, poly(D,L, lactic-co-glycolic acid) NPs (mPEG-PLGA NPs), all were synthesised in house.  Methods The TA HPLC method was adopted from a reported method (Gupta, 1982) with some modification. An Econosil column, C18 10 µm, 250 x 4.6 mm (Alltech Associates Inc, PA, USA) was used as the stationary phase. The mobile phase consisted of a composition of acetonitrile (ACN) and 20 mM phosphate buffer solution (pH 4.2) in the proportion of 50:50 v/v, filtered through a 0.45 µm membrane filter prior to use. The flow rate of 1.5 mL/min and the injection volume of 20 µL were used. The assay validation was conducted with TA standards dissolved in two media: the ACN:mobile phase 1:1 v/v (extracting media) and phosphate buffer              saline pH 7.4 containing 0.05 % w/v sodium                azide (PBS-NaN3, release media). To obtain a maximum sensitivity, TA was measured at its wavelength of maximum absorption: 242 nm. The temperature of the system was maintained at 25 °C. The HPLC assay of TA was validated           for selectivity, .linearity, precision, recovery (accuracy), sensitivity  and stability of TA during the assay.  Selectivity The peak purity of TA in two different media (ACN:mobile phase 1:1 and PBS-NaN3) containing TA and degradation products were analysed to determine the selectivity of the assay.   Linearity Linearity of the assay was determined in two types of media for TA loading and release study i.e. ACN:mobile phase 1:1 v/v and PBS-NaN3, respectively. The calibration curve was constructed for each type of medium by plotting peak areas against the concentration of TA standard solutions.  Recovery (accuracy) The accuracy of the HPLC assay was studied by examining the levels of recovery of TA in the presence of empty NPs made of three different polymers by the same method used for TA loading determination. The recovery was calculated by comparing the actual TA concentration detected with the initial concentration.   Figure 1. Structure of triamcinolone acetonide (Dollery, 1999). Assay method validation of  triamcinolone………… Majalah Farmasi Indonesia, 20(4), 2009 180 Sensitivity Blank solvents were injected six times consecutively into the HPLC column and the standard deviation of average noise levels in the expected retention time were determined. For the TA loading determination assay, blank solvent was defined as the loading media after being filtered from blank NPs, while in the release study, blank solvent was the PBS-NaN3. The theoretical LOD and LOQ were then calculated by the following formula:   3×=curvencalibratioofionconcentratversusheightofslopelevelsnoiseofdeviationndardtasLOD  10×=curvencalibratioofionconcentratversusheightofslopelevelsnoiseofdeviationndardtasLOQ  Stability assay of TA in aqueous environment  The TA solutions with known concentrations were placed in 1.5 mL vials for sampling and kept under the experimental temperatures.  Samples were taken at 3, 6, 9, 24, and 72 hours after initial incubation and determined by the HPLC assay. The potency of TA was determined according to the following formula:  100(%) ×=productsradationgdeandTAofareaspeaktotalTAofareapeakTAofPotency                       Results and Discussion To determine the TA concentration, an HPLC assay developed by Gupta  (1982) was adopted with some modification. Selectivity in the HPLC assay development was assessed to determine that the peaks can be well-separated (Figure 2). The retention time of TA and its degradation product in release medium (PBS-NaN3), which is speculated to be 6β-hydroxyltriamcinolone (Sieh, 1982), were approximately 3.7 and 2.9 minutes respectively, showing that they were completely separated (Figure 3.(c)).              Figure 2. Purity factor of TA peak on HPLC assay.   Table I. Precision of the HPLC method for TA determination  Analytes Average of AUC (mAu*sec) RSD (%) 1.03 µg/mL of TA solution in ACN: mobile phase 1:1 27.17 1.25 30.90 µg/mL of TA solution in ACN:mobile phase 1:1 764.33 1.61 0.53 µg/mL of TA in PBS-NaN3 13.12 0.53 5.27 µg/mL of TA in PBS-NaN3 130.00 0.08  Christofori Maria Ratna Rini Nastiti Majalah Farmasi Indonesia, 20(4), 2009 181In the release medium, the buffer substances and the sodium azide (NaN3, a preservative) eluted faster than TA (at 1.6 minutes), suggesting that those compounds did not interfere with the TA analysis. Correlation coefficient r2 was calculated for the calibration curve of area under the curve (AUC) against the TA concentration in both media (Figure 4). The detector’s response was found to be linear with r2 of 0.9999 and 1 for ACN:mobile phase 1:1 media and PBS-NaN3, respectively. This suggests that the concentration of TA in the samples can be determined against the standard in the concentration range of calibration curve.                                       a                   b             c               d  Figure 3. (a) Typical HPLC chromatogram of TA standard in ACN:mobile phase 1:1. (b) Typical HPLC chromatogram of TA extraction from loaded NPs in ACN: mobile phase 1:1. (c) Typical HPLC chromatogram of TA (peak 1) and  degradation product (peak 2) in PBS-NaN3 at 37 °C over 24 hours. (d) Typical HPLC chromatogram of PBS-NaN3 (release medium).  Table II: Recovery of TA in the presence of empty NPs in mobile phase  Parameters Empty PDLLA NPs Empty PLGA NPs Empty mPEG PLGA Recovery (%) 99.15 ± 2.79 99.69 ± 1.97 99.91 ± 2.41 Slope 0.9849 0.9941 0.9921 Correlation coefficient 0.9999 0.9999 0.9999 95 % CI slope 0.9738 - 0.9960 0.9813 - 1.0070  0.9798 – 1.0045 95 % CI Y-intercept -0.5187 to 0.5050 -0.6809 to 0.5036 -0.5961 to 0.5427  Assay method validation of  triamcinolone………… Majalah Farmasi Indonesia, 20(4), 2009 182 Precision for the HPLC assay is shown in Table I. The relative standard deviation (RSD) of the injections was obtained below the nominal acceptable level of 2 %, indicating that the injection of assay was precise for TA analysis. The recovery of TA was investigated for determination of TA loading to assess the interference of the NP matrix in the assay. The results are presented in Table II.  In the presence of empty PDLLA NPs, the 95 % confident interval (CI) of the slope was 0.9738-0.9960, not including                 1 (Table II). It indicates that the TA was not totally recovered by the assay method in the presence of the PDLLA matrix. However, the average of recovery was in the                value of  99.69 ± 1.97 %, with a slope and the correlation coefficient of 0.9849 and 0.9999, respectively. The 95 % CI of Y-intercept goes through zero, therefore, this level of recovery was considered acceptable. The recovery levels in the presence of empty PLGA NPs and empty 4.8 % mPEG-PLGA NPs revealed 95 % CI of slope including              1 and 95 % CI of Y-intercepts going through zero. These data indicate that the assay is unbiased and produces a satisfactory recovery. The recovery of TA in the           release media was not carried out since there was no extraction method prior to TA determination by HPLC in the release study.  In order to assess the sensitivity of the assay, the theoretical LOD and LOQ              were determined both in the loading determination and in the release study (Table III). To determine whether the stability of TA in the aqueous environment could be maintained during the assay, an investigation of the stability of TA in the aqueous medium was carried out. In this study, the potency of TA in aqueous solutions                was determined over 72 hours after storage at three different experimental temperatures:                4 °C, 25 °C, and 37 °C.  The data of stability study are shown in Figure 5 and 6. y = 23.675x - 1.8577R2 = 0.99990200400600800100012000 10 20 30 40Concentration (µg/mL)AUC (mAu*sec)   y = 24.922x - 0.06R2 = 102004006008000 10 20 30Concentration (mg/ml)AUC (mAu*sec)                                       a                                                                   b Figure 4. Standard curve for TA in ACN:mobile phase 1:1 (a) and  TA in PBS-NaN3 (b).  Table III. LOD/LOQ of the HPLC assay for TA determination    LOD (µg/mL) LOQ (µg/mL) Loading determination: - PDLLA NPs - PLGA NPs - mPEG PLGA NPs  0.090 0.094 0.118  0.299 0.315 0.394 Release study: 0.053 0.180  Christofori Maria Ratna Rini Nastiti Majalah Farmasi Indonesia, 20(4), 2009 183TA stability in the extraction medium (ACN:mobile phase 1:1 v/v)  was analysed using the HPLC assay. The results showed that TA degraded slightly faster at higher temperatures (Figure 5).  Although Gupta (1982) reported that TA is likely to be unstable at pH above 5.5 in an aqueous medium, a slow process of the degradation was also observed in the aqueous medium with pH approximately 7.  The stability of TA in the release medium was assessed at 4 °C, 25 °C, and            37 °C using HPLC assay. To investigate whether the stability of TA is affected by microbial activity, the study was conducted in the release media, with and without                 a preservative. It was found that the change of potency of TA appeared to be insignificant affected by the inclusion of preservative in the release media over 72 hours (Figure 6). It may suggest that microbial activity does not influence the degradation process of TA. In the release medium, TA was degraded the fastest at 37 °C, as predicted. After 72 hours storage at 37 °C, the degradation was increased dramatically up to 43 %. A substantial decomposition of TA was found at room temperature, with TA  potency  reduced to 85 % over 72 hours, while at 4 °C the TA showed less than 10 % degradation (6.4 %).   Figure 5. Potency of TA over 72 hours. Samples with a known concentration of TA in water were kept under experimental temperatures of 4 °C, 25 °C, and 37 °C for 72 hours.      Figure 6. Potency of TA at 72 hours. Samples with a known concentration of TA in PBS  and PBS–Na azide (PBS-NaN3) were kept under experimental temperatures of 4 °C, 25 °C, and 37 °C for 72 hours.    Assay method validation of  triamcinolone………… Majalah Farmasi Indonesia, 20(4), 2009 184 Conclusions The assay method of TA showed linear response of the detector suggesting that the concentration of TA in the            samples can be determined against the standard in the concentration range of calibration curve. The precision and                   level of recovery were considered to be acceptable, and the method was selective and sensitive.   Acknowledgements The author would like to acknow-ledge the Australian Development Scholar-ships for sponsoring author’s mastery study. The author also wishes to express sincere gratitude to Dr. Yan Chen, Assoc.Prof. Dr. Heather Benson, and Mr. Mike Boddy for their excellent guidance, supervision, continual technical support and invaluable encouragement in the laboratory. References  Ciulla, T. A., Chriswell, M. H., Danis, R. P., Fronheiser, M., Yuan, P.,  Cox, T. A.,  Csaky K. G.,  Robinson, M. R., 2003, Choroidal neovascular membrane inhibition in a laser treated rat model with intraocular sustained release triamcinolone acetonide microimplants. Br. J. Ophthalmol;87:1032-1037. Dollery, C.,1999, Therapeutic drugs. 2nd ed. Edinburgh: Churchill Livingstone Derendorf, H., Rohdewald, P., Hochhaus, G., Mollmann, H., 1986, HPLC determination of glucocorticoid alcohols, their phosphates and hydrocortisone in aqueous solutions and biological fluids. J. Pharm Biomed Anal, 4, 2, 197-206. Gupta, V., 1982, Stability of triamcinolone acetonide solution as determined by high performance liquid chromatography. J. Pharm Sci., 72,12,1453-1456. Jonas, J. B., Kreissig, I., Degenring R., 2005, Intravitreal triamcinolone acetonide for treatment of intraocular proliferative, exudative, and neovascular diseases. Prog Retin Eye Res., 24, 5, 587-611 Krause, H. J., Schwarz, A., Rohdewald. P., 1985, Polylactic acid nanoparticles, a colloidal drug delivery system for lipophilic drugs. Int. J. Pharm, 27, 2-3, 145-155. Kreuter, J., 1994, Nanoparticles. In: Kreuter, J., editor. Colloidal drug delivery systems. New York: Marcel Dekker Matysová, L, Hájková R, Sicha, J., Solich, P., 2003., Determination of methyparaben, propylparaben, triamcinolone acetonide, and its degradation product in topical cream by RP-HPLC. Anal. Bioanal Chem, 376,4, 440-443 Obata, R., Iriyama, A., Inoue, Y., Takahashi, H., Tamaki, Y., Yanagi, Y.,2007,  Triamcinolone acetonide suppresses early proangiogenic response in retinal pigment epithelial cells after photodynamic therapy in vitro. Br. J. Ophthalmol, 91, 1, 100-4. Rechtman, E., Allen, V. D., Danis, R. P., Pratt, L. M., Harris, A., Speicher M A.,2003, Intravitreal triamcinolone for choroidal neovascularization in ocular histoplasmosis syndrome. Am. J. Ophthalmol.,136, 4, 739-741. Sieh D. H. 1982, Triamcinolone acetonide. in: Florey K, editor. Analytical profiles of drug substances. New Jersey: Academic Press; p. 619-623.   * correspondence: Christofori Maria Ratna Rini Nastiti Faculty of Pharmacy, Sanata Dharma University, Yogyakarta  e-mail : ratnarini@staff.usd.ac.id 

Assay method validation of triamcinolone  acetonide (TA) to support the investigation of  TA-loaded nanoparticles

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Assay method validation of triamcinolone acetonide (TA) to support the investigation of TA-loaded nanoparticles

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