Isolation and structure elucidation of Tracheospasmolytic Compounds from Vitex trifolia L. Leaves

ALAM, Gemini, Promotor Prof.Dr. Ibnu Gholib Gandjar, DEA.,Apt

2005 | Disertasi | S3 MIPA (Ilmu Farmasi)

Vitex trifolia L. (Verbenaceae) adalah salah satu tanaman yang telah digunakan sebagai obat tradisional di Indonesia untuk mengobati gangguan saluran pernafasan, demam, sakit kepala, diuretik dan anti serangga. Penelitian ini ditujukan untuk mendapatkan senyawa aktif trakeospasmolitik dari ekstrak daun V. trifolia. Isolasi senyawa trakeospamolitik dari daun V. trifolia didasarkan atas bioassay-guided isolation. Fraksinasi dilakukan menggunakan pelarut organik yang sesuai untuk memisahkan campuran senyawa dari ekstrak hingga diperoleh 6 fraksi (A-G); Fraksi G merupakan fraksi paling aktif. Selanjutnya, fraksi G difraksinasi dengan VLC didapatkan 6 fraksi (F1-F6); Fraksi F5 menunjukkan aktivitas paling besar. Fraksi F5 dipisahkan dengan kromatografi kolom menggunakan Lobar B-prep column hingga diperoleh 3 fraksi aktif (F5b, F5c, dan F5d), setiap fraksi tersebut mengandung satu senyawa utama. Tiga senyawa murni diperoleh melalui pemurnian dengan KLT Preparatif; senyawa 1 dan 3 adalah senyawa golongan diterpenoid dan 2 flavonoid. Berdasarkan data spektroskopi (UV, IR, MS, dan NMR) dan pustaka, senyawa 1 telah diidentifikasi sebagai viteosin-A atau (8R,10S)-6β-asetoksi-9α-hidroksi-labda-13Z-en-15,16-olid, senyawa 2 sebagai viteksikarpin or 3’,5-dihidroksi-3,4’,6,7-tetrametoksiflavon, and senyawa 3 vitetrifolin-E atau (6S,7R,8S,9R)-6-asetoksi-5(10),14-halimadien- 7,13-diol. Semua senyawa (1–3) menunjukkan aktivitas trakeospasmolitik. Viteosin- A (1.3 x 10-4 M, n=3) menghambat 27% kontraksi trakea yang diinduksi histamin (10-7 – 10-3 M). Apabila konsentrasi dinaikkan tiga kali lipat (4.0 x 10-4 M, n=3), penghambatan viteosin-A meningkat menjadi 47%. Viteksikarpin juga menghambat kontraksi trakea lebih baik dari pada viteosin-A, karena viteksikarpin mampu menghambat 33% kontraksi pada konsentrasi yang sama (1.3 x 10-4 M). Bila konsentrasi viteksikarpin dinaikkan (4.0 x 10-4 M), penghambatan kontraksi mencapai 97%. Sedangkan vitetrifolin-E (1.3 x 10-4 M) hanya menghambat 22% kontraksi trakea, sedangkan pada konsentrasi 4.0 x 10-4 M kontraksi trakea dihambat sebanyak 86%. Mekanisme aksi tiga senyawa tersebut adalah antagonisme non-kompetitif. Efek antialbumin menggunakan trakea marmut yang telah disensitisasi menunjukkan bahwa vitetrifolin-E menghambat kontraksi trakea lebih kecil dari viteksikarpin (83%); aktivitas penghambatannya berada diantara viteosin-A (26%) dan viteksikarpin (93%). Mekanisme aksi ketiga senyawa tersebut kemungkinan adalah meningkatkan stabilitas membran sel mast. Aktifitas trakeospasmolitik sediaan infus daun V. trifolia menunjukkan penghambatan kontraksi otot polos trakea marmut yang telah diinduksi dengan histamin. Analisis kuantitatif dengan metode KLT-Densitometri, kadar viteksikarpin pada sediaan infus 30% adalah 0,52 ± 0,005 % b/v. Pada infus tersebut (100 μl infus 30%) kontraksi trakea yang diinduksi oleh histamin dapat dihambat sebanyak 34%.

Vitex trifolia L. (Verbenaceae) is one of the plants that are used in Indonesian traditional medicines for the treatment of respiratory disorders, colds, headaches, diuretic and as a mosquito repellent. Plants of the genus of Vitex are rich in diterpenoids, flavonoids, and iridoids, which might be responsible for their effectiveness in traditional medicine. The present investigations focused on the tracheospasmolytic active compounds in V. trifolia leaves extract. Isolation of tracheospasmolytic compounds from the leaves of V. trifolia carried out by a bioassay-guided isolation. Fractionations using suitable organic solvents were used to separate the mixture of compounds from crude extract to give six fractions (A-G); fraction G was the most active to inhibit tracheal contraction. A VLC fractionation of fraction G resulted in six (F1-F6) active fractions; F5 was the most active one. Further separation of F5 by Lobar B-prep column resulted in 3 active fractions (F5b, F5c, and F5d), each of them contained one major compound. Purification was done by preparative TLC. Three pure compounds were isolated; compound 1 and 3 are diterpenoids and 2 belong to flavonoid class of compounds.Based on the spectroscopic data (UV, IR, MS and NMR) and comparison with literature, compound 1 was identified as viteosin-A or (8R,10S)-6β-acetoxy-9α-hydroxy-labda-13Z-en-15,16-olide, compound 2 as vitexicarpin or 3’,5-dihydroxy-3,4’,6,7-tetramethoxyflavone, and compound 3 as vitetrifolin-E or (6S,7R,8S,9R)-6-acetoxy-5(10),14-halimadien-7,13-diol. All these compounds show tracheospasmolytic activity on guinea-pig tracheal smooth muscle contracted by histamine. Viteosin-A (1.3 x 10-4 M, n=3) inhibits 27% of the trachea contraction induced by histamine (10-7 – 10-3 M). When the concentration is raised to 3 fold (4.0 x 10-4 M, n=3), viteosin-A inhibits 47%. Vitexicarpin inhibits the trachea contraction better than that viteosin-A, as vitexicarpin is able to inhibit 33% of the contraction at the equal concentration of 1.3 x 10-4 M. When the concentration of vitexicarpin is raised to 4.0 x 10-4 M, an almost complete inhibition is obtained (97%). A bit different to the first two compounds, vitetrifolin-E (1.3 x 10-4 M) only inhibited the tracheal contraction by 22%, but when the concentration was raised to 4.0 x 10-4 M the tracheal contraction was tremendously inhibited by 86%.The mechanism of action of the three active compounds was a non-competitive antagonism. Anti-albumin effects in a model using trachea from sensitized male guinea-pig have shown that vitetrifolin-E inhibited tracheal contraction a bit less than that of vitexicarpin (83%); its activity was in between of those of viteosin-A (26%) and vitexicarpin (93%). Mode of action of the three isolated compounds was possibly improving the mast cell membrane stability. Tracheospasmolytic activity of infusion preparations of V. trifolia leaves was inhibiting of tracheal guinea pig smooth muscle contraction induced by histamine. A quantitative analysis of vitexicarpin on 30% infusion preparations of V. trifolia leaves by TLC-Densitometry was 0.52 ± 0.005 % w/v. This infusion (100 μl of infusion 30%) inhibited 34% tracheal contraction induced by histamine.

Kata Kunci : Obat Tradisional,Ekstrak Daun Vitex trifolia,Tracheospasmolytic