Identifikasi komponen antioksidan buah Andaliman (Zanthoxylum acanthopodium DC.)

SURYANTO, Edi, Promotor Prof.Dr. Tranggono, M.Sc

2006 | Disertasi | S3 Ilmu Pertanian (Ilmu Pangan)

Buah andaliman (Zanthoxylum acanthopodium DC.) dimanfaatkan sebagai bumbu masakan yang biasanya digunakan dalam makanan tradisional di Sumatera Utara. Buah andaliman juga dikenal sebagai obat tradisional untuk pengobatan beberapa jenis penyakit. Penelitian ini bertujuan mengisolasi dan mengidentifikasi senyawa dalam ekstrak andaliman yang mempunyai aktivitas antioksidatif dan antifotooksidatif. Buah andaliman diekstraksi secara sekuensial dengan menggunakan heksana, aseton dan etanol masing-masing selama 24 jam. Ekstrak andaliman selanjutnya dipisahkan dan diisolasi dengan kromatografi kolom dan campuran etil asetat-metanol digunakan sebagai fasa gerak dan silika gel G-60 sebagai fasa diam yang dielusi secara gradien. Senyawa yang terisolasi ditentukan strukturnya dengan metoda ultraviolet (UV), infra merah (IR), resonansi magnetik inti (1H and 13C-NMR), and spektrometer massa (MS). Aktivitas antioksidan dari ekstrak andaliman, fraksi-fraksi, subfraksi dan senyawa murni pada konsentrasi 50-500 ppm dievaluasi dengan daya reduksi dan penangkap radikal bebas 1,1-difenil-2- pikrilhidrazil (DPPH), sedangkan aktivitas antifotooksidatif digunakan asam linoleat dan minyak kacang tanah yang mengandung 100 ppm eritrosin sebagai sensitiser. Selain itu, ketiga ekstrak juga dievaluasi stabilitasnya terhadap panas pada 100 oC selama 15, 30, 60 dan 120 menit dan pemanasan pada 100, 120, 140 dan 180 oC selama 60 menit. Selanjutnya perlakuan ketiga ekstrak yang disinari cahaya fluoresen (4000 lux) dan cahaya ultraviolet C (200-280 nm) selama 5 jam. Ekstrak yang mempunyai aktivitas tertinggi selanjutnya dievaluasi pada otooksidasi dan fotooksidasi ikan mas (Cyprinus carpio) giling mentah dan masak. Untuk otooksidasi, diperlakukan pada konsentrasi (0, 200, 400, 600 dan 1000 ppm) yang disimpan dalam tanpa cahaya pada suhu 5 oC selama 10 hari, sedangkan fotooksidasi digunakan konsentrasi 0, 100, 200, dan 400 ppm dalam 3 ppm riboflavin sebagai sensitiser yang disinari cahaya fluoresen (4000 lux) selama 56 jam pada suhu 5 oC. Oksidasi lipida diukur dengan angka asam tiobarbiturat (TBA). Penambahan ekstrak sekuensial heksana aseton etanol (ESHAE) pada berbagai konsentrasi menunjukkan daya reduksi yang kuat daripada ekstrak sekuensial heksana aseton (ESHA) dan ekstrak heksana (EH). Ekstrak ESHAE pada konsentrasi 500 ppm menunjukkan sama dengan 200 ppm BHT, tetapi lebih tinggi daripada α-tocopherol. Pengaruh ESHAE menunjukkan aktivitas penangkap radikal bebas DPPH paling kuat diikuti oleh ESHA dan EH. Ekstrak ESHAE menunjukkan aktivitas penangkap radikal terkuat daripada kontrol positif, BHT dan α-tokoferol dalam sistem DPPH. Ekstrak ESHAE memiliki stabilitas tinggi terhadap panas (100 oC), cahaya fluoresen dan cahaya ultraviolet. Pengaruh antifotooksidatif dari ESHAE lebih tinggi diikuti ESHA dan EH. Asam linoleat yang diperlakukan dengan 500 ppm ESHAE dan 500 ppm α- tokoferol menunjukkan angka peroksida lebih rendah dibandingkan dengan kontrol selama 5 jam disinari cahaya fluoresen pada suhu kamar. ESHAE menunjukkan perbedaan efektivitas dengan α-tokoferol (p<0.05). Sejalan dengan itu, ESHAE juga menunjukkan pengaruh penstabil paling kuat daripada α- tokoferol dalam minyak kacang tanah murni (p<0,05), sedangkan ekstrak EH dan ESHA bahkan menunjukkan peningkatan angka peroksida dan sebagai profotooksidatif. Ekstrak ESHAE selanjutnya dipisahkan menjadi 5 fraksi utama dengan kromatografi kolom. Semua fraksi menunjukkan aktivitas sebagai penangkap radikal bebas. Fraksi II, III dan V menunjukkan aktivitas paling kuat diikuti oleh I dan V. Akan tetapi, fraksi II, II dan V pada konsentrasi 200 ppm tidak berbeda secara signifikan (p<0,05). Ketiga fraksi menunjukkan aktivitas penangkap radikal lebih besar daripada BHT dan α-tokoferol. Semua fraksi menunjukkan pengaruh sebagai antifotooksidatif pada penyinaran cahaya fluoresen (4000 lux) selama 5 jam. Fraksi II menunjukkan efektivitas paling kuat diikuti I, IV dan V. Akan tetapi fraksi II menunjukkan efektivitas penstabil oksigen singlet tidak berbeda nyata dengan fraksi III (p<0,05). Pengaruh penstabil fraksi II dan III menunjukkan paling kuat daripada α-tokoferol. Fraksi II selanjutnya direkromatografi, senyawa yang telah dimurnikan dikarakterisasi dengan metoda spektrometer UV, IR, NMR dan MS, senyawa antioksidatif yang terisolasi dari ekstrak andaliman diidentifikasi sebagai 3-benzoiloksi-17-(1-etil-pentil)-6-en-16- asam karbosilat-3-etoksi-propil ester dan selanjutnya disebut Endalin. Aktivitas penangkap radikal dari Endalin lebih rendah daripada BHT dan α-tokoferol, sedangkan pengaruh antifotooksidatif secara signifikan menurunkan pembentukan peroksida dibandingkan kontrol selama 5 jam disinari cahaya fluoresen. Akan tetapi, Endalin pada level 200 ppm menunjukkan aktivitas antifotooksidatif yang lebih rendah daripada α-tokoferol (p<0,05). Ekstrak ESHAE pada konsentrasi 200 ppm dan 1000 ppm tidak menujukkan perbedaan pengaruh antioxidatif dengan 200 ppm BHT dalam ikan mas mentah, sedangkan dalam ikan masak menunjukkan perbedaan efektivitas dengan 200 ppm BHT. Ekstrak ESHAE pada 100, 200 dan 400 ppm secara signifikan menurunkan oksigen singlet dalam ikan mas mentah yang disinari cahaya fluoresen 4000 lux sedangkan untuk ikan mas masak menunjukkan perbedaan pengaruh antifotooksidatif. Penambahan 400 ppm ESHAE menunjukkan pengaruh antifotooksidatif yang sama dengan BHT (p<0,05). Hasil penelitian ini menemukan bahwa ESHAE dari buah andaliman mengandung komponen-komponen yang mempunyai antioksidatif dan antifotooksidatif terhadap daya reduksi, radikal bebas DPPH, asam linoleat, minyak kacang tanah dan daging ikan mas. Senyawa yang teridentifikasi dari ekstrak andaliman mempunyai pengaruh antioksidatif dan antifotooksidatif sebagai senyawa 3-benzoiloksi-17-(1-etil-pentil)-6-en-16-asam karbosilat-3- etoksi-propil ester.

Andaliman (Zanthoxylum acanthopodium DC.) is a fruit commonly used for seasoning of some traditional food in North Sumatera. It is also well known as a folk medicine source to cure several kind of illness. The objectives of the research were to isolate and to identify some responsible compounds in andaliman extract having both antioxidative and antiphotooxidative activities. Andaliman fruit was sequentially extracted, with hexane, acetone and ethanol for 24 hours, respectively. The extracts of andaliman fruit were further separated and isolated with column chromatography, using ethyl acetate-methanol as mobile phase and silica gel G-60 as stationary phase by gradient elution. The isolated compounds were elucidated by ultraviolet (UV), infrared (IR), nuclear magnetic resonance (1H and 13C-NMR), and mass spectrometry (MS) techniques. Antioxidant activity of each extracts, fractions, sub fractions and pure compound of andaliman fruits at 50-500 ppm level were evaluated using reducing power, 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical. On the other hand, antiphotooxidative activity test was conducted using linoleic acid and peanut oil as substrates each containing 100 ppm erythrosine as a photosensitizer. In addition, the third extract was evaluated by heating at several high temperatures i.e. at 100 oC for 15, 30, 60, and 120 minutes or at 100, 120, 140 and 180 oC for 60 minutes. The light exposure was done under 4000 lux fluorescent lamp and ultraviolet C (200-280 nm) light for 5 hours. Extract with highest activity was further evaluated by autooxidation and photooxidation using raw and cooked fleshes of common carp (Cyprinus carpio). For autooxidation treatment, the extract was tested in 0, 200, 400, 600, and 1000 ppm concentration levels in common carp fleshes, stored in the dark for 10 days at 5 oC. Furthermore, the photooxidation was carried out in 0, 100, 200, and 400 ppm concentration of the extract with 3 ppm riboflavin as sensitizer and held under 4000 lux fluorescent light for 56 hours at 5 oC. Lipid oxidation was accessed with TBA number. The addition of ethanol extract (ESHAE) at different concentration exhibited excellent reduction power which was higher than those of acetone extract (ESHA) and hexane extract (EH). The ethanol extract (ESHAE) at 500 ppm concentration showed an equal result with 200 ppm BHT, however, higher than that of α-tocopherol. The effect of using ESHAE exhibited the highest scavenging activity in DPPH radical, followed by ESHA and EH, respectively. ESHAE had stronger radical scavenging activity than the positive control, BHT and α-tocopherol in DPPH system. It was indicated that ESHAE at 200 ppm possessed extremely high stability in heating at 100 oC, under fluorescence and ultraviolet lights. Ethanol extract (ESHAE) was found to have the highest antiphotooxidative effect, followed by those of ESHA and EH, respectively. Linoleic acid treated will ESHAE at 500 ppm concentration and 500 ppm α-tocopherol showed lower peroxide value compare to the control samples at 5 hours light exposure in room temperature. ESHAE showed higher activity in its antiphotooxidative effect than that of α-tocopherol as a positive control (p<0.05). Similarly, ethanol extract revealed also stronger quencher effect than α-tocopherol in peanut oil, whereas EH and ESHA even demonstrated an increased in peroxide value and as prophotooxidative agents (p<0,05). The ethanolic extracts of andaliman fruits was further separated into five major fractions with column chromatography. All fractions were verified having activities as free radical scavengers. Fraction II, III and V exhibited higher activity, and it was followed by I and IV. However, the third showed no significant difference. Three fractions exhibited the greatest radical scavenging activity than those of α-tocopherol and BHT (p<0,05). An anti-photooxidative effect under fluorescent light illumination (4000 lux) for 5 hours was shown by all fractions. Fraction II showed the strongest effect and followed in order by I, IV and V (p<0,05). However, fraction II were discovered having properties as quencher of singlet oxygen effectively as well as fraction III. The quenching effect of fractions II and III were much higher than that of α-tocopherol (p<0,05). This ethanolic fraction II was further rechromatograped. The UV, IR, NMR, and MS spectrometry techniques were used to characterize its purified compound. The antioxidative compound, isolated from andaliman extract, was identified as 3- benzoyloxy-17-(1-ethyl-pentyl)-6-en-16-carboxylic acid-3-ethoxy-propyl ester and further named as Endalin. The radical scavenging activity of Endalin was less than that of BHT and α-tocopherol, whereas effect of its anti-photooxidative significantly decreased its peroxide formation capacity compared to the control samples in 5 hours fluorescent light illumination. However, Endalin at a level of 200 ppm showed much lower antiphotooxidative activity than α-tocopherol (p<0,05). Ethanol extract (ESHAE) at 200 and 1000 ppm concentration showed no difference in their antioxidative effect with 200 ppm BHT when applied in raw muscles of common carp, whereas application in cooked tissues showed contradictory effect with 200 ppm BHT. Treatment with ethanol extract at 100, 200, and 400 ppm significantly reduced singlet oxygen in raw muscle tissues, whereas for cooked tissues showed difference effect. The addition of ethanol extract at concentration of 400 ppm into the tissues exhibited the same antiphotooxidative effect with BHT. These results suggest that the ethanolic extract of andaliman fruit contains some compounds having antioxidative and antiphotooxidative activities on reducing power, free radical DPPH, linoleic acid, peanut oil and muscles of common carp. In addition, pure compound of andaliman extract was found having both antioxidative and antiphotooxidative effects and identified as a 3- benzoyloxy-17-(1-ethyl-pentyl)-6-en-16-carboxylic acid-3-ethoxy-propyl ester compound.

Kata Kunci : Antioksidatif dan Antifotooksidatif,Ekstrak Buah Andaliman