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Abstrak :
Paparan sinar UV yang berlebihan merupakan salah satu faktor ekstrinsik penyebab terjadinya penuaan dini, yaitu akibat terbentuknya radikal bebas dan Reactive Oxygen Species (ROS). Rubus fraxinifolius Poir. terutama pada bagian daunnya terbukti secara in vitro memiliki sifat antipenuaan dengan cara mencegah aktivitas enzim elastase. Tujuan dari penelitian ini adalah menganalisis kadar total fenol, kadar total flavonoid, aktivitas antioksidan DPPH (1,1-difenil-2-pikrilhidrazil), FRAP (Ferri Reducing Antioxidant Power) dan ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) serta aktivitas penghambatan elastase dari ekstrak (hasil optimasi jenis daun dan optimasi metode ekstraksi) dan fraksi daun Rubus fraxinifolius, selanjutnya fraksi ter-aktif diformulasikan dalam sediaan gel. Penelitian diawali dengan optimasi jenis daun terlebih dahulu dengan cara ekstraksi terhadap daun tua dan daun muda secara Soxhlet, dan dievaluasi aktivitas antioksidan dan aktivitas penghambatan elastase. Selanjutnya dilakukan optimasi metode ekstraksi secara Soxhlet, Microwave Assisted Extraction (MAE) dan Ultrasound Assisted Extraction (UAE) kemudian dievaluasi aktivitas antioksidan dan aktivitas penghambatan elastase. Ekstrak teraktif selanjutnya dilakukan fraksinasi dengan n-heksana, etil asetat, dan aquadest. Fraksi teraktif dilakukan pengujian antioksidan dan aktivitas penghambatan elastase, kemudian diidentifikasi menggunakan LC-MSMS. Berdasarkan hasil optimasi jenis daun, ekstrak daun tua memiliki kinerja yang lebih baik dibandingkan dari daun muda dalam hal aktivitas antioksidan dan aktivitas penghambatan enzim elastase. Sedangkan hasil optimasi metode ekstraksi, UAE memberikan hasil terbaik untuk selanjutnya difraksinasi dan fraksi aquadest memberikan hasil terbaik aktivitas antioksidan dan aktivitas penghambatan enzim elastase. Formulasi gel fraksi aquadest dari metode ekstraksi secara UAE menunjukkan pada formula 1 dengan konsentrasi fraksi daun Rubus 3% memberikan hasil yang lebih baik dan stabil selama masa penyimpanan 12 minggu. ......Excessive exposure to UV light is one of the extrinsic factors that causes premature aging, which is due to the formation of free radicals and Reactive Oxygen Species (ROS). Rubus fraxinifolius Poir. especially in the leaves have antiaging in vitro properties by preventing the activity of elastase enzyme. The purpose of this study was to analyze total phenol levels, total flavonoid levels, antioxidant activity of DPPH (1,1-diphenyl-2-pikrilhidrazyl), FRAP (Ferri Reducing Antioxidant Power) and ABTS (2,2'-azino-bis(3- ethylbenzothiazoline-6-sulfonic acid) also elastase inhibitory activity of the extract (from optimizing leaf types and optimizing extraction methods) and Rubus fraxinifolius leaf fractions, then the most active fractions are formulated in Gel preparations. The research begins with optimizing leaf types first, by extraction old leaves and young leaves used Soxhlet method, and evaluated their antioxidant activity and elastase inhibition activity. Furthermore, optimization of extraction methods (Soxhlet, Microwave Assisted Extraction/ MAE and Ultrasound Assisted Extraction/ UAE) was carried out and then evaluated antioxidant activity and elastase inhibitory activity. Active extract then fractionated with n-hexane, ethyl acetate, and aquadest. The most active fraction was tested for antioxidant and elastase inhibitory activity, then identified using LC-MSMS. Based on the optimization of leaf types, old leaf extract has better performance than young leaves in terms of antioxidant activity and elastase enzyme inhibitory activity. While the optimization results of extraction method UAE gave the best results. Fractionation process gave aquadest fraction the best results for antioxidant activity and elastase enzyme inhibitory activity. The gel formulation of the aquadest fraction from the UAE extraction method showed that formula 1 with a concentration of 3% Rubus leaf fraction gave better and more stable results during a storage period of 12 weeks.

Abstrak :

Rubus fraxinifolius dan R. rosifolius merupakan tanaman Rubus yang dapat ditemukan di daerah pegunungan Indonesia. Kedua tanaman memiliki morfologi buah yang mirip yaitu berbentuk berry merah dan edible, serta mengandung senyawa golongan triterpenoid, polifenol dan flavonoid.  Beberapa spesies Rubus dilaporkan memiliki aktivitas sebagai antiaging yaitu antielastase, antioksidan, dan antitirosinase. Penelitian ini bertujuan untuk menelaah aktivitas antiaging secara in vitro pada ekstrak dan isolat dari tanaman R. fraxinifolius dan R. rosifolius. Batang, buah, dan daun kedua tanaman diekstraksi menggunakan alat Soxhlet serta dilakukan skrining aktivitas anti elastase dan antioksidan.  Selanjutnya terhadap ekstrak terpilih dilakukan pemisahan, fraksinasi dan isolasi senyawa. Isolat yang didapat diidentifikasi menggunakan spektrometri FTIR, ¹H-NMR, ¹³C-NMR, DEPT, HSQC, HMQC, HMBC, dan LC-MS, serta diuji aktivitas antielastase, antitirosinase dan sitotoksisitas pada sel fibroblast secara in vitro. Hasil ekstraksi bertingkat menunjukkan bahwa ekstrak metanol daun R. fraxinifolius memiliki aktivitas antielastase dan antioksidan tertinggi dengan masing-masing IC₅₀ 57,45 dan 4,33 µg/ml. Terhadap fraksi metanol daun R. fraxinifolius (DFM) dilakukan pemisahan menggunakan kromatografi cair vakum dan diperoleh 11 fraksi. Uji antielastase fraksi menunjukkan fraksi paling aktif adalah DFM8. Selanjutnya dilakukan isolasi lebih lanjut terhadap DFM8 dan diperoleh 3 isolat. Hasil elusidasi struktur menunjukkan bahwa ketiga isolat merupakan suatu triterpen pentasiklik tipe ursan. Hasil telaah data pengujian DEPT, HMQC, HSQC, HMBC serta IR dan MS disimpulkan senyawa DFM 8a adalah asam 2,3-glikol, 19α-hidroksi-12-ursen-23,28-dioat (C₃₂H₄₈O₇, BM 544); DFM8b asam 2,3-propanandiol, 19α-hidroksi-12-ursen-28-oat (C₃₃H₅₂O₅, BM 528,38); dan DFM8c asam 2,3-glikol-19α-hidroksi-23,24-nor-12-ursen-28-oat (C₃₀H₄₆O₅, BM 486,33). Ketiga senyawa hasil isolasi ini merupakan senyawa baru dan belum pernah ditemukan sebelumnya. Uji aktivitas antielastase senyawa DFM8a, DFM8b, dan DFM8c memiliki IC₅₀ berturut-turut adalah 122,199; 98,22; dan 54,33 µg/ml, serta antitirosinase dengan IC₅₀ 207,8; 221,5; dan 335,9 µg/ml. Uji toksisitas menunjukkan bahwa ekstrak DFM, fraksi DFM8, dan isolat DFM8b tidak toksik terhadap sel fibroblas NIH/3T3.

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Rubus fraxinifolius and R. rosifolius are Rubus genus, which can be found in the mountain of Indonesia. Both plants have similar fruit morphology: red and edible berries and contain triterpenoid, polyphenols, and flavonoids. Some species of Rubus are reported to have antiaging activity, antielastase, antioxidant, and antityrosinase. This study aims to examine the in vitro antiaging activity of extracts and isolated compounds from R. fraxinifolius and R. rosifolius. The stems, fruits, and leaves of both plants were extracted and screened for antielastase and antioxidant activity. Furthermore, the selected extracts were separated, fractionated, and isolated to yield isolates. The obtained isolates were identified using FTIR spectrometry, ¹H-NMR, ¹³C-NMR, DEPT, HSQC, HMQC, HMBC, and LC-MS, and also were tested for antielastase, antityrosinase, and cytotoxicity activities in fibroblast cells. The continuous extraction results showed that the methanol extract of R. fraxinifolius leaves had the highest antielastase and antioxidant activity with IC₅₀ 57.45 ppm and 4.33 ppm, respectively. The methanol fraction of R. fraxinifolius (DFM) leaves were separated using vacuum liquid chromatography and obtained 11 fractions. The antielastase assay of fractions gave the most active fraction was DFM8. Then, further isolation of DFM8 was carried out, and three isolates were obtained. The structural elucidation showed that the three isolates were ursane-type of pentacyclic triterpenes. The results of DEPT, HMQC, HSQC, HMBC, IR and MS spectrometry test concluded that the compound DFM 8a was 2,3-glycol, 19α-hydroxy-12-ursen-23,28-dioic acid (C₃₂H₄₈O₇, MW 544); DFM8b 2,3-propanandiol, 19α-hydroxy-12-ursen-28-oic acid (C₃₃H₅₂O₅, MW 528.38); and DFM8c 2,3-glycol-19α-hydroxy-23,24-nor-urs-12-en-28-oic acid (C₃₀H₄₆O₅, MW 486.33). All isolated compounds are new compounds and have never been found before. The IC₅₀ of antielastase activity of DFM8a, DFM8b, and DFM8c were 122.199; 98.22; and 54.33 ppm, respectively, and the IC₅₀ of antityrosinase activity were 207.8; 221.5; and 335.9 ppm, respectively. Toxicity tests showed that the DFM extract, the DFM8 fraction, and the DFM8b were not toxic to NIH/3T3 fibroblast cells.