Pemeriksaan Sebatian Polifenolik Dalam Kalus Cincau Hijau (Premna oblongifolia) Yang Diaruh Kombinasi Pengawalatur Pertumbuhan Berbeza
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1 Undergraduate Research Journal for Biomolecular Sciences and Biotechnology ISSN Pemeriksaan Sebatian Polifenolik Dalam Kalus Cincau Hijau (Premna oblongifolia) Yang Diaruh Kombinasi Pengawalatur Pertumbuhan Berbeza Muhammad Amran Afrizal 1, Zuraidah Abd Rahman 2 & Che Radziah Che Mohd Zain 1 * 1 Pusat Pengajian Biosains dan Bioteknologi, Fakulti Sains dan Teknologi, Universiti Kebangsaan Malaysia, Bangi, Selangor, Malaysia *mel-e pengarang rujukan: cradziah@ukm.edu.my Abstrak: Premna oblongifolia atau lebih dikenali sebagai cincau hijau adalah spesies tumbuhan yang tergolong dalam keluarga Verbenaceae. Ekstrak daun tumbuhan ini mampu untuk merawat demam, mengurangkan sakit perut, dan tekanan darah tinggi. Keupayaan tumbuhan ini merawat penyakit berkait dengan kandungan sebatian polifenoliknya yang berfungsi sebagai antioksidan dan anti-radang. Oleh itu, kajian ini dijalankan untuk menentukan kepekatan pengawalatur pertumbuhan yang optimum untuk memaksimumkan sintesis sebatian polifenolik (flavonoid dan fenolik) dari kalus tumbuhan tersebut secara in vitro. Sebanyak 15 rawatan dikenakan terhadap kalus iaitu BAP tunggal, kombinasi BAP dan NAA, dan kombinasi BAP dan Kinetin. Pengukuran sebatian polifenolik dilakukan pada bulan pertama dan kedua. Penghasilan sebatian fenolik tertinggi didapati daripada rawatan kombinasi 3.0 mg/l BAP dan 0.5 mg/l NAA iaitu sebanyak mg GA/g pada bulan pertama. Manakala, kalus yang menghasilkan sebatian flavonoid tertinggi adalah daripada rawatan kombinasi 3.0 mg/l BAP dan 2.0 mg/l NAA iaitu sebanyak mg QE/g juga pada bulan pertama. Jumlah kandungan sebatian fenolik dan flavonoid yang dihasilkan oleh rawatan kombinasi BAP dan NAA adalah lebih tinggi berbanding rawatan kombinasi BAP dan Kinetin mahupun rawatan BAP tunggal. Kata kunci: fenolik; flavonoid; BAP; NAA; Kn Abstract: Premna oblongifolia or better known as the green grass jelly is a species that belongs to the family Verbenaceae. The leaves extract of this plant can treat fever, reduce abdominal pain, and high blood pressure. Its ability to treat diseases are related to the polyphenolic content that serves as an antioxidant and antiinflammatory. Thus, a study is carried out to determine the suitable concentration of growth regulators to maximize the synthesis of polyphenolic compounds (flavonoids and phenolic) from the plant s callus in vitro. A total of 15 treatment applied to the callus which is single BAP, the combination of BAP with NAA and combination of BAP with Kinetin. Measurement of polyphenolic compounds is done in a first and second month. The highest phenolic compounds production is obtained from the treatment of combination of 3.0 mg/l BAP with 0.5 mg/l NAA which is mg GA/g. Callus that produces the highest flavonoid compounds is the treatment of combination of 3.0 mg/l BAP with 2.0 mg/l NAA which is mg QE/g. Total phenolic and flavonoid content produced in the treatment of combination of BAP with NAA shows the highest measurement compared to the treatment of BAP with Kinetin and single BAP. Keywords: fenolic; flavonoid; BAP; NAA; Kn 177
2 1. Pengenalan Spesies P. oblongifolia ialah tumbuhan yang dikenal dalam penghasilan cincau hijau daripada ekstrak daunnya. Tumbuhan ini boleh didapati di Asia Tenggara, China, Australia dan Afrika. Antara khasiatnya adalah merawat demam, mengurangkan sakit perut, dan menurunkan tekanan darah tinggi. Tambahan pula, sebatian alkaloidnya bersifat sitotoksik dan mampu bertindak sebagai anti-kanser (Indah & Laeli 2011). Keupayaan tumbuhan ini merawat penyakit-penyakit tersebut adalah kerana kandungan sebatian polifenoliknya yang bertindak sebagai antioksidan. Antioksidan bertindak menghalang tindak balas radikal bebas atau spesies oksigen reaktif yang merosakkan komponen-komponen penting di dalam sel seperti degradasi DNA, dinding sel dan sebatiansebatian kimia yang diperlukan oleh sel untuk menjalankan metabolisma (Beer et al. 2002). Fenolik atau polifenol ditafsirkan sebagai metabolik sekunder yang mempunyai struktur gelung aromatik berkumpulan hidroksil. Ia dihasilkan melalui tapak jalan shikimat dan tapak jalan malonat asetat sekaligus wujud secara heterogenus di dalam sel. Tumbuhan memerlukan sebatian polifenolik untuk pigmentasi, pertumbuhan dan kerintangan terhadap patogen (Systar et al. 2012). Sebatian polifenolik disintesis sebagai tindak balas terhadap tekanan ekologi dan fisiologi seperti jangkitan patogen, serangan daripada serangga, dan sinaran ultralembayung yang menyebabkan kecederaan (Nagendran et al. 2005). Flavonoid merupakan subkelas fenolik yang diedarkan secara meluas di dalam tisu tumbuhan. Flavonoid bertanggungjawab bersama dengan karotenoid dan klorofil bagi membentuk pigmentasi berwarna biru, ungu, kuning, oren dan merah. Ia sering didapati daripada buah-buahan yang mempunyai warna-warna tersebut. Umumnya, lebih banyak kumpulan hidroksil sebatian flavonoid tersebut, lebih kuat keupayaannya bertindak sebagai antioksidan. Flavonoid bertindak mengikat kepada ion logam seterusnya menghalang pembentukkan radikal bebas (Marzena & Mateusz 2012). Kajian klinikal telah dibuktikan oleh Fitriana et al. (2014) dengan melihat keupayaan cincau hijau yang mengandungi sebatian flavonoid merawat pesakit wanita yang mempunyai tekanan darah tinggi. Curtis et al. (2013) turut melaporkan flavonoid dapat menurunkan ketegangan arteri seterusnya menjadi alternatif perubatan bagi mengurangkan risiko penyakit jantung dan diabetes. Manipulasi pembentukkan metabolik sekunder dalam tumbuhan ubatan secara in vitro sudah lama digunapakai. Kaedah ini termasuklah penggunaan pengawalatur pertumbuhan yang berbeza dari segi jenis dan kepekatan sama ada auksin dan sitokinin mahupun kombinasi keduaduanya. Jumlah penghasilan metabolik sekunder di dalam tumbuhan adalah berbeza mengikut spesies mahupun jenis eksplan yang digunakan (Lorena et al. 2012). Justeru, kajian dijalankan bagi menentukan kepekatan pengawalatur pertumbuhan tunggal atau kombinasi yang dapat memaksimumkan kadar penghasilan sebatian fenolik dan flavonoid di dalam kalus cincau hijau. Kultur kalus penting bagi mendapatkan sebatian fenolik dan flavonoid di mana sebahagian tisu tumbuhan dikultur secara in vitro bagi penginduksian kalus dan seterusnya sebatian tersebut boleh diekstrak tanpa mengorbankan keseluruhan tumbuhan. 2. Bahan dan Kaedah 2.1 Sumber tumbuhan dan penjanaan kalus Kalus P. oblongifolia yang diinduksi adalah daripada batang pokok tersebut dengan menggunakan kaedah pengkulturan in vitro di atas medium Murashige & Skoog (MS) bersama-sama dengan pengawalatur pertumbuhan 1.0 mg/l BAP dan 3.0 mg/l NAA. Pengkulturan kalus telah dijalankan di Makmal Kultur Tisu Tumbuhan yang terletak di Pusat Penyelidikan Bioteknologi dan Nanoteknologi MARDI, Serdang, Selangor Darul Ehsan. 178
3 2.2 Rawatan pengawalatur pertumbuhan Kalus rapuh seberat 1 g telah disubkultur dari media asal ke atas medium Llyod & McCown Woody Plant Medium (WPM)yang mempunyai kepekatan BAP tunggal, kombinasi BAP dan Kn, serta kombinasi BAP dan NAA seperti yang ditunjukkan dalam Jadual 1. Kultur kalus dieramkan di dalam bilik kultur pada suhu C yang mempunyai keamatan cahaya 18 µmol m -2 s -1 selama dua bulan. Jadual 1. Formula media dengan rawatan kombinasi pengawalatur pertumbuhan yang berbeza WPMO terhadap kalus P. oblongifolia (cincau hijau). WPM + BAP (3.0 mg/l) WPM + BAP (5.0 mg/l) Kandungan media WPM + BAP (3.0 mg/l): Kn pada kepekatan 0.5 mg/l, 1.0 mg/l, 2.0 mg/l WPM + BAP (5.0 mg/l): Kn pada kepekatan 0.5 mg/l, 1.0 mg/l, 2.0 mg/l WPM + BAP (3.0 mg/l): NAA pada kepekatan 0.5 mg/l, 1.0 mg/l, 2.0 mg/l WPM + BAP (5.0 mg/l): NAA pada kepekatan 0.5 mg/l, 1.0 mg/l, 2.0 mg/l 2.3 Penyediaan ekstrak kalus Kalus P. oblongifolia ditimbang dari setiap rawatan sebanyak 3 g, kemudian dimasukkan ke dalam mortar dan digiling hingga lumat. Sebanyak 8 ml metanol 70% dicampurkan dan kalus digiling lagi. Ekstrak kalus kemudiannya dipindahkan ke dalam tiub falcon bersaiz 15mL dan diempar pada 8000 rpm selama 6 minit pada suhu bilik. Akhir sekali, supernatan diambil dan hampas dibuang (Subramaniam & Nagarajan 1969). Supernatan mengandungi sebatian fenolik yang sedia untuk dibaca menggunakan spektrofotometer. 2.4 Penentuan jumlah kandungan fenolik Jumlah kandungan fenolik ditentukan melalui kaedah Folin-Ciocalteu reagen Maurya & Sigh (2010). Sebanyak 0.5 ml hasil ekstrak kalus dimasukkan ke dalam tabung uji dan 2.5 ml reagen folin-ciocalteu ditambah. Seterusnya 2 ml 7.5% natrium karbonat ditambah dan ditutup dengan parafilem, larutan dibiarkan selama 30 minit pada suhu bilik. Bacaan diambil menggunakan spektrofotometer pada 760 nm dan diplotkan berdasarkan piawai asid galik bagi menentukan sebatian fenolik. Jumlah kandungan fenolik ini akan diunitkan sebagai mg GA/g. 2.5 Penentuan jumlah kandungan flavonoid Jumlah kandungan flavonoid ditentukan melalui kaedah Fernandes et al. (2012). Sebanyak 1 ml ektrak kalus dimasukkan ke dalam tabung uji yang mengandungi 4 ml air suling. Sebanyak 0.3 ml 5% natrium nitrat ditambah, selepas 5 minit 0.3 ml 10% aluminimum klorida ditambah. Kemudian pada minit ke-enam, sebanyak 2 ml 1M natrium hidroksida ditambah dan seterusnya air suling sehingga larutan mencecah 10 ml. Bacaan spektrofotometer dibuat pada 510 nm dan nilai bacaan ini digunakan bagi memplotkan graf piawai quasertin bagi mendapatkan nilai kandungan falvonoid. Jumlah kandungan flavonoid diunitkan sebagai mg QE/g. 3. Hasil 179
4 Kepekatan sebatian fenolik (mg GAE/g) Undergrad. Res. J. Biomol. Sci. Biotech. 1, Jumlah kandungan fenolik Rajah 1 menunjukkan kesan rawatan pengawalatur pertumbuhan terhadap penghasilan sebatian fenolik daripada kultur kalus P. oblongifolia. Kalus yang dirawat dengan rawatan kombinasi BAP dan NAA memberi kesan yang memberangsangkan bagi penghasilan sebatian fenolik berbanding rawatan kombinasi BAP dan Kn serta rawatan BAP tunggal. Selepas sebulan pengkulturan, kalus daripada rawatan 3.0 mg/l BAP dan 0.5 mg/l NAA menghasilkan sebatian fenolik tertinggi iaitu sebanyak 3.34 mg GA/g. Namun begitu, jumlah kandungan fenolik dalam rawatan ini berkurangan kepada 2.25 mg GA/g selepas 2 bulan pengkulturan. Selain itu, rawatan 5.0 mg/l BAP dan 2.0 mg/l NAA juga menghasilkan kandungan fenolik yang kedua tinggi pada tempoh pengkulturan sebulan dengan jumlah kandungan fenolik sebanyak 3.10 mg GA/g, dan kandungan fenolik dalam rawatan ini berkurangan pada bulan kedua dengan bacaan sebanyak 2.90 mg GA/g. Majoriti rawatan dalam kajian ini mencatatkan penurunan sebatian fenolik dari bulan pertama hingga ke bulan kedua Rajah 1. Kesan rawatan pengawalatur pertumbuhan terhadap penghasilan sebatian fenolik kultur kalus P. oblongifolia. 3.2 Jumlah kandungan flavonoid Kepekatan BAP/Kn/NAA (mg/l) Bulan pertama Bulan kedua Rajah 2 menunjukkan kesan rawatan pengawalatur pertumbuhan terhadap penghasilan sebatian flavonoid kultur kalus cincau hijau. Pada bulan pertama, rawatan 3.0 mg/l BAP dan 2.0 mg/l NAA mencatatkan kadar sebatian flavonoid tertinggi iaitu sebanyak 2.50 mg QE/g. Namun bacaan kandungan flavonoid semakin menurun pada bulan kedua dalam kebanyakan rawatan. Walaubagaimanapun, hanya rawatan 3.0 mg/l BAP dan 2.0 mg/l Kin mencatatkan kenaikan iaitu bulan pertama mencatatkan 1.46 mg QE/g dan bulan kedua adalah terus meningkat kepada 2.40 mg QE/g. 180
5 Kepekatan Sebatian Flavonoid (mg QE/g) Undergrad. Res. J. Biomol. Sci. Biotech. 1, Kepekatan BAP/Kn/NAA (mg/l) Bulan pertama Bulan kedua Rajah 2. Kesan rawatan pengawalatur pertumbuhan terhadap penghasilan sebatian flavonoid kultur kalus cincau hijau. 4. Perbincangan Kombinasi rawatan BAP (iaitu sitokinin) dengan NAA (iaitu auksin) menunjukkan sintesis sebatian polifenolik terbanyak berbanding rawatan kombinasi BAP dengan Kn mahupun rawatan tunggal BAP sahaja. Secara semulajadi, sebatian fenolik semula jadi biasanya berkonjugasi dengan mono atau polisakarida yang membentuk polimer seperti dinding sel. Ia tidak larut dan membentuk kompleks dengan komponen sel yang lain. Dua kumpulan fenolik dinding sel adalah lignin dan asid hidrosinamik (Nagendran Balasundran et al. 2005). Pengawalatur pertumbuhan auksin dan sitokinin penting dalam proliferasi sel dan penghasilan metabolik sekunder dalam tumbuhan. Sitokinin bukan sahaja bertindak menggalakkan pembahagian sel malah turut berperanan menstabilkan membran daripada dirosakkan oleh radikal bebas. Pengawalatur pertumbuhan auksin diperlukan oleh sel yang dikultur secara in vitro bagi pembahagian sel yang mana ia diperlukan dalam replikasi DNA seterusnya pembahagian sel (Yang et al. 2008). Secara keseluruhannya, kadar sebatian flavonoid daripada rawatan BAP dengan Kn lebih rendah berbanding rawatan BAP dengan NAA. Kajian kajian ini selari dengan kajian yang telah dikemukakan oleh Suat Hian Tan (2010) yang turut mendapati bahawa penggunaan kepekatan Kn terhadap kalus akan menurunkan sintesis sebatian flavonoid kultur kalus. Ini membuktikan aktiviti biologi yang lemah dalam prolifeasi kalus seterusnya menurunkan penghasilan sebatian polifenolik berbanding rawatan kombinasi BAP dengan NAA. 5. Kesimpulan Hasil menunjukkan potensi kalus P. oblongifolia untuk dijadikan sumber alternatif bioaktif sebatian fenolik secara in vitro. Tambahan pula, dapatan yang memberangsangkan ini membolehkan kajian seterusnya dijalankan dengan lebih mendalam tentang jenis-jenis sebatian polifenolik yang dihasilkan oleh kalus ini serta penambahbaikkan protokol penghasilan sebatian polifenolik secara in vitro. 181
6 Penghargaan Penghargaan kepada Dr. Che Radziah Che Mohd Zain selaku penyelia dan penyelia bersama Dr. Zuraidah Abd Rahman. Seterusnya kepada pihak MARDI bahagian Pusat Penyelidikan Bioteknologi dan Nanoteknologi dan kakitangan yang membekalkan kemudahan peralatan makmal sepanjang penyelidikan dijalankan. Rujukan Beer. D. D., Joubert. E., Gerlderblom. C. A. W., & Manley. M Phenolic compound: A Review of Their Possible Role as In Vitro Antioxidants of Wine. South African Journal of Enology and Viticulture 23: Curtis. P. J., Potter. J., Kroon. P. A., Wilson. P., Dhatariya. K., Sampson. M., & Cassidy, A Vascular function and atherosclerosis pregression after 1 year of flavonoid intake in statin-treatment postmenopausal women with type 2 diabetes. The American Journal of Clinical Nutrition 97: Fernandes. A. J., Ferreira. M. R., Randau. K. P., Tatiane. P. S., & Soares. L. A Total flavonoid content in the raw material and aqueous extreactives from Bauhinia monandra Kurz. The Scientific World Journal ID: Fitriana. S., Leily. A., & Karina. R. E Green grass jelly drink (Premna oblongifolia Merr.) can reduce blood pressure of adult women suffered from mild and moderate hypertension. Journal Gizi dan Pangan 9(3): Indah. H., & Laeli. K Enzymatic extraction of low methoxyl pectin as a potential anti-cancer from green cincau (Premna oblongifolia). Prosiding Seminar Nasional Sains Dan Teknologi 22: Lorena. P., Juan. J. C., Rosa. M. C., & Marta. E. G Phenolic compound in relation to differentiation in cell and tissue cultures of Larrea divaricate (Cav.). Plant Science 194: 1-7 Maurya. S., & Sigh. D Quantitative analysis of total phenolic content in Adhatoda vasica Nees extracts. International Journal of Farmtech Research 2(4): Marzena. S., & Mateusz. K Flavonoid and their properties to form chelate complexes. Biotechnology and Food Science 76(1): Nagendran. B., Kalyana. S., & Samir. S Phenolic compounds in plants and agri industrial by-products: Antioxidant activity, occurrence, and potential uses. Food Chemistry 99: Subramaniam. S. S., & Nagarajan. S Flavonoid of the seed of Crotalaria retusa and C. striata. Current Science 38: Systar. O., Brestic. M., Rai. M., & Bo. H. S Plant phenolic compounds for food, pharmaceutical and cosmetics production. Journal of Medicinal Plants Research 6(13): Tan. H. S., Radzali Musa., Arbakariya Ariff., & Mahmood Maziah Effect of plant growth regulators on callus, cell suspension and cell line selection for flavonoid production from Pegaga (Centella asiatica L. urban). American Journal of Biochemistry and Biotechnology 6(4): Yang. J., Gong. Z. C., & Tan. X Induction of callus and extraction of alkaloid from Yi Mu Cao (Leonurus heterophylus Sw.) culture. African Journal of Biotechnology 7:
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