ISOLASI SENYAWA MARKER DARI EKSTRAK AIR DAUN KELOR (MORINGAN OLEIFERA LAMK.)

Kelor (MoringaoleiferaLamk.) adalah tanaman termasuk dalam famili Moringaceae yang telah lama digunakan dalam pengobatan beberapa penyakit secara tradisional. Penggunaan secara empiris tersebut telah dibuktikan secara ilmiah. Penelitian ini bertujuan untuk mengisolasi senyawa marker dari ekstrak air daun kelor. Penelitian ini dimulai dari pembuatan ekstrak, karakterisasi simplisia, dan penapisan fitokimia. Pembuatan ekstrak dilakukan dengan cara daun diblender dengan penambahan aquades kemudian disaring. Filtrat yang diperoleh dikumpulkan, kemudian dikeringkan menggunakanalat freeze dryer sampai diperoleh ekstrak kering.Ekstrak air daun kelor difraksi nasi dengan metode ekstraksi cair-cair menggunakan pelarut etil asetat. Fraksi etil asetat disubfraksinasi dengan menggunakan kromatografi kolom klasik. Pemurnian dilakukan dengan menggunakan kromatografi lapis tipis preparatif dan uji kemurnian dilakukan dengan menggunakan KLT pengembangan tunggal dan KLT dua dimensi. Isolat dikarakterisasi menggunakan KLT dengan penampak bercak spesifik dan pereaksi geser. Dari hasil pemurnian didapatkan senyawa murni dengan bentuk amorf. Berdasarkan data spektroskopi UV diduga isolat yang diperoleh merupakan flavonol, dimana terdapat OH pada posisi C3, C7, dan C4', serta tidak adanya orto di-OH pada cincin B

Elisitasi Kultur Sel Temulawak (Curcuma xanthorrhiza Roxb) untuk Produksi Senyawa Aktif Xantorizol

Temulawak (Curcuma xanthorrhiza Roxb.) merupakan salah satu tanaman asli Indonesia yang telah digunakan untuk tujuan pengobatan. Xantorizol, senyawa seskuiterpenoid dari temulawak, telah banyak diteliti aktivitasnya. Kandungan senyawa xantorizol dari tanaman ini sangat kecil dan waktu panen relatif lama. Untuk mengoptimalkan produksi xantorizol, teknik kultur jaringan tanaman dapat digunakan sebagai salah satu alternatif. Penelitian ini ditujukan untuk meningkatkan kadar xantorizol dari kultur suspensi sel temulawak menggunkan elisitor. Kultur kalus yang telah diinisiasi pada media padat diinduksi menjadi suspensi sel dengan media cair. Kultur suspensi sel yang berumur dua minggu dan dielisitasi dengan ekstrak ragi. Kultur dipanen pada minggu pertama dan kedua setelah perlakuan dan dikeringkan. Sampel kering diekstraksi dengan etil asetat dan dianalisis dengan KCKT. Hasil analisis menunjukkan bahwa kultur yang dielisitasi dengan ekstrak ragi 100 ppm dapat menstimulasi pembentukan xantorizol sebesar 0,186%.Kata kunci: Curcuma xanthorrhiza Roxb., ekstrak ragi, kultur suspensi sel, temulawak.AbstractTemulawak (Curcuma xanthorrhiza Roxb.) is the one of indigenous plants in Indonesia that has been used for medicinal purpose. Xanthorrhizol, a sesquiterpenoid compound from temulawak, was studied for various activities. Xantorrhizol content in this plant is very low and relatively have long time for harvest. For optimize the production of xanthorrhizol, tissue culture technique could be used as an alternative. The aim of this research was carried out by to enhance the production of xanthorrhizol from cell suspension cultures using elicitors. The initiated callus cultures from solid medium, was induced to suspension cell cultures in liquid medium. The suspension cell culture was grown for two weeks and elicited with yeast extract. The cultures were harvested on the first and second weeks after elicited. Dry sample was extracted by ethyl acetate as a solvent and analyzed by HPLC. The results showed for elicitated culture by yeast extract 100 ppm could stimulate production of xanthorrhizol by 0.186%.Keywords: Curcuma xanthorrhiza Roxb., yeast extract, cell suspension culture, temulawak

Phytochemical and Biosynthetic Studies of Lignans, with a Focus on Indonesian Medicinal Plants

In this thesis phytochemical and biosynthetic studies of lignans are described. The focus is on the Indonesian medicinal plants Phyllanthus niruri and Piper cubeba and on two Linum species, Linum flavum and L. leonii, native to European countries. Both Indonesian plants are used in jamu. Jamu is the Indonesian traditional herbal medicine, practised for many centuries in the Indonesian community to maintain good health and to treat diseases. The manufacturing of jamu is shifting more and more from household scale to the bigger industries. As the economical and clinical value of jamu nowadays increases in Indonesia, there is a need for further scientific proof and well conducted research. Jamu has to be developed in order to assure its efficacy and safety. Chapter 2 reviews the research carried out on jamu and jamu plants, covering a broad range of aspects including phytochemistry, pharmacology, toxicologicy and clinical studies. In addition, ethical issues such intellectual property right (IPR), benefit sharing, and conservation are addressed. Phyllanthus niruri is an important medicinal plant for jamu. All parts of the plant are used, among others to treat gonorrhea, syphilis, nephralgia, diarrhea, fever, and tetanus. The leaves serve to treat epilepsy, malaria, constipation, hypertension, and menstrual disorders. Lignans seem to be an important group of secondary metabolites, responsible for the biological activity of P. niruri. We initiated cell cultures of P. niruri that were able to produce lignans. The lignan profiles of cell suspensions, callus cultures, aerial plant parts, roots and seeds were compared and significant differences, both qualitatively and quantitatively, were found (chapter 3). Two compounds that were not found in P. niruri before were isolated from the cell suspension cultures: a new dihydrocubebin-dimethylether and urinatetralin, a new lignan from P. niruri, but reported earlier from P. urinaria. Feeding 0.5 mM of ferulic acid or 0.5 mM of caffeic acid, being early precursors of lignan biosynthesis, resulted in an increase up to 0.7 mg g-1 DW of the dihydrocubebin-dimethylether (control value 0.1 mg g-1 DW) and up to 0.3 mg g-1 DW of urinatetralin (control value 0.2 mg g-1 DW) in the suspension cultures. Lignans are also found in another important medicinal plant for jamu, Piper cubeba. The berries of this plant are used to treat gonorrhea, dysentery, syphilis, abdominal pain, diarrhea, enteritis and asthma. The lignan profile of berries (the particular plant part used in jamu), leaves and stalks were investigated and compared using gas chromatography (GC), gas chromatography coupled to mass spectrometry (GC-MS), and high pressure liquid chromatography (HPLC) (chapter 4). Thirteen lignans were identified in the berries, fifteen in the leaves and five in the stalks. Our further phytochemical investigation of P. cubeba berries and leaves focused on the essential oil composition (chapter 5). The essential oil of the berries is commonly used as a constituent of cosmetics as well as for medicinal purposes. Antimicrobial, antiherpes simplex, antifungal, cardiovascular, and gastroprotective are mentioned in the latter context. Hydrodistillation of the berries of P. cubeba yielded 11.8% (w/w) and the leaves 0.9% (v/w) oil. In total 105 components could be identified (GC, GC-MS) in the berries, dealing with 63.1% of the oil. In the leaves, 63 components could be identified, corresponding with 78.0% of the oil. The total amount of monoterpenes was comparable in both oils (17.2% and 17.0%, for berries and leaves, respectively). The main monoterpenes in the berries and leaves oil were α-thujene, α-pinene, sabinene, and limonene. In the oxygenated monoterpene fractions trans-sabinene hydrate was the main component. α-Copaene, β-elemene, E-caryophyllene, and caryophyllene were the main sesquiterpenes in the berries oil. E-caryophyllene, and γ-cadinene were the main sesquiterpenes in the leaves oil. Based on the similarity of the lignan and essential oil profiles we conclude that, in addition to the berries, also the leaves may be used for medicinal purposes. This knowledge can be used for the further development of (rationally designed) phytomedicines from P. cubeba. Studies on the lignan biosynthesis were performed with the European plant, Linum flavum. L. flavum cell cultures accumulate a high amount of coniferin (12% on a dry weight basis). Cell suspension cultures of leaves from this plant were treated with glucosyltransferase inhibitors in order to enhance the production of the lignan 6-methoxypodophyllotoxin (6-MPT) and to reduce the coniferin production (chapter 6). These two compounds originate from the common precursor coniferyl alcohol by different biosynthetic branches. Enzymatic transformation of this substrate by coniferyl alcohol glucosyltransferase (CAGT) yields coniferin. It was hypothized that by inhibiting this step more precursor would be available for the biosynthetic branch leading to the formation of lignans. Na2EDTA inhibited the production of coniferin up to 88% and on the other hand enhanced of the 6-MPT production up to 0.6 mg g-1 DW, 3.2-fold more than in untreated cultures. The inhibition of the coniferin production related to an inhibition of the CAGT activity, as shown in cell homogenates incubated with coniferyl alcohol. This indicates that Na2EDTA inhibits CAGT and therefore reduces the production of coniferin. The mechanism of inhibition is not clear as yet. To further study the role of CAGT in the biosynthesis of lignans, we partially purified this enzyme from cell suspension cultures of L. flavum. A complete purification of CAGT, however, appeared to be impossible because of its unstable nature. We tried to clone the CAGT from cell suspension cultures of L. flavum in Escherichia coli (chapter 7). The total RNA isolation revealed that the quality of RNA was sufficient to synthesize the cDNA. A forward and a reverse degenerated primer were designed based on the most conserved region of 100 glucosyltransferases from various species. These glucosyltransferases were aligned using MegAline software from DNASTAR Inc. The conserved region, called the PSPG box, is highly characteristic and present in all GTs involved in natural product biosynthesis. This domain may also define the active site of GTs of animals and microorganisms. The PSPG box is considered to represent the nucleotide diphosphate binding site. Several GT-encoding genes are suitable candidates to be inserted into a variety of plants with the aim of improving food, crop quality as well understanding the biosynthesis of valuable natural products for medicine. Three different potential GTs were cloned from cell suspension cultures of L. flavum in E. coli. The gene sequence of three different products was elucidated. Two of them were complete sequences (ORF): CAGT A and CAGT B. We were able to find the conserved region (PSPG box) for the third one. Alignment of CAGT A and B with the sequences from the plant GTs obtained from databases showed 50% and 40% homology, repectively. The conserved region (PSPG box) of CAGT A and B showed 80% and 89% homology respectively. Phylogenetic analysis revealed that CAGT A and B belong to the same subfamily together with other phenylpropanoid glucosyltransferases. Although the expression of these GTs is not yet finished, it may be concluded that at least one of the two CAGT is involved in the biosynthetic step from coniferyl alcohol to coniferin in L. flavum cell suspension cultures. In chapter 8, we describe the production of justicidin B, a cytotoxic aryltetraline lignan in cell and hairy root cultures of Linum leonii. The hairy root cultures were obatained by genetic transformation using the agropine-type strain Agrobacterium rhizogenes 15834. The products encoded by rol A and rol C genes were found to have a synergistic effect on root induction and to induce increased sensitivity to auxin. The transformation of these genes from A. rhizogenes into the hairy root was checked by PCR (polymerase chain reaction). Proof of transformation was given by the PCR products showing that rol A and rol C genes were present in the hairy roots of L. leonii. Genetically modified hairy roots produced 5-fold higher yields of justicidin B (10.8 mg g-1 DW) compared to untreated callus. This suggests that this technique may be used to enhance the accumulation of justicidin B. In addition to the production, we investigated the cytotoxic effect of justicidin B in three chronic human myeloid leukemia-derived cell lines (LAMA-84, K-562 and SKW-3), that show a lower responsiveness to cytotoxic drugs due to a strong expression of the fusion oncoprotein BCR-ABL (a non-receptor tyrosine kinase). IC50 values of justicidin B were 1.1, 6.1 and 1.5 µM for the chronic myeloid leukemia (LAMA-84), pre-B-cell lymphoma (K-562) and chronic lymphoid leukemia (SKW) cell lines respectively. These IC50 were comparable to the anticancer drug etoposide (a semi-syntehetic lignan derivative). We conclude that the phytochemical studies of the two selected Indonesian medicinal plants as well as the production of lignans in cell suspension cultures provide further scientific approach for the development of jamu. Furthermore the genetic engineering studies of two Linum species contribute to medicinal plant research with respect to a better understanding of the lignan biosynthesis. The biotechnology approach may also be applied to use medicinal plants as a source for drugs discovery

Isolasi Senyawa Aktif Lignan dari Buah Lada Hitam (Piper nigrum L.) dan Daun Sirih (Piper betle L.)

Buah lada hitam (Piper nigrum L.) dan daun sirih (Piper betle L.) telah banyak digunakan secara tradisional untuk mengobati beberapa jenis penyakit. Beberapa senyawa metabolit sekunder yang terkandung pada kedua tanaman tersebut diduga bertanggungjawab terhadap efek farmakologi, salah satu golongan metabolit sekunder tersebut adalah lignan. Penelitian ini bertujuan untuk mengisolasi senyawa lignan dari buah lada dan daun sirih. Serbuk simplisia dari daun sirih dan buah lada hitam diekstraksi dengan ekstraksi sinambung menggunakan pelarut metanol. Ekstrak difraksinasi dengan ekstraksi cair-cair menggunakan pelarut air-diklorometan (1:1) dan kromatografi cair vakum. Pemurnian dilakukan dengan menggunakan metode kromatografi lapis tipis preparatif. Isolat dikarakterisasi dengan menggunakan kromatografi gas-spektroskopi massa (KG-SM). Dari buah lada hitam telah berhasil diisolasi dan diidentifikasi dua senyawa lignan berupa hinokinin dan satu senyawa lignan lain yang memiliki ciri fragmen 135 dan 286 pada KG-SM. Sedangkan daun sirih memberikan data kromatografi untuk golongan lignan tetapi belum dapat dikonfirmasi dengan data KG-SM.Kata kunci : buah lada hitam, daun sirih, lignan, tanaman obat Indonesia Black pepper fruits and betel leaves are widely used traditionally to cure several illnesses. Secondary metabolites of both plants are believed to be responsible for their pharmacological effect; one of the secondary metabolites groups is lignan. The goal of this research is to isolate lignans from betel leaves and black pepper fruits. Crude drugs of betel leaves and pepper fruits were extracted with Soxhlet apparatus, using methanol. The extract was fractionated by liquid-liquid extraction using dichloromethane-water (1:1) and vacuum liquid chromatography. Purification was conducted by preparative thin layer chromatography. Isolated compounds were characterized by gas chromatography-mass spectra (GC-MS). Two lignans were isolated from black pepper fruits and identified with GCMS. First known as hinokinin, and another has MS fragment 135 and 268, which are specific for lignan compounds. Betel leaves showed chromatography data to lignan groups but cannot confirm yet by GC-MS.Keywords: black pepper fruits, betel leaves, lignan, Indonesian Medicinal Plan

ANALYSIS OF SECONDARY METABOLITES OF CALLUS OF RAMBUTAN Nephelium lappaceum L

Rambutan plant (Nephelium lappaceum L.) is a member of the Sapindaceae family. The rambutan plant is one of the natural ingredients that can be developed as traditional medicine. Rambutan peel has the potential for good antioxidant and anticancer activity. Rambutan fruit does not grow every time it needs efforts to produce the active substance in rambutan, using plant tissue culture techniques. The use of the correct variety of mediums and hormones at the right concentration is the key to thriving tissue culture. Explants derived from rambutan leaves were planted precisely on solid media Murashige and Skoog (MS) and WoddyPlant Medium (WPM) containing Indole-3-Butyric Acid (IBA) and Kinetin. After seven days, the callus was subcultured, then after 35 days, the subculture callus was collected and dried. Dry callus and rambutan leaves (Wild type) were macerated with n-hexane, ethyl acetate, and ethanol. The concentrated extract was then applied to a GF 254 silica gel plate with the mobile phase Toluene-Acetone (7: 3) and n-hexane-EthylAsetate (3: 7). The results showed that the concentration of IBA 2 ppm and kinetin three ppm was the best combination because it produced callus. TLC results of rambutan leave with plant tissue culture containing flavonoids and triterpenoids. This study provides new information regarding the induction of rambutan callus and can become the basis for producing active metabolites in rambutan with cell suspension culture development.  Tanaman rambutan (Nephelium lappaceum L.) Merupakan salah satu anggota famili Sapindaceae. Tanaman rambutan merupakan salah satu bahan alami yang berpotensi untuk dikembangkan sebagai obat tradisional. Kulit rambutan mempunyai potensi aktivitas antioksidan dan antikanker yang baik. Buah rambutan tidak tumbuh setiap saat maka perlu upaya untuk memproduksi zat aktif dalam rambutan, salah satunya dengan menggunakan teknik kultur jaringan tanaman. Penggunaan variasi medium dan hormone yang tepat pada konsentrasi yang tepat merupakan kunci sukses kultur jaringan. Eksplan yang berasal dari daun rambutan ditanam secara tepat pada media padat Murashige dan Skoog (MS) dan Woddy Plant Medium (WPM) yang mengandung Indole-3-Butric Acid (IBA) dan Kinetin. Setelah 7 hari kalus disubkultur, kemudian setelah 35 hari kalus subkultur dikumpulkan dan dikeringkan. Kalus kering dan daun rambutan (Wlid type) dimaserasi dengan n-heksan, etilasetat dan etanol, kemudian ekstrak pekat diaplikasikan pada plate silika gel GF 254 dengan fasa gerak Toluen-Aseton (7: 3) dan n-heksan-EtilAsetat (3: 7). Hasil penelitian menunjukkan bahwa konsentrasi IBA 2 ppm dan kinetin 3 ppm merupakan kombinasi terbaik karena menghasilkan kalus. Hasil Kromatografi lapis tipis (KLT) daun rambutan dengan kultur jaringan tanaman mengandung flavonoid dan triterpenoid. Hasil penelitian ini memberikan informasi baru mengenai induksi kalus rambutan dan bisa menjadi dasar produksi metabolit aktif dalam rambutan dengan pengembangan ke arah kultur suspensi sel

Phytochemical Study of Cell Culture Jatropha Curcas

Jatropha curcas belongs to the Euphorbiaceae family which has potential economically. This plant has been reported to contain toxic compounds such as curcin and phorbol ester and its derivatives. These compounds may become a problem if J. curcas will be explored as a source of biofuel. In order to provide safety plants, the research on the study of phytochemical and initiation of cell and organ culture have been carried out. J curcas which has been collected from different regions in Indonesia showed to contain relatively the same profile of chemical contents. Dominant compounds that were detected by GCMS are hidrocarbon such as 2-heptenal, decadienal, hexsadecane, pentadecane, cyclooctane etc, fatty acid such as oktadecanoate acid, etthyl linoleate, ethyl stearate, heksadecanoate acid and steroid such as stigmasterol, fucosterol, sitosterol. No phorbol ester and its derivatives have been detected yet by the GCMS method. Callus and suspension cultures of J. curcas have been established to be used for further investigation

Produksi Senyawa Metabolit Sekunder Melalui Kultur Jaringan dan Transformasi Genetik Artemisia Annua L.

Produksi metabolit sekunder pada tanaman biasanya menghasilkan kadar yang rendah. Metode bioteknologi telah terbuktidapat meningkatkan produksi beberapa metabolit sekunder pada tanaman. Untuk meningkatkan perolehan metabolit sekunder telah digunakan teknik kultur jaringan dan transformasi genetik dengan induksi Agrobacterium rhizogenes. Penelitian ini bertujuan untuk meningkatkan kandungan metabolit sekunder dari kultur kalus dan akar rambut dari tanaman Artemisia annua hasil transformasi genetik menggunakan A. rhizogenes. Kultur kalus dan akar rambut hasil transformasi genetika mengandung senyawa artemisinin lebih tinggi dibanding dengan kultur kalus dan akar tanpa transformasi.Kata Kunci : Artemisia annua, kultur kalus, akar rambut Agrobacterium rhizogenes, artemisinin. The production of secondary metabolites of plant is usually low. Biotechnological methods have been proved to enhance the production of some of plant's secondary metabolites. To enhance the production of secondary metabolites, cell cultures and genetically transformed plants which were induced by Agrobacterium rhizogenes have been used. This research aimed to enhance the secondary metabolite content from A. rhizogenes transformed callus and hairy roots cultures of Artemisia annua. Genetically transformed callus and hairy root cultures of A. annua contained higher artemisinin content compared to untransformed callus and root cultures.Keywords : Artemisia annua, callus cultures, hairy roots, Agrobacterium rhizogenes, artemisinin

ANALISIS KEPENTINGAN JEPANG MEMBERIKAN BANTUAN LUAR NEGERI MELALUI OFFICIAL DEVELOPMENT ASSISTANCE (ODA) TERHADAP NEGARA VIETNAM

This research aims to analyze Japan's interests in providing foreign assistance through Official Development Assistance (ODA) to Vietnam. ODA has become an important instrument in Japan's foreign policy, and Vietnam has been one of the main recipients of this aid. In this context, this research looks in detail at the reasons behind Japan's decision to provide ODA assistance to Vietnam and the implications and impact on bilateral relations between the two countries. The research method used is a qualitative approach by collecting data through literature studies and policy analysis. This approach enables a comprehensive understanding of Japan's interests in providing ODA assistance to Vietnam. The analysis results show that Japan has various strategic interests in providing ODA assistance to Vietnam. Therefore, it is important to consider the long-term impact of Japanese ODA assistance on Vietnam's development and transformation as an economically independent country. In its conclusion, this research identifies Japan's strategic interests in providing ODA assistance to Vietnam