PHYTOCHEMICAL STUDY AND EVALUATION OF ELICITATION EFFECT ON PRODUCTION OF SECONDARY METABOLITES OF MORUS ALBA VAR. SHALUN (MORACEAE) ROOT CULTURES

• Main Author: Fitriani, Rizki
• Format: Dissertations
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/46395
• Institution: Institut Teknologi Bandung

Morus which is known as mulberry is an important genus of the family Moraceae. This genus is widely distributed in Asia, Europe, North and South America, and Africa. Some species of this genus have been widely cultivated in Asian countries, such as India, China and Indonesia for its leaves which serve as indispensable food for silkworms. However, in most European countries, including Turkey and Greece, mulberries are grown for fruit production. The fruits are used in juices, liquors, natural dyes, and in the cosmetics industry. In the traditional Chinese medicine, the root barks of Morus alba have been used for treatment of hepatitis, diabetes, arthritis, rheumatism and other disorders for thousands years. The remarkable effects and sufficient resource of mulberry trees have attracted a lot of researchers to investigate this plant and other species of Morus. Previous phytochemical investigation showed that Morus produced either phenolic compounds, mainly stilbenes, 2-arylbenzofurans, flavonoids, and Diels-Alder adducts, or non phenolic compounds, such as terpenoids and steroids. Some of these phenolic compounds exhibited important and various bioactivities, such as cytotoxicity, antioxidant, antimicrobial, anti-inflammatory, antifungal, and antiviral The source of biologically important secondary metabolites was conventionally obtained from the intact plants. However, recent development in biotechnological approach has shown the possibility of plant tissue cultures as an alternative supply for producing bioactive secondary metabolites. Moreover, the production of some secondary metabolites of Morus tissue cultures is reported in higher amount when treated by elicitors. Based on these backgrounds, thus, the aim of this study was conducting a phytochemical study of Morus alba var. shalun root culture, examining the cytotoxic properties of the isolated compounds against P-388 murine leukemia cells as well as revealing the relationship between structure and cytotoxic activity, and evaluating the effect of elicitor addition on secondary metabolite production in M. alba var. shalun root culture. The method used in this study consisted of two steps. The first step was a phytochemical study of M. alba var. shalun root cultures which included developing root culture of M. alba var. shalun in MS (Murashige-Skoog) liquid media supplemented with 1 ppm IBA followed by isolation of secondary metabolites from liquid media and M. alba var. shalun root cultures. Isolation of secondary metabolites involved several stages of work, including extraction, fractionation and purification of compounds using various chromatographic techniques. The structures of the isolated compound were determined based on spectroscopic data, which included 1D-NMR (1H and 13C), 2D-NMR (HSQC and HMBC), and high resolution mass spectra. The cytotoxic effect of the isolated compounds was evaluated against murine leukemia cell P-388 using MTT [3-(4,5- dimethylthiazo-2-yl)-2,5-diphenyltetrazolium bromide] method. The second step was an evaluation of the elicitation effect on the production of secondary metabolites in the root cultures of M. alba var. shalun. At this step, the root cultures were developed in MS liquid media with the addition of elicitor as sample and the root cultures without addition of the elicitors were prepared as the control samples. The sample and control root cultures were extracted using methanol and analyzed by HPLC-MS. In the phytochemical investigation, five new Diels-Alder type adducts have been isolated, trivially named as morushalunin (2), morushalunin A (1), morushalunin B (3), morushalunin C (4), and morushalunin D (5). These new compounds are identified together with five known phenolic compounds, including one stilbene derivative (oxyresveratrol (6)), one 2-arylbenzofuran derivative (moracin M (7)), and three Diels-Alder type adducts (sorocein A (8), mulberofuran T (9), and mulberofuran K (10)). The discovery of five new Diels-Alder type adducts is chemically important to the phytochemistry of Morus. The presence of five new compounds as well as five known ones showed that M. alba var. shalun root cultures has the ability to produce secondary metabolites with structural diversity, even produce secondary metabolites that have never been reported from natural plants. The cytotoxicity of the isolated compounds against murine leukemia P-388 cells showed that the eight compounds of the Diels-Alder adduct were categorized as highly cytotoxic active against P-388 murine leukemia cells with IC50 values below 2.0 ?g/mL. Oxiresveratrol (6) and moracin M (7) which act as precursors of the Diels-Alder adduct possessed IC50 values of 3.3 ?g/mL and 2.0 ?g/mL respectively. This shows that the isolated Diels-Alder adduct exhibited a better activity in inhibiting the growth of murine leukemia P-388 cells compared to its precursors. The study of structure-activity relationship showed that the addition of methyl cyclohexene ring as a result of Diels-Alder cycloadition reaction, the presence of isoprenyl groups, and the increasing of the number of hydroxyl groups in the Diels-Alder adducts seemed to contribute significantly to the increase of cytotoxic properties of murine leukemia P-388 cells. Evaluation of the elicitation effect using salicylic acid, CuCl2, and methyl jasmonate elicitor on the production of secondary metabolites in M. alba var. shalun root culture is the first report. Among the three elicitors, the most effective elicitor in increasing secondary metabolites production in M. alba var. shalun root culture is salicylic acid because it can increase the level of production almost all compounds, even can induce the formation of new compounds. Thus, this result proved the hypothesis that the addition of elicitor to M. alba var. shalun root culture can increase the production of secondary metabolites and even induce the production of other secondary metabolites which are not produced by the control of the root cultures.