Waraporn Putalun

Anti-solasodine glycoside single-chain Fv antibody stimulates biosynthesis of solasodine glycoside in plants

We constructed a recombinant antibody fragment—single chain fragment-variable (scFv) antibody—derived from hybridoma cell lines to control the concentration of solasodine glycosides in hairy root cultures of Solanum khasianum transformed by the anti-solamargine (As)-scFv gene. The properties of the As-scFv protein expressed in Escherichia coli were almost identical to those of the parent monoclonal antibody (MAb). Up to 220xa0ng recombinant As-scFv was expressed per milligram of soluble protein in transgenic hairy root cultures of S. khasianum. The concentration of solasodine glycosides was 2.3-fold higher in the transgenic than in the wild-type hairy root, as reflected by the soluble As-scFv level and antigen binding activities. These results suggested that the scFv antibody expressed in transgenic hairy roots controlled the antigen level, thus representing a novel plant breeding methodology that can produce secondary metabolites.

Rapid separation of solasodine glycosides by an immunoaffinity column using anti-solamargine monoclonal antibody

Immunoaffinity column using anti-solamargine monoclonal antibody for separation of solasodine glycosides was established. This method was specific for solasodine glycosides which was detected by thin layer chromatography and the western blotting. Total solasodine glycosides have been separated directly from the crude extract of Solanum khasianum fruit by the newly established immunoaffinity column.

Methyl jasmonate elicitation enhances glycyrrhizin production in Glycyrrhiza inflata hairy roots cultures.

Hairy roots were induced by infecting stems and leaves of Glycyrrhiza inflata with Agrobacterium rhizogenes ATCC 15834. The optimization of growth and glycyrrhizin accumulation of G. infl ata hairy roots was studied. Sucrose (6%, w/v) was optimal for growth and glycyrrhizin accumulation in G. infl ata hairy roots. Effects of elicitors like chitosan, methyl jasmonate, and yeast extract on glycyrrhizin production were studied. Methyl jasmonate (100 μM) was most efficient in enhancing glycyrrhizin production up to almost 109 μg/g dry weight on day 5 of elicitation. These results indicate that application of elicitors can enhance the capacity of G. inflata hairy roots to produce glycyrrhizin.

Stability studies of saponins in Bacopa monnieri dried ethanolic extracts.

Bacopa monnieri (L.) Wettst. (Brahmi) is currently used as a drug and food supplement for memory improvement. However, studies on the physical and chemical stability of the extract components, especially on the lead compound important for pre-formulation, have not yet been reported. In this study, the stabilities of the crude extract and the diluted crude extract were investigated at various temperatures using saponin glycosides, bacopaside I and bacoside A3 as markers for quantitative analysis. The stability testing of bacopaside I and bacoside A3 standard solution was performed at various temperatures and pH values. The quantity of both compounds under all conditions was analyzed using HPLC techniques. The moisture adsorption of the crude extract was determined at 5, 40, 60 and 80 degrees C at 75 % relative humidity using gravimetric methods. The results revealed that the crude extract quickly adsorbed moisture up to 54 % w/w at both 40 and 80 degrees C, while it only slowly adsorbed moisture at 5 degrees C. The amounts of intact bacopaside I and bacoside A3 in the crude extract decreased drastically at 80 degrees C, slowly at 40 and 60 degrees C, and remained unchanged at 5 degrees C during the period of investigation. Moreover, the amount of both compounds in the standard solution dropped sharply at a pH of 1.2 but slowly at pH 6.8 and 9.0, respectively. The pre-formulation data could be further used for improvement of the final product quality.

Artemisinin Production by Shoot Regeneration of Artemisia annua L. Using Thidiazuron

An efficient in vitro method for multiple shoot bud induction and regeneration has been developed in Artemisia annua L. using leaf and stem explants in various concentrations and combinations of plant growth regulators to evaluate the frequency of regeneration. The sources of explants as well as plant growth regulators in the medium were found to influence the multiple shoot induction. The result shows that the stem segment cultured on Murashige and Skoog (MS) medium supplemented with 0.1 mg/l thidiazuron (TDZ) gave a perfect shoot formation (100%) and good shoot multiplication (57 shoots/explant) after 2 weeks of culture. Healthy regenerated shoots were elongated and rooted in MS medium without hormones. The artemisinin content in plants regenerated from stem explants using 0.1 mg/l TDZ was (3.36 ± 0.36) μg/mg dry weight and two-fold higher than that of in vitro grown plants of the same age [(1.73 ± 0.23) μg/mg DW]. This system exhibited a potential for a rapid propagation of shoots from the stem explant and makes it possible to develop a clonal propagation of A. annua.

Isoflavonoid production in a hairy roots culture of Pueraria candollei.

A hairy roots culture of Pueraria candollei was established using Agrobacterium rhizogenes ATCC15834 and grown in half-strength Murashige and Skoog (MS) medium. The highest production of total isoflavonoids was found to be (36.48 ± 4.09) mg/g dry wt [(3.39 ± 0.20) mg/g dry wt puerarin, (29.91 ± 3.74) mg/g dry wt daidzin, (1.65 ± 0.09) mg/g dry wt genistin, (0.76 ± 0.03) mg/g dry wt daidzein, and (0.76 ± 0.03) mg/g dry wt genistein, respectively]. The total isoflavonoid content in hairy roots of P. candollei was 5.18-fold higher than that of the native tuber. Effects of sucrose content and medium type on growth and isoflavonoid production were investigated. 5% (w/v) Sucrose was an optimum content for the growth and isoflavonoid accumulation in P. candollei hairy roots. Half-strength MS medium had the highest effect for biomass production whereas woody plant medium had mostly stimulated isoflavonoid content in hairy roots

IMMUNOAFFINITY COLUMN FOR ISOLATION OF BIOACTIVE COMPOUNDS USING MONOCLONAL ANTIBODIES

ABSTRACT Anti-solamargine and anti-ginsenoside Rb1 monoclonal antibodies were used for preparation of an immunoaffinity column. Total solasodine glycosides were separated directly from the crude extract of Solanum khasianum fruit, by the established immunoaffinity column. This method was specific for solasodine glycosides, which was detected by thin layer chromatography and eastern blotting. An immunoaffinity column, using anti-ginsenoside Rb1 monoclonal antibody, has made possible a single-step separation of ginsenoside Rb1 from a crude extract of ginseng roots (Panax ginseng).

Fingerprinting of Natural Product by Eastern Blotting Using Monoclonal Antibodies

We succeeded in developing the fingerprint of natural product by eastern blotting using monoclonal antibodies. After developing and separating them on a TLC plate, solasodine glycosides are oxidized by NaIO4 and reacted with a protein to give conjugates which are recognized with anti-solamargine monoclonal antibody (MAb). Anti-solamargine MAb having wide cross-reactivity can stain and detect all solasodine glycosides by fingerprint. Different sensitivity between solamargine and solasonine was observed. The detection limit was 1.6u2009ng of solasonine. The hydrolysed products of solamargine were determined by fingerprint of eastern blotting compared to their Rf values depending on the sugar number. Fingerprint by eastern blotting using anti-ginsenoside Rb1 MAb distinguished the formula containing ginseng prescribed in traditional Chinese medicine. By double-staining of ginsenosides it is possible to suggest that the staining color shows the pharmacological activity, such as the purple bands indicate ginsenosides having stimulation activity, and the blue color indicated compound like ginsenosides possessed the depression affect for the central nervous system (CNS), respectively.

WESTERN BLOTTING OF STEROIDAL ALKALOID GLYCOSIDES USING MONOCLONAL ANTIBODY AGAINST SOLAMARGINE

A method for the determination of solasodine glycosides using western blotting was investigated. Solasodine glycosides, separated by silica gel thin-layer chromatography, were transferred to a polyvinylidene difluoride membrane. The membrane was treated with sodium periodate solution followed by bovine serum albumin, resulting in a solasodine glycoside-bovine serum albumin conjugate. Individual spots were stained with monoclonal antibody against solamargine. Immunostaining of solasodine glycosides was more sensitive compared with other staining methods. The newly established western blotting method is extended to the distribution of solasodine glycosides in plants.