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Redalyc.Phenolic Compound Production and Biological Activities from in Vitro Regenerated Plants of Gherkin ( Cucumis Anguria Electronic Journal of Biotechnology E-ISSN: 0717-3458 [email protected] Pontificia Universidad Católica de Valparaíso Chile Thiruvengadam, Muthu; Chung, Ill-Min Phenolic compound production and biological activities from in vitro regenerated plants of gherkin ( Cucumis anguria L.) Electronic Journal of Biotechnology, vol. 18, núm. 4, 2015, pp. 295-301 Pontificia Universidad Católica de Valparaíso Valparaíso, Chile Available in: http://www.redalyc.org/articulo.oa?id=173341112006 How to cite Complete issue Scientific Information System More information about this article Network of Scientific Journals from Latin America, the Caribbean, Spain and Portugal Journal's homepage in redalyc.org Non-profit academic project, developed under the open access initiative Electronic Journal of Biotechnology 18 (2015) 295–301 Contents lists available at ScienceDirect Electronic Journal of Biotechnology Phenolic compound production and biological activities from in vitro regenerated plants of gherkin (Cucumis anguria L.) Muthu Thiruvengadam, Ill-Min Chung ⁎ Department of Applied Bioscience, College of Life and Environmental Science, Konkuk University, Seoul, South Korea article info abstract Article history: Background: The effect of polyamines (PAs) along with cytokinins (TDZ and BAP) and auxin (IBA) was induced by Received 8 January 2015 the multiple shoot regeneration from leaf explants of gherkin (Cucumis anguria L.). The polyphenolic content, Accepted 2 May 2015 antioxidant and antibacterial potential were studied from in vitro regenerated and in vivo plants. Available online 30 May 2015 Results: Murashige and Skoog (MS) medium supplemented with 3% sucrose containing a combination of 3.0 μM TDZ, 1.0 μMIBAand75μM spermidine induced maximum number of shoots (45 shoots per explant) was Keywords: achieved. Regenerated shoots elongated in shoot elongation medium containing 1.5 μMGA3 and 50 μM Biological activities μ Cucumis anguria spermine. The well-developed shoots were transferred to root induction medium containing 1.0 M IBA and μ Multiple shoot induction 50 M putrescine. Rooted plants were hardened and successfully established in soil with a 95% survival rate. Plant growth regulators Twenty-five phenolic compounds were identified by ultra-performance liquid chromatography (UPLC) Polyamines analysis The individual polyphenolic compounds, total phenolic and flavonoid contents, antioxidant and Polyphenolic content antibacterial potential were significantly higher with in vitro regenerated plants than in vivo plants. Conclusions: Plant growth regulators (PGRs) and PAs had a significant effect on in vitro plant regeneration and also a biochemical accumulation of flavonols, hydroxybenzoic and hydroxycinnamic acid derivatives in C. anguria. Due to these metabolic variations, the antioxidant and antibacterial activities were increased with in vitro regenerated plants than in vivo plants. This is the first report describing the production of phenolic compounds and biological activities from in vitro and in vivo regenerated plants of C. anguria. ©2015Pontificia Universidad Católica de Valparaíso. Production and hosting by Elsevier B.V. All rights reserved. 1. Introduction Anthraquinones and saponins that are present in C. anguria are used for antibacterial and antifungal activity [3]. Plant secondary Cucumis anguria L. commonly known as bur cucumber, bur gherkin, metabolites are unique sources for pharmaceuticals, food additives, and West Indian gherkin is an important cucurbit vegetable and flavors, and industrially important biochemicals. Plant growth conventional medicinal crop. It is mainly cultivated and used in Africa, regulators (PGRs) are one of the most important factors affecting Brazil, Cuba, India, United States and Zimbabwe [1]. The fruits and cell growth, differentiation and metabolite formation [4].Thetype leaves of gherkin are consumed as boiled, fried, stewed, pickled and and concentration of PGRs especially auxins and/or cytokinins have also used as fresh in salads and hamburgers [2]. Additionally, the been shown to modify the shikimate/phenylpropanoid pathways in fruits, roots and seeds of gherkins are used for traditional medicine to the biochemical synthesis of phenolic acids, probably by regulating treat stomach ache, jaundice, hemorrhoids and preventing stone the enzyme activity of phenylalanine ammonia lyase and chalcone formation in the kidney. Since there is a growing worldwide demand synthase [5]. The capacity for plant cell, tissue and organ cultures for pickled gherkins, many food companies have started to explore to produce and accumulate many of the same valuable chemical opportunities for producing gherkins [2]. Phytochemists have reported compounds as the parent plant in nature has been recognized since a number of important medicinal components of cucurbitacin B, the inception of in vitro technology. Previously, many valuable cucurbitacin D and cucurbitacin G from C. anguria; the cucurbitacin B compounds are produced by in vitro plant cell cultures [6,7,8]. have been used for cancer prevention [2].Gherkinconsistsofmany Polyamines (PAs), a diamine putrescine, a triamine spermidine useful compounds such as flavonoids, tannins, alkaloids, saponins and a tetraamine spermine, are ubiquitous in the animal and plant and steroids that contained a high level of antioxidant activity. kingdom. PAs have been associated with many important cellular processes such as cell division, protein synthesis, DNA replication, response to abiotic stress, regulation of rhizogenesis, embryogenesis, ⁎ Corresponding author. fl E-mail address: [email protected] (I.-M. Chung). senescence, oral development and fruit ripening [9]. The exogenous Peer review under responsibility of Pontificia Universidad Católica de Valparaíso. application of PAs has shown a positive effect in the micropropagation http://dx.doi.org/10.1016/j.ejbt.2015.05.005 0717-3458/© 2015 Pontificia Universidad Católica de Valparaíso. Production and hosting by Elsevier B.V. All rights reserved. 296 M. Thiruvengadam, I.-M. Chung / Electronic Journal of Biotechnology 18 (2015) 295–301 of several species such as Withania somnifera [10] and Glycine max 2.4. Transplantation and acclimatization [11]. PAs have been successfully used in a variety of morphogenic processes in horticultural crops [12]. Very few studies reported in Rooted plantlets were washed thoroughly with tap water to remove cucurbits the effect of exogenous PAs have been enhanced for somatic agar and transplanted to plastic pots containing a mixture of autoclaved embryogenesis in Momordica dioica [9], multiple shoot regeneration sand, red soil, and vermiculite (1:1:1 v/v/v). Potted plants were grown from leaf explants of Momordica charantia [13] and shoot tip explants in a growth chamber (Sanyo, Tokyo, Japan) at 85% relative humidity of Cucumis sativus [14].Anefficient plant regeneration protocol is a for 2–3 weeks, and then moved to a greenhouse for 3 weeks before prerequisite for biotechnological breeding of economically important transferring to the field. The survival percentage was calculated after crops like cucurbits. Recently, we established the plant regeneration 4 weeks in the greenhouse. After, 4 weeks of acclimatization, plants from petiole derived callus [15], and direct somatic embryogenesis were transplanted to field. from hypocotyl and leaf explants of gherkin [2]. However, there is no report describing the effect of PGRs and PAs on in vitro regeneration 2.5. Sample preparation in gherkin. The objective of this investigation was to evaluate the synergistic effect of PAs (spermidine, spermine, and putrescine) Nine-week-old leaf derived from in vitro regenerated plants and with different PGRs for high-frequency multiple shoot regeneration in vivo-grown seed-derived from plants (one-month-old) of C. anguria systematically, via direct organogenesis using leaf explants. In were oven dried at 50°C to constant weight. The dried fine powder addition, the variations of total polyphenolic content, individual samples (0.1 g) were extracted with 10 mL 80% methanol in a polyphenols, antioxidant and antibacterial activities on in vitro and sonication bath for 30 min. The methanolic extracts were then filtered in vivo regenerated plants of C. anguria. under vacuum through Whatman No. 1 filter paper, and the filtrates were dried under vacuum using Rotary evaporator of crude extract. The extracts obtained were dissolved in the methanol immediately 2. Materials and methods used for the determination of total phenolics, flavonoids, antioxidant and antibacterial activity. 2.1. Plant material 2.6. Determination of total phenolic (TPC) and flavonoid (TFC) contents Seeds of gherkin (C. anguria L.) were obtained from Nunhems Seeds Pvt. Ltd. (Bangalore, India). Seeds were surface sterilized following our The TPC and TFC contents of the samples were analyzed by following previous reports [2], and were inoculated with the hormone free MS our previous procedure [18]. The concentration of the TPC and TFC was [16] basal salt mixture + B5 vitamins [17] (MSB5) supplemented with determined as mg of gallic acid and quercetin equivalent respectively. 0.8% (w/v) agar and 3% (w/v) sucrose. The seedlings were grown fl μ -2 -1 under white uorescent light (40 moL·m ·s ) at a photoperiod of 2.7. Extraction of phenolic compounds and analysis by ultra-performance 16/8 h of light/dark and temperature 25 ± 2°C. The leaf explants were liquid chromatography (UPLC) trimmed into appropriate sizes (0.5 cm2) to obtain 10 d old in vitro seedlings. One gram of dried plant
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