Phenolic Compound Production and Biological Activities from in Vitro Regenerated Plants of Gherkin (Cucumis Anguria
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EJBT-00103; No of Pages 7 Electronic Journal of Biotechnology xxx (2015) xxx–xxx Contents lists available at ScienceDirect Electronic Journal of Biotechnology 1 Phenolic compound production and biological activities from in vitro regenerated 2 plants of gherkin (Cucumis anguria L.) 3Q3 Muthu Thiruvengadam, Ill-Min Chung ⁎ 4 Department of Applied Bioscience, College of Life and Environmental Science, Konkuk University, Seoul, South Korea 5 article info abstract 6 Article history: Background: The effect of polyamines (PAs) along with cytokinins (TDZ and BAP) and auxin (IBA) was induced by 17 7 Received 8 January 2015 the multiple shoot regeneration from leaf explants of gherkin (Cucumis anguria L.). The polyphenolic content, 18 8 Accepted 2 May 2015 antioxidant and antibacterial potential were studied from in vitro regenerated and in vivo plants. 19 9 Available online xxxx Results: Murashige and Skoog (MS) medium supplemented with 3% sucrose containing a combination of 3.0 μM 20 TDZ, 1.0 μMIBAand75μM spermidine induced maximum number of shoots (45 shoots per explant) was 21 10 Keywords: achieved. Regenerated shoots elongated in shoot elongation medium containing 1.5 μMGA and 50 μM 22 11 Biological activities 3 μ 23 12 Cucumis anguria spermine. The well-developed shoots were transferred to root induction medium containing 1.0 M IBA and μ 24 13 Multiple shoot induction 50 M putrescine. Rooted plants were hardened and successfully established in soil with a 95% survival rate. 14 Plant growth regulators Twenty-five phenolic compounds were identified by ultra-performance liquid chromatography (UPLC) 25 15 Polyamines analysis The individual polyphenolic compounds, total phenolic and flavonoid contents, antioxidant and 26 16 Polyphenolic content antibacterial potential were significantly higher with in vitro regenerated plants than in vivo plants. 27 Conclusions: Plant growth regulators (PGRs) and PAs had a significant effect on in vitro plant regeneration and 28 also a biochemical accumulation of flavonols, hydroxybenzoic and hydroxycinnamic acid derivatives in 29 C. anguria. Due to these metabolic variations, the antioxidant and antibacterial activities were increased with 30 in vitro regenerated plants than in vivo plants. This is the first report describing the production of phenolic 31 compounds and biological activities from in vitro and in vivo regenerated plants of C. anguria. 32 33 34 ©2015Pontificia Universidad Católica de Valparaíso. Production and hosting by Elsevier B.V. All rights reserved. 353638 37 39 1. Introduction Anthraquinones and saponins that are present in C. anguria are 56 used for antibacterial and antifungal activity [3]. Plant secondary 57 40 Cucumis anguria L. commonly known as bur cucumber, bur gherkin, metabolites are unique sources for pharmaceuticals, food additives, 58 41 and West Indian gherkin is an important cucurbit vegetable and flavors, and industrially important biochemicals. Plant growth 59 42 conventional medicinal crop. It is mainly cultivated and used in Africa, regulators (PGRs) are one of the most important factors affecting 60 43 Brazil, Cuba, India, United States and Zimbabwe [1]. The fruits and cell growth, differentiation and metabolite formation [4].Thetype 61 44 leaves of gherkin are consumed as boiled, fried, stewed, pickled and and concentration of PGRs especially auxins and/or cytokinins have 62 45 also used as fresh in salads and hamburgers [2]. Additionally, the been shown to modify the shikimate/phenylpropanoid pathways in 63 46 fruits, roots and seeds of gherkins are used for traditional medicine to the biochemical synthesis of phenolic acids, probably by regulating 64 47 treat stomach ache, jaundice, hemorrhoids and preventing stone the enzyme activity of phenylalanine ammonia lyase and chalcone 65 48 formation in the kidney. Since there is a growing worldwide demand synthase [5]. The capacity for plant cell, tissue and organ cultures 66 49 for pickled gherkins, many food companies have started to explore to produce and accumulate many of the same valuable chemical 67 50 opportunities for producing gherkins [2]. Phytochemists have reported compounds as the parent plant in nature has been recognized since 68 51 a number of importantUNCORRECTED medicinal components of cucurbitacin B, the inception ofPROOFin vitro technology. Previously, many valuable 69 52 cucurbitacin D and cucurbitacin G from C. anguria; the cucurbitacin B compounds are produced by in vitro plant cell cultures [6,7,8]. 70 53 have been used for cancer prevention [2].Gherkinconsistsofmany Polyamines (PAs), a diamine putrescine, a triamine spermidine 71 54 useful compounds such as flavonoids, tannins, alkaloids, saponins and a tetraamine spermine, are ubiquitous in the animal and plant 72 55 and steroids that contained a high level of antioxidant activity. kingdom. PAs have been associated with many important cellular 73 processes such as cell division, protein synthesis, DNA replication, 74 response to abiotic stress, regulation of rhizogenesis, embryogenesis, 75 ⁎ Corresponding author. fl 76 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 77 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. Please cite this article as: Thiruvengadam M, Chung I-M, Phenolic compound production and biological activities from in vitro regenerated plants of gherkin (Cucumis anguria L.), (2015), http://dx.doi.org/10.1016/j.ejbt.2015.05.005 2 M. Thiruvengadam, I.-M. Chung / Electronic Journal of Biotechnology xxx (2015) xxx–xxx 78 of several species such as Withania somnifera [10] and Glycine max 2.4. Transplantation and acclimatization 135 79 [11]. PAs have been successfully used in a variety of morphogenic 80 processes in horticultural crops [12]. Very few studies reported in Rooted plantlets were washed thoroughly with tap water to remove 136 81 cucurbits the effect of exogenous PAs have been enhanced for somatic agar and transplanted to plastic pots containing a mixture of autoclaved 137 82 embryogenesis in Momordica dioica [9], multiple shoot regeneration sand, red soil, and vermiculite (1:1:1 v/v/v). Potted plants were grown 138 83 from leaf explants of Momordica charantia [13] and shoot tip explants in a growth chamber (Sanyo, Tokyo, Japan) at 85% relative humidity 139 84 of Cucumis sativus [14].Anefficient plant regeneration protocol is a for 2–3 weeks, and then moved to a greenhouse for 3 weeks before 140 85 prerequisite for biotechnological breeding of economically important transferring to the field. The survival percentage was calculated after 141 86 crops like cucurbits. Recently, we established the plant regeneration 4 weeks in the greenhouse. After, 4 weeks of acclimatization, plants 142 87 from petiole derived callus [15], and direct somatic embryogenesis were transplanted to field. 143 88 from hypocotyl and leaf explants of gherkin [2]. However, there is no 89 report describing the effect of PGRs and PAs on in vitro regeneration 2.5. Sample preparation 144 90 in gherkin. The objective of this investigation was to evaluate 91 the synergistic effect of PAs (spermidine, spermine, and putrescine) Nine-week-old leaf derived from in vitro regenerated plants and 145 92 with different PGRs for high-frequency multiple shoot regeneration in vivo-grown seed-derived from plants (one-month-old) of C. anguria 146 93 systematically, via direct organogenesis using leaf explants. In were oven dried at 50°C to constant weight. The dried fine powder 147 94 addition, the variations of total polyphenolic content, individual samples (0.1 g) were extracted with 10 mL 80% methanol in a 148 95 polyphenols, antioxidant and antibacterial activities on in vitro and sonication bath for 30 min. The methanolic extracts were then filtered 149 96 in vivo regenerated plants of C. anguria. under vacuum through Whatman No. 1 filter paper, and the filtrates 150 were dried under vacuum using Rotary evaporator of crude extract. 151Q6 The extracts obtained were dissolved in the methanol immediately 152 97 2. Materials and methods used for the determination of total phenolics, flavonoids, antioxidant 153 and antibacterial activity. 154 98 2.1. Plant material 2.6. Determination of total phenolic (TPC) and flavonoid (TFC) contents 155 99 Seeds of gherkin (C. anguria L.) were obtained from Nunhems Seeds 100 Pvt. Ltd. (Bangalore, India). Seeds were surface sterilized following our The TPC and TFC contents of the samples were analyzed by following 156 101 previous reports [2], and were inoculated with the hormone free MS our previous procedure [18]. The concentration of the TPC and TFC was 157 102 [16] basal salt mixture + B5 vitamins [17] (MSB5) supplemented with determined as mg of gallic acid and quercetin equivalent respectively. 158 103 0.8% (w/v) agar and 3% (w/v) sucrose. The seedlings were grown 104 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 159 105 16/8 h of light/dark and temperature 25 ± 2°C. The leaf explants were liquid chromatography (UPLC) 160 106 trimmed into appropriate sizes (0.5 cm2) to obtain 10 d old in vitro 107 seedlings. One gram of dried plant powder was extracted as described in the 161 procedure [8].Thefiltrate was used for analysis using a Thermo Accela 162 163 108 2.2. Multiple shoot induction UPLC (Thermo, New York, USA) system. The separation was achieved using a HALO C18 (2.7 μm, 2.1 × 100 mm) column and the absorbance 164 was measured at 280 nm. The mobile phases were 0.1% glacial acetic 165 109Q4 The shoot bud initiation media which consisted of MSB5 166 110 medium with 3% (w/v) sucrose and solidified with 0.8% (w/v) acid in distilled water (solvent A) and 0.1% glacial acetic acid in 167 111 agar supplemented with different concentrations (1.0–4.0 μM) of acetonitrile (solvent B).