Differential Production of Phenylpropanoid Metabolites in Callus

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Bioactive Constituents, Metabolites, and Functions

Differential production of phenylpropanoid metabolites in callus cultures of Ocimum basilicum L. with distinct in vitro antioxidant activities and in vivo protective effects against UV stress Munazza Nazir, Duangjai Tungmunnithum, Shankhamala Bose, Samantha Drouet, Laurine Garros, Nathalie Giglioli-Guivarc’h, Bilal Haider Abbasi, and Christophe Hano J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b05647 • Publication Date (Web): 25 Jan 2019 Downloaded from http://pubs.acs.org on January 27, 2019

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Journal of Agricultural and Food Chemistry

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Research article for Journal of Agricultural and Food Chemistry

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Differential production of phenylpropanoid metabolites in callus cultures of Ocimum basilicum L. with distinct in vitro antioxidant activities and in vivo protective effects against UV stress

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Munazza Nazir†‡, Duangjai Tungmunnithum¶#⊥, Shankhamala Bose§, Samantha Drouet¶#, Laurine Garros¶#∥, Nathalie Giglioli-Guivarc’h§, Bilal Haider Abbasi†§¶#*, Christophe Hano¶#*

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†Department

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¶Laboratoire

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§ Biomolécules

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‡Department of Botany, University of Azad Jammu &Kashmir Muzaffarabad-13230 Azad Kashmir

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⊥Department

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#COSM’ACTIFS,

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∥Institut

of Biotechnology, Quaid-i-Azam University, Islamabad-45320, Pakistan

de Biologie des Ligneux et des Grandes Cultures (LBLGC), Plant Lignans Team, EA 1207, INRA USC1328, Université d’Orléans, F 28000 Chartres, France et Biotechnologies Végétales (BBV), EA2106, Université de Tours, Tours, France

Pakistan of Pharmaceutical Botany, Faculty of Pharmacy, Mahidol University, 447 SriAyuthaya Road, Rajathevi, Bangkok 10400, Thailand Bioactifs et Cosmétiques, CNRS GDR3711, 45067 Orléans Cedex 2, France

de Chimie Organique et Analytique (ICOA) UMR7311, Université d’Orléans-CNRS, 45067 Orléans Cedex 2, France

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*These two authors have equal contribution as senior author

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Correspondance

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Email : [email protected]; [email protected]

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Phone & fax: +33 77 698 41 48 (BHA), +33 2 37 30 97 53 (CH)

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ACS Paragon Plus Environment


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Abstract

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Ocimum basilicum L. (Purple basil) is a source of biologically active antioxidant compounds,

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particularly phenolic acids and anthocyanins. In this study, we have developed a valuable protocol

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for the establishment of in vitro callus cultures of O. basilicum and culture conditions for the

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enhanced production of distinct classes of phenylpropanoid metabolites such as hydroxycinnamic

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acid derivatives (caffeic acid, chicoric acid, rosmarinic acid) and anthocyanins (cyanidin and

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peonidin). Callus cultures were established by culturing leaf explants on Murashige and Skoog

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medium augmented with different concentrations of plant growth regulators (PGRs) [thidiazuron

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(TDZ), α-naphthalene acetic acid (NAA) and 6-benzyl amino purine (BAP)] either alone or in

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combination with 1.0 mg/l NAA. Among all the above mentioned PGRs, NAA at 2.5 mg/l led to

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the highest biomass accumulation (23.2 g/l DW), along with total phenolic (TPP; 210.7 mg/l) and

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flavonoid (TFP; 196.4 mg/l) production, respectively. HPLC analysis confirmed the differential

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accumulation of phenolic acid [caffeic acid (44.67mg/g DW), rosmarinic acid (52.22 mg/g DW)

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and chicoric acid (43.89mg/g DW)] and anthocyanins [cyanidin (16.39mg/g DW) and peonidin

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(10.77mg/g DW)] as a function of the PGRs treatment. The highest in vitro antioxidant activity,

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was determined with the ORAC assay as compared to the FRAP assay, suggesting the prominence

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of the HAT over the ET-based mechanism for the antioxidant action of callus extracts.

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Furthermore, in vivo results illustrated the protective action of the callus extract to limit the

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deleterious effects of UV-induced oxidative stress, ROS/RNS production and membrane integrity

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in yeast cell culture. Altogether, these results clearly demonstrated the great potential of in vitro

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callus of O. basilicum as a source of human health-promoting antioxidant phytochemicals.

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Keywords: Ocimum basilicum L., callus culture, α-naphthalene acetic acid, phenylpropanoid

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metabolites, rosmarinic acid, anthocyanins.


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1. Introduction

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Ocimum basilicum L., belongs to family Lamiaceae which is well-known with its common name

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“Basil”, and is an important medicinal plant found mainly in South Asian region, India, and Africa1.

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The genus Ocimum consists of about 200 species of shrubs and herbs and few of them are being

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cultivated all around the world, as a natural herbal medicine2. Basil has been used as a culinary

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herb since prehistoric times and its essential oils have been employed in food, flavor, and fragrance

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for cosmetic industries3. Ocimum species have a key position not only as an aromatic or ornamental

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plant which applied for many soft cape purposes, but also as a medicinal plant due to its high

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substances from volatile oils and plentiful secondary metabolites4.

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The purple basil, an aromatic annual plant with erect growth habit, is one of the important cultivar

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of this species with traditional sweet basil flavor5. It accumulates a wide variety of

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phenylpropanoids including hydroxycinnamic derivatives (caffeic acid, rosmarinic acid and

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chicoric acid) and anthocyanins (cyanidin and peonidin)6-8. These compounds are known to show

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vital roles in the plant interactions with its environment and provide tolerance against biotic and/or

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abiotic stresses9. The antioxidant activity of these compounds is of great interest for human health9.

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“Antioxidant” is a generic term used for a group of a large array of molecules with distinct

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structures that can exert their action through the cession of electron(s), hence acting as reducing

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agents, but can differ in their action mode10. Antioxidants can neutralize radicals through two main

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mechanisms: the electron transfer (ET) mechanism or the hydrogen atom transfer (HAT)

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mechanism, that both led to the same result11. To determine the antioxidant activity and its action

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mode of one compound, several in vitro assays have been developed. Among these protocols, the

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ferric reducing antioxidant power (FRAP) is an ET-based reaction, whereas the oxygen radical

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absorbance capacity (ORAC) operates through an HAT mechanism11. The use of these two in vitro



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assays applied to the same extract can therefore determine both its antioxidant capacity but also

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give some important information about the molecular mechanism involved. In the development of

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many human ailments including cancers, atherosclerosis and neurodegenerative diseases, free-

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radical pathways have been implicated. An example of deleterious action of oxidative stress is its

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involvement in the formation of advanced glycation end-products (AGEs), resulting from non-

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enzymatic reactions based on the Maillard reaction12. AGEs resulted in protein dysfunctions and

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have implicated in the development of Alzheimer and cardiovascular diseases12. For more detailed

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mechanism study, in vivo or in cellulo models are of interest to confirm these in vitro assays. For

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this purpose, baker yeast (Saccharomyces cerevisiae), a true eukaryote, is an attractive and

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persistent model with a well understood mechanism of defense and adaptation to oxidative stress13.

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Ocimum species standout among the most economically critical beneficial plants on the globe 14.

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With the advent of modern and high throughput technologies, pharmaceutical industries are

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focusing on utilizing plant-derived compounds to develop medicine with precise mode of action.

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The apparent obstacle in the application of Lamiaceae members in pharmaceutical industry is due

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to their genetic makeup and biochemical complexity15. Propagation through seeds is the usual

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method for this family, however, there are some limitations of this method in proliferation on a

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large scale including specify natural cross pollination of plant, poor seed viability, low seed

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germination, high level of content variability and poor extraction procedures16, 17. These are some

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major drawbacks and obstacles in using whole plant for treatment therapy. In vitro plant tissue

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culture procedures can be used to overcome these shortcomings of traditional methods. Huge

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number of in vitro studies have been reported on Lamiaceae species, including the genus Ocimum,

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using diverse explants as nodal portions18, young inflorescence19, axillary buds20 and leaf

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explants21.


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The compounds attained from the in vitro cell, tissue and organ culture might be precisely used as

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medicines, deprived of any variations, or may endure extra semi-synthetic modulation22. Enhanced

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production of plant secondary metabolites can be ensured by establishing uniform callus cultures

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of plant, irrespective to external environmental alternations23. Production of natural phenolic acids,

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essential oils and anthocyanins via tissue culture provide a clear advantage in terms of both yield

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and biomass conservation24, 25.

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Thus, the goal of current study was to establish an effective and efficient protocol to produce in

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vitro callus cultures derived from O. basilicum, which accumulate significant levels of

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hydroxicinnamic acid derivatives and anthocyanins under the impact of various plant growth

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regulators. The rationalization of the antioxidant properties of the extracts derived from these callus

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cultures were studied using both in vitro (ORAC, FRAP, anti-AGEs formation) and in vivo (yeast

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cell cultures) assays.



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2. Materials and Methods

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2.1. Chemicals and Reagents

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Analytical grade extraction solvents were used in the present study and were obtained from Thermo

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Scientific. Standards were from Sigma-Aldrich. All chemicals for antioxidant (FRAP, ORAC and

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anti-AGEs) activities were from Sigma-Aldrich.

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2.2. Seeds Collection and Germination

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O. basilicum seeds were gained from the National Agriculture Research Center (NARC)

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Islamabad, Pakistan. For surface disinfection of seeds, previously described method was

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followed26. Viable seeds were carefully chosen and treated for 30 seconds with 0.1 % mercuric

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chloride solution, followed by 60 seconds exposure to ethanol (70%) and subsequent washing with

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purified water thrice to remove unwanted and unnecessary particles. Murashige and Skoog (MS)

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media comprising of agar (8 g/l) and sucrose (30 g/l) was inoculated with decontaminated seeds.

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Preceding to autoclave, pH was maintained between 5.6 and 5.8. Growth chamber was used with

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controlled temperature (25°C ± 2°C) and photoperiod of 16h/8h (light/dark cycle) was employed

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for proper germination of inoculated seeds.

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2.3. Establishment of Callus Culture

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For establishment of callus culture, in vitro derived plantlets (28 days old) were used as explant

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source. Leaf explants (ca 0.5 cm2) were excised and then placed on MS media, augmented with

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different concentrations (0.1-20.0 mg/l) of plant growth regulators (PGRs) [thidiazuron (TDZ), α-

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naphthalene acetic acid (NAA) and 6-benzyl amino purine (BAP)] either alone or in combination

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with 1.0 mg/l NAA, as shown in Table 1. Controlled growth conditions were sustained at 25 ± 2

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°C for 16/8h light/dark in the growth chamber. Experiments were performed thrice with explants


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for each PGR treatment. Data was collected on callus initiation day, and the callus was further sub-

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cultured using same PGRs conditions for subsequent 4 weeks. Following this, the callus was

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harvested after 1st sub-culture for the determination of fresh weight (FW) and dry weight (DW),

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respectively.

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2.4. Sample Extraction and Hydrolysis

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Samples were first extracted by ultrasound and then hydrolyzed by adopting previously

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documented protocol27. Freeze dried samples from each treatment (2g) was thoroughly mixed with

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10 ml HPLC grade methanol (Thermo Scientific) at 50 °C for 1 hour followed by 30 min sonication

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and vortexing. Ultrasonic bath USC1200TH (Prolabo; inner dimension: 300 mm x 240 mm x 200

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mm) was used for ultrasonication, with an electrical power of 400W (i.e. acoustic power of

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1W/cm2) working at a frequency of 45 kHz. Centrifugation was done at 10000 rpm for 10 min

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followed by repeated sonication and vortexing and the resultant supernatant was then separated for

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hydrolysis. For this purpose, concentrated HCl was added to the supernatant to perform hydrolysis

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in strong acidic medium for a complete hydrolysis of glycosides. During preliminary experiments,

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different HCl concentrations, temperature and duration were tested (data not shown). The extract

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was hydrolyzed with HCl (2.5M final concentration), heated at 80 °C for 2 hours. The hydrolyzed

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sample was cooled in the dark and the reaction medium was then neutralized with NaOH, filtered

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through a 0.45 μm filter, and 50 ml methanol was used for dilution. A 20 μl aliquot was used for

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HPLC analysis.

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2.5. Determination of Total Phenolic and Flavonoid Contents

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Previous method was used for the estimation of total phenolic content (TPC) using Folin-Ciocalteu

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(FC) reagent28. Extracted sample (20 µl) from each PGR treatment was incubated for 5 min at

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normal temperature after putting into 96 well microplate with sodium carbonate (90 µl) and Folin-



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Ciocalteu (FC) reagent (90 µl) and then the absorbance (Halo DR-20, UV-VIS spectrophotometer,

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Dynamica Ltd, Victoria, Australia) of the reaction mixture was measured at 725 nm. For positive

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and negative controls, gallic acid (1 mg/ml) and methanol (20 µl) were used respectively. TPC was

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expressed as gallic acid equivalents (GAE/g DW) and it also serve as standard for plotting

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calibration curve (0–50 µg/ml, R2=0.998). Total phenolic production (TPP) was assessed in mg

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gallic acid/l and measured as:

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Total Phenolic Production (mg/l) = DW (g/l) × TPC (mg/g).

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Total flavonoid content (TFC) was estimated by following previously reported protocol29. Briefly,

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the extracted samples (20 µl) were mixed with 1M potassium acetate (10 µl) and 10 µl of AlCl3

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(10%, w/v) in a 96 well micro-plate and then 160 µl of purified water was added to it to make a

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final volume of 200 µl. After half an hour, absorbance was taken at 415 nm with UV-Visible

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spectrophotometer. Calibration curve (0-40 µg/ml, R2=0.998) was plotted by using quercetin as

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standard and TFC was expressed as quercetin equivalents (QE/g DW), with their production (TFP)

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being expressed in mg quercetin/l and calculated as:

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Total Flavonoid Production (mg/l) = DW (g/l) × TFC (mg/g).

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2.6. DPPH Antioxidant Assay

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Free radical scavenging assay (FRSA) was performed according to the previously described

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procedure30. Briefly, 20 µl of sample and 180 µl of DPPH (2, 2-Diphenyl-1-picrylhydrazyl) reagent

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were incubated in the dark for 60 min. DMSO (20 µl) and final concentrations of ascorbic acid (40,

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20, 10, 5µg/ml) were taken as negative control. With spectrophotometer, readings were taken at

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517 nm. FRSA activity calculations were done by using the following formula:

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% scavenging DPPH free radical = 100 × (1-AE/AD)


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Where, AE represents absorbance of the mixture with sample addition and AD displays the

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absorbance without adding anything at 517 nm.

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2.7. High Performance Liquid Chromatography (HPLC) Analysis

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Main metabolites quantification was done by HPLC via standard grade chemicals. Separation was

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performed according to a technique documented previously31. To perform separation, Hypersil PEP

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300 C18 column (250 x 4.6 mm, 5 µm) equipped with a guard column Alltech (10 x 4.1 mm) was

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utilized at 35 °C and Varian high-performance liquid chromatography system (equipped with

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Varian Prostar 230 pump, Metachem Degasit degasser, Varian Prostar 410 autosampler and Varian

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Prostar 335 Photodiode Array Detector), driven by Galaxie version 1.9.3.2 software, was used for

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the detection of compounds at 320 and 520 nm wavelengths. The mobile phase was composed of

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A = HCOOH / H2O, pH = 2.1, B = CH3OH (HPLC grade solvents). Throughout one-hour run,

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mobile phase composition varied with a nonlinear gradient 8 % B (0 min), 12 % B (11 min), 30 %

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B (17 min), 33 % B (28 min), 100 % B (30–35 min), 8 % B (36 min) at a flow rate of 1 ml/min. A

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10-minute re-equilibration time was used among individual runs. Quantification was done based

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on assessment of holding times and reliable reference standards. The comparative standards of

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caffeic acid, chicoric acid, rosmarinic acid, cyanidin and peonidin were purchased from Sigma

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Aldrich. All the samples examination was done in triplicates and the outcomes were voiced as

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µg/mg DW of the sample.

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2.8. Method Validation

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Five-point calibration curves were made by means of diluted solutions of each authentic

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commercial standard (Sigma Aldrich). Each sample was inserted thrice and arithmetic means was

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calculated. To generate linear regression equations plotting was done by the peak areas (y) against

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the inserted quantities (x) of standard compounds. Coefficients of determination (R2) was used for



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linearity verification. Based on the signal-to-noise ratios (S: N) 3:1 and 10:1, were used for

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determination of the limits of detection (LOD) and the limits of quantification (LOQ), respectively.

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Accuracy was observed by measuring recovery rates using the “purple” and “green” O. basilicum

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callus extracts. Sample of each extract was standardized and divided into two equal mass halves,

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one was spiked with a recognized stock solutions volume. Both parts were examined by HPLC in

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three repeats ensuing the method designated. To calculate rates of recovery, subsequent formula

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was used:

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Recovery rate (%) = [(amount in spiked part) (amount in non-spiked part)/spiked amount] x 100.

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Technique accuracy was assessed by decisive the intraday and interday differences computed from

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data gained from repetitive of standard mixture injections. Five replicates per day were used for

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intraday variation while three continuous days injections were used for interday variation

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evaluation. The repeatability using five continuous injections of the same extracted sample were

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used to evaluate accuracy. Relative standard deviation (RSD %) was used to express precision.

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2.9. Ferric Reducing Antioxidant Power (FRAP) Assay

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Ferric reducing antioxidant power (FRAP) was evaluated as designated by Benzie and Strain32.

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Briefly, extracted samples (10 μl) were mixed with FRAP (190 μl) solution [composed of 10 mM

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TPTZ; 20 mM FeCl3, 6H2O and 300 mM acetate buffer pH 3.6; ratio 1:1:10 (v/v/v)]. Reaction

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mixtures were then stored for 15 min at room temperature (25 ± 1 °C). Absorption was measured

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with BioTek ELX800 absorbance microplate reader (BioTek Instruments) at 630 nm. Tests were

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performed in triplicate and the antioxidant capacity was expressed as Trolox C equivalent

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antioxidant capacity (TEAC).

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2.10. Oxygen Radical Absorbance Capacity (ORAC) Assay

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Previously described method was used for oxygen radical absorbance capacity (ORAC) 11. Briefly,

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an amount of 190 μl of fluoresce prepared in 75 mM phosphate buffer (pH 7.4) was mixed with 10

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μl extracted sample and then incubated for 20 minutes at 37 ± 1 °C with orbital shaking. Then, by

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adding 20 µl of 119.4 mM 2, 2’-azobis-amidinopropane (ABAP, Sigma Aldrich), the fluorescence

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intensity was measured for every 5 mins during 2.5 h at 37 °C using a fluorescence

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spectrophotometer (Bio-Rad) set with an excitation wavelength at 485 nm and emission

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wavelength at 535 nm. Assays were completed thrice. Trolox C equivalent antioxidant capacity

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(TEAC) was used to express capacity of antioxidant.

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2.11. Anti-AGEs Formation Activity

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The inhibitory capacity of advanced glycation end products (AGEs) formation was determined as

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described by Kaewseejan and Siriamornpun33. Extracts were prepared at a concentration of 50

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µg/ml in DMSO (Dimethyl sulfoxide) mixed with 20 mg/ml BSA (Sigma Aldrich). Both were

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prepared in phosphate buffer and 1 ml of 0.1 M phosphate buffer containing 0.02 % (w/v) sodium

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azide at pH 7.4. The amount of fluorescent AGE formed was measured after incubation of mixture

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at 37 °C for 5 days in the dark by using a fluorescent spectrometer (Bio-Rad VersaFluor) with an

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excitation wavelength set at 330 nm and emission wavelength set at 410 nm. For each extract, the

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percentage of anti-AGEs formation was showed as percentage inhibition relative to the

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corresponding control (addition of the same volume of DMSO).

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2.12. Yeast Cell Cultures and UV-Induced Oxidative Stress

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Saccharomyces cerevisiae INVSc1 (MATahis3D1 leu2 trp1-289 ura3-52 MATα his3D1 leu2 trp1-

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289 ura3-52) (Invitrogen) wild-type strain was routinely maintained on YPDA nutrient medium

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(2% glucose, 1% yeast extract, 2% peptone, 0.003% adenine hemisulfate, pH 6.5; Sigma Aldrich).



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Flasks were inoculated at an absorbance value of 0.1 at 630 nm from the same starter culture and

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incubated at 28 °C with orbital shaking at 120 rpm in the dark. Cell numbers for each condition

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were determined by using a modified Malassez hemocytometer slide. O. basilicum callus extracts

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were prepared in DMSO (0.1 % final concentration of DMSO was applied on yeast cell

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suspensions) and added at a final concentration of 50 µg/ml to the yeast cell suspension. Two

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different extracts of O. basilicum were used: “purple” (TDZ 5mg/l) containing high content of

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anthocyanins and “green” (NAA 2.5 mg/l) containing low content of anthocyanins. Control yeast

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cells received the same volume of DMSO. Cells were then incubated at 28 °C with orbital shaking

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at 120 rpm in the dark until an optimal growth phase was reached (absorbance value at 630 nm of

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0.3–0.4). For the wash test, cells were collected by centrifugation (5 min at 3,000 rpm), washed

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three times with fresh YPDA medium, and cells were re-suspended in the same volume of fresh

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YPDA medium before UV treatment. Oxidative stress was then induced by UV treatment. The

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irradiated yeast cells were subjected to a UV dose of 60 J/m2 in the dark with a 30-W general

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electric germicidal lamp and incubated for 24 hrs at 28 °C with orbital shaking at 120 rpm in the

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dark. Non-irradiated cells were grown under the same conditions.

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2.13. Estimation of Yeast Cell Viability

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Cell viability was determined as the ability to produce colony-forming unit (CFU). UV treatment

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after definite time, aliquots of yeast cell suspension were removed and coated on yeast-selective

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OGYE agar medium (Lab M) after suitable dilution. After 4 days incubation at 28 ºC, counting of

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colonies were done (no further colonies appeared after that time).

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2.14. Detection of Reactive Oxygen/Nitrogen Species

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Dihydrorhodamine-123 (DHR-123) fluorescent dye13, was used to determine the level of reactive

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species. Overnight growth of yeast cells was carried out in the presence of O. basilicum extracts or


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DMSO (control cells), then washed twice with PBS, re-suspended in PBS with 0.4 µM DHR-123

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and then incubated at 30 °C for 10 min in the dark. After washing twice with PBS, the fluorescence

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signal was detected using BioRad VersaFluor Fluorimeter (λex = 505 nm, λem = 535 nm).

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2.15. Evaluation of Membrane Lipid Peroxidation

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Thiobarbituric acid reactive substances (TBARS) were used for the quantification of Lipid

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peroxidation. Modified previous method was used for TBARS determination34. Cell suspension

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samples (0.5 ml) were removed at specific time (up to 3 hrs), after the addition of Cd(NO3)2 or

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Cu(NO3)2 and added to 1 ml of TBA reagent [0.25 M HCl, 15% (wt/vol) trichloroacetic acid,

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0.375% (wt/vol) TBA]. Assay was initiated after mixing of the substance that ended lipid

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peroxidation. To remove cell debris, samples were heated for 15 min followed by chilling and then

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centrifuged at 1000 rpm for 5 min. By using a BioTek ELX800 absorbance microplate reader

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(BioTek Instruments) absorbance was taken at 535 nm against a reference solution. Standard curve

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was prepared by using 1,1,3,3, tetramethoxypropane for calculations of TBARS concentrations in

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samples.

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2.16. Statistical Analysis

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All experiments were performed in a coordinated way, with three (biological) replicates for each

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treatment and repeated thrice (technical replicates). For statistical analysis, Origin software (v8.5)

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was used and analytical data was shown as mean ± SD using Microsoft Excel Program.

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3. Results and Discussion

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3.1. Callogenesis Initiation and Biomass Accumulation in Callus Culture of Purple Basil

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In vitro culture is a successful technique for large-scale production and rapid multiplication of

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medicinal plants species35. In the present study, different plant growth regulators were found to

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have substantial effect on callus induction frequency and biomass accumulation. Callus was

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initiated at the cut ends of leaves in MS medium supplemented with various concentrations (0.1,

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1.0, 2.5, 5.0, 10, 20 mg/l) and/or combinations of PGRs (NAA, TDZ, BAP). Varied responses were

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noted in initial callus induction days for each PGR treatment (Figure 1). The best callus induction

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response was observed with NAA concentrations ranging from 0.1 to 5.0 mg/l, with minimum of

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7 days required for callus initiation (Table 1). Using TDZ, a minimum of 10 days was required,

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whereas with BAP, a minimum 28 days before callus induction was noted. Similar findings have

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been reported before for NAA which induced high callus induction frequency as compared to other

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PGRs36. Likewise, NAA supplementation to the culture medium of Silybum marianum L. also

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exhibited increased callus induction26.

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Among all the tested PGRs, optimum results of biomass accumulation were showed by NAA,

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followed by TDZ, while minimum response was observed for BAP alone in callus culture (Figure

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2A, 2B). Maximum callus biomass, on both fresh weight (FW) and dry weight (DW) basis, was

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detected on MS medium supplemented with 2.5 mg/l of NAA (405 g/l FW and 23.2 g/l DW). This

303

indicates that NAA alone is very efficient to stimulate biomass accumulation in purple basil cell

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culture, which is in harmony with several reports on other medicinal plant species37- 41.

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3.2. Estimation of Total Phenolic, Total Flavonoid Contents and Free Radical Scavenging

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Activity


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Results illustrated in Figure 2C indicated that as a consequence to PGRs treatments (NAA, TDZ,

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BAP, NAA + TDZ and NAA + BAP), highest TPC levels were detected at 2.5 mg/l NAA (11.56

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mg/g DW) followed by 1.0 mg/l TDZ (11.04 mg/g DW) while, minimum TPC (0.2 mg/g DW)

310

levels were detected at high concentrations (20.0 mg/l) of BAP. These findings are in harmony

311

with the previously published reports which showed that NAA at a lower concentration results in

312

highest level of TPC in calli of Citrullus colocynthis L.42. From the observed data, a positive

313

relationship can be drawn between the total phenolic content and biomass accumulation in callus

314

cultures of O. basilicum. Highest total phenolic production (TPP; 210.7 mg/l) was also noted on

315

2.5 mg/l NAA (Figure 2D). Similar results were observed for Canscora decussata (Roxb.) Schult43.

316

Likewise, PGRs promoted biomass accumulation and resulted in quantifiable changes in the

317

formation of major elements in shoot culture of O. basilicum44. Analogous results were reported

318

before for phenolic content of purple basil45. Like total phenolic content (TPC) and their production

319

(TPP), total flavonoid content (TFC) and total flavonoid production (TFP) were also dependent on

320

PGRs treatments and biomass accumulation (Figure 2E, 2F). An increase in TFC was observed

321

with increased PGRs concentrations up to optimum concentration of 2.5 mg/l NAA (8.4 mg/g DW),

322

following which an increase in concentration of PGRs led to a sharp decrease in TFC (Figure 2E).

323

Maximum TFP (196.4 mg/l) was observed at 2.5 mg/l NAA concentration (Figure 2F). Our results

324

are in agreement with an earlier report for total flavonoid content in Artemisia absinthium L.46.

325

Though, many investigators reported comparable results of phytochemical analysis for cell cultures

326

of many other plants species 17, 47- 48. But to our knowledge this is the first report on TPC and TFC

327

estimation in purple basil cell cultures.



328

DPPH assay was then used to broadly estimate the free radical scavenging activities of each sample

329

extracts (Supplementary Figure 1). Basil leaf derived callus at different PGRs concentrations

330

showed increased free radical scavenging activity when treated with NAA and/or TDZ. Maximum

331

activity (93%) was observed with NAA concentration of 2.5 mg/l.

332

3.3. HPLC Analysis

333

Quantification of the main phytochemicals was achieved by HPLC analysis. Typical HPLC of

334

green (NAA 0.1 mg/l) and purple callus (TDZ 2.5 mg/l) are presented in Table 3. Caffeic acid,

335

rosmarinic acid, chicoric, cycanidin and peonidin were identified as the main phytochemicals by

336

comparison with authentic standards. To ensure the accuracy and precision of the method used to

337

quantify hydroxycinnamic acid derivatives (caffeic acid, chicoric acid and rosmarinic acid) and

338

anthocyanins (cyanidin and peonidin), the HPLC method was validated (Figure 3). The validation

339

parameters are presented in Table 2. The calibration curves of the peak areas (y) against the injected

340

quantities of hydroxycinnamic acid derivatives at 320 nm and anthocyanins at 520 nm were linear

341

over the wide range analyzed (50–1000 µg/ml) with R2 > 0.999. The LODs were low as 1.8 ng for

342

rosmarinic acid and 3.1 ng for caffeic acid, the LOQs ranged from 5.2 ng for rosmarinic acid and

343

10.2 ng for caffeic acid. The intra- and inter-day variations presented RSDs between 0.21%

344

(rosmarinic acid) and 1.54% (peonidin), and the RSDs of 5 repeats of the 2 selected extract samples

345

were less than 1.56% (peonidin). The measured minor variations reflected the high accuracy of the

346

HPLC method, whereas, the recovery rates between 95.1% (peonidin) and 102.3% (caffeic acid)

347

indicated the precision of the method.

348

Based on the TPC, TFC and DPPH analysis, samples of leaf derived callus grown on NAA, TDZ

349

and their combination were selected to quantify the accumulation in caffeic acid, rosmarinic acid,

350

chicoric acid, cyanidin and peonidin. Their accumulation in response to different PGRs is presented


Page 16 of 46

Page 17 of 46


351

in Table 3. In general, increased NAA concentration resulted in increased accumulation of

352

hydroxycinnamic acid derivatives. Comparable results were observed in different medicinal plants,

353

showing highest level of secondary metabolites accumulation in response to increased

354

concentrations of auxins49-51. TDZ supplementation greatly enhanced the accumulation of

355

hydroxycinnamic acid derivatives but also of anthocyanins resulting in an intense purple coloration

356

of the callus. Combination of both PGRs did not resulted in any additive effect on the accumulation

357

of these phytochemicals. Meyer and Staden52 have previously reported a substantial increase in

358

anthocyanins biosynthesis in callus cultures of Oxalis linearis Jacq. following TDZ application.

359

Among the identified phytochemicals, rosmarinic acid is considered as one of the key phenolic

360

compound found in basil species. Many authors previously reported rosmarinic acid content in

361

some intact Lamiaceae plants53,

362

production, although at a rate (0.01% DW) lower than other species55. The high quantity of

363

rosmarinic acid detected in our study ranged from approximately 21.47 mg/g DW (0.1 mg/l NAA)

364

to 52.22 mg/g DW (5.0 mg/l TDZ). These contents were in the range of results available for sweet

365

basil ranging from 0.1 to 100 mg/g DW56- 60. Furthermore, comparable contents of rosmarinic acid

366

were also reported in leaf derived callus culture of Ocimum species56. Similarly, higher content of

367

rosmarinic acid was found in in vitro callus cultures of holy basil as compared to field-grown plant

368

organs61. Study also showed that the yield of rosmarinic acid in callus culture of Salvia fructicosa

369

Mill. was 2.1% DW62. Furthermore, around 1 mg/g DW of rosmarinic acid was accumulated in

370

callus culture of O. basilicum and it was slightly more than the concentration in intact plants63.

371

Here, the considerable biomass accumulation (Figure 2A, 2B) associated to this high production

372

of rosmarinic acid was observed following TDZ application indicated the practicability of in vitro

373

leaf derived callus of purple basil as a potent production system of this compound.

54 .

O. basilicum can be proficiently used for rosmarinic acid



374

The amount of chicoric acid obtained from the callus culture ranged from the maximum level

375

(43.89 mg/g DW) in 5 mg/l TDZ to the minimum level (9.17 mg/g DW) in 0.1 mg/l NAA. These

376

results are comparable to the variations experienced previously to produce chicoric acid in O.

377

bascilicum cultured in vitro64. The highest biosynthesis of caffeic acid (44.67 mg/g DW) was

378

recorded in response to callus culture grown on 5.0 mg/l TDZ while the lowest (6.43 mg/g DW)

379

was noted when callus was grown on 0.1 mg/l of TDZ. Since caffeic acid is a precursor of

380

rosmarinic acid, this lower minimum level of caffeic acid may be related to more turnover of caffeic

381

acid to rosmarinic acid65, 66.

382

Purple basil is also known as a key source of cyanidin (major) and peonidin (minor) based pigments

383

51, 67.

384

different PGRs concentrations. Best results were observed following 5.0 mg/l TDZ addition to the

385

culture medium (Table 3). Minimum cyanidin (0.14 mg/g DW) and peonidin (0.09 mg/g DW) were

386

detected on 0.1 mg/l NAA augmented medium, whereas maximum cyanidin (16.39 mg/g DW) and

387

peonidin (10.77 mg/g) were observed on 5.0 mg/l TDZ. Comparable contents were reported in

388

sweet cherries (Prunus avium L.)31. In basil, Phippen and Simon67 reported on FW basis,

389

anthocyanin contents ranging from 6.5 to 18.7 mg/100g, depending on the variety. In the present

390

case, calculated in the less variable DW basis, our results show that in vitro cultures of basil could

391

also be considered as an attractive production system of these pigments.

392

3.4. Antioxidant Activities of Purple Basil Callus Extracts

393

The in vitro antioxidant activity of the selected O. basilicum callus extracts was further investigated

394

by three different methods: the ORAC and FRAP assays as well as their potential to inhibit both

395

vesperlysine- and pentosidine-like AGEs. ORAC represent a HAT reaction mechanism, which is

396

most related to human biology, whereas the FRAP mechanism is totally electron transfer rather

In the current study, contents of cyanidin greatly increased when calli were treated with


Page 18 of 46

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397

than mixed SET and HAT10,

11,

398

distinctive dominant mechanisms with changed antioxidants. AGEs formation is a good example

399

of the deleterious action of oxidative stress and have several human diseases11, 12 .

400

The results from these in vitro antioxidant assays are presented in Table 4. The highest antioxidant

401

activity, expressed in Trolox C equivalent antioxidant capacity (TEAC), was determined with the

402

ORAC assay as compared to the FRAP assay, suggesting the prominence of the HAT over the ET-

403

based mechanism for the antioxidant action of these extracts. The ORAC values ranged from

404

418.89 mM (0.1 mg/l NAA) to 962.54 mM (5.0 mg/l TDZ) TEAC, whereas the FRAP values

405

ranged from 417.88 mM (1 mg/l NAA + 0.1 mg/l TDZ) to 656.04 mM (5.0 mg/l TDZ) TEAC

406

(Table 4). From these results, it clearly appeared that “purple” callus of basil (5.0 mg/l TDZ) was

407

more potent source of natural antioxidant than the “green” ones (0.1 mg/l NAA). Interestingly, a

408

higher correlation existed between ORAC and phenolic acid contents (PCC = 0.925, p = 0.010 for

409

caffeic acid) than for anthocyanin contents (PCC = 0.845, p = 0.024 for cyanidin) (Table 5). An

410

inverse relationship was observed for FRAP, with a higher correlation between FRAP and

411

anthocyanin contents (PCC = 0.899, p = 0.014 for cyanidin), than between FRAP and phenolic

412

acid contents (PCC = 0.868, p = 0.020 for chicoric acid) (Table 5). A higher linear relation between

413

ORAC and total phenolic was previously observed, as compared to a linear relation between

414

anthocyanin contents and ORAC68.

415

Our analysis showed that O. basilicum callus extracts were able to inhibit the formation of both

416

vesperlysine and pentosidine-like AGEs (Table 4). TDZ treated purple-colored callus showed

417

highest capacities to inhibit both type of AGEs with a maximum inhibition of 58.76 % and 57.99

418

% toward the formation of vesperlysine- and pentosidine-like AGEs formation, respectively (Table

419

4). However, correlations linking the inhibition of AGEs formation and the main phytochemicals

so combination of these two methods can be very useful in



420

accumulated in the callus revealed that anthocyanins were not the primary contributors to these

421

inhibitions. Indeed, inhibition of vesperlysine-like AGEs formation was primary linked to the

422

presence of caffeic acid (PCC = 0.948, p = 0.007), whereas, analysis pointed chicoric acid (PCC =

423

0.958, p = 0.006) as the main contributor for the inhibition of pentosidine-like AGEs formation

424

(Table 5). In human, pentosidine-like AGEs are primarily observed in plasma and erythrocytes

425

whereas vesperlysine-like AGEs are mainly localized in the lens of diabetic subjects69. Therefore,

426

the identification of specific inhibitors of these AGEs could be of primary importance to direct a

427

therapeutic action toward a specific complication.

428

To better mimic the in vivo conditions, we next selected the two sample extracts from purple (5.0

429

mg/l TDZ, hereafter called Ob_purple extract) and green (1 mg/l NAA, hereafter called Ob_green

430

extract) O. basilicum callus to evaluate their protective action against UV-induced oxidative stress

431

in yeast cells. At first, the growth index, growth curves and viability were determined (Table 6;

432

supplementary Figure 2). The growth and viability of yeast cells grown in presence of the extracts

433

(50 µg/ml) evidenced the absence of deleterious effect of these two extracts since no significant

434

differences were noted in comparison to the control cells.

435

To study the antioxidant activity of these extracts, oxidative stress was induced by UV treatment

436

using previously described method70. To differentiate the action of cellular vs extracellular

437

antioxidants, a PBS washing was realized before UV treatment. As expected, the growth of the

438

yeast cells was strongly inhibited and drop of their viability were observed following UV-induced

439

oxidative stress. However, yeast cell cultures supplemented with the O. basilicum extracts showed

440

a significant improvement of their growth and viability as compared to control cells. This evidence

441

support a probable protective effect of the extracts against UV- induced oxidative stress. This

442

protective effect was observed in both PBS washed and non-washed conditions evidencing the


Page 20 of 46

Page 21 of 46


443

intervention. The highest protective effect was noted with the non-PBS washed yeast cell treated

444

with the Ob_purple extract. However, a slight (non-significant) decrease in cell viability and a

445

stronger (significant) decrease in growth was noted with cell treated with this Ob_purple extract

446

following PBS wash which indicates a possible intervention of extracellular antioxidants. On the

447

contrary, no significant effect of PBS wash was observed with the Ob_green extract, affirming the

448

primary action of cellular (intracellular or membrane-associated) antioxidants (Table 6).

449

We also evaluated the production of reactive oxygen and nitrogen species (ROS and RNS) and the

450

formation of lipid peroxidation (Figure 4). We used DHR-123 previously used in yeast as an

451

indicator of ROS/RNS production and membrane integrity in yeast13. The results confirmed the

452

previous trends i.e. a protective effect of both O. basilicum callus extract with a highest protective

453

effect of Ob_purple extract and a decrease of this protective effect following PBS wash (Figure

454

4A). These trends were also confirmed by the measurements of lipid membrane peroxidation

455

evaluated as TBARS production (Figure 4B).

456

A similar protective effect against oxidative stress was formerly detected on yeast cells treated with

457

thiamine13 and melatonin70. To the best of our information, this the first time that this system is

458

used to characterize a plant extract. Interestingly, the different effect of the two extracts following

459

PBS wash was attributed by the presence of anthocyanins. We hypothesized that it may be because

460

of their positive charge anthocyanins are not able to cross nor interact strongly with the cellular

461

membranes, and therefore remained in the culture medium acting as non-cellular antioxidant or

462

UV filters. In contrast, we hypothesized that phenolic acids were able to cross and/or interact with

463

the membranes. In our hands, we were able to detect caffeic acid, rosmarinic acid and chicoric acid

464

in yeast cells following PBS washes but not cyanidin nor peonidin (data not shown). Future



465

researches dedicated to the elucidation of the molecular mechanism involved in this cellular

466

antioxidant action of O. basilicum extract component(s) will be conducted.

467

Abbreviations

468

BAP; 6-benzyl amino purine

469

DW; Dry weight

470

DMSO; Dimethyl sulfoxide

471

DPPH; 2, 2-Diphenyl-1-picrylhydrazyl

472

ET; Electron transfer

473

FRSA; Free radical scavenging activity

474

FW; Fresh weight

475

HAT; Hydrogen atom transfer

476

MS; Murashige and Skoog

477

NAA; α-naphthalene acetic acid

478

ORAC; Oxygen Radical Absorbance Capacity

479

TDZ; Thidiazuron

480

TFC; Total flavonoid content

481

TFP; Total flavonoid production

482

TPC; Total phenolic content

483

TPP; Total phenolic production

484 485 486 487


Page 22 of 46

Page 23 of 46


488

Acknowledgements

489

This research was partially supported by Cosmetosciences, a global training and research program

490

devoted to the cosmetic industry. Positioned in the heart of the Cosmetic Valley, this program led

491

by University of Orléans is funded by the Région Centre-Val de Loire. BHA acknowledges

492

research fellowship of Le Studium-Institute for Advanced Studies, Loire Valley, Orléans, France.

493

DT appreciatively acknowledges the support of French government via the French Embassy in

494

Thailand in the form of Junior Research Fellowship Program 2018.

495

Conflict of Interest

496

The authors declare no conflict of interest

497

Contribution of Authors

498

MN performed in vitro experiments and carried out all biochemical assays. SB, SD, LG and DT

499

contributed to HPLC analysis and antioxidant assays. CH performed HPLC and antioxidant

500

analyses, interpretation, and contributed to the writing and reviewing manuscript. BHA

501

apprehended the idea, helped and supervised the research and reviewed the paper critically.

502 503 504 505 506 507 508 509



510

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F.; Ammar, S.; Bouzid, S.; Harzallah-Skhiri, F., Phenolic composition and antioxidant and

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antimicrobial activities of extracts obtained from Crataegus azarolus L. var. aronia (Willd.) Batt.

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ovaries calli. J Bot 2014, 2014.

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48. Jain, P.; Rashid, A. Stimulation of shoot regeneration on Linum hypocotyl segments by

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thidiazuron and its response to light and calcium. Biol Plant 2001, 44, 611-613.

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49. Al Abdallat, A.; Sawwan, J.; Al Zoubi, B. Agrobacteriumtumefaciens-mediated transformation

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of callus cells of Crataegusaronia. Plant Cell Tissue Organ Cult 2011, 104, 31-39.

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50. Danya, U.; Udhayasankar, M.; Punitha, D.; Arumugasamy, K.; Suresh, S., In vitro regeneration

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of Tecomella undulata (Sm.) Seem-an endangered medicinal plant. Int J Plant Animal Environ Sci

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2012, 2, 44-49.

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51. Fazal, H.; Abbasi, B. H.; Ahmad, N., Optimization of adventitious root culture for production

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of biomass and secondary metabolites in Prunella vulgaris L. Appl Biochem Biotechnol 2014, 174,

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2086-2095.

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52. Meyer, H.; Van Staden, J., The in vitro production of an anthocyanin from callus cultures of

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Oxalis linearis. Plant Cell Tissue Organ Cult 1995, 40, 55-58.

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53. Sahraroo, A.; Babalar, M.; Mirjalili, M.H.; Moghaddam, M.R.F.; Ebrahimi, S.N., In-vitro

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callus induction and rosmarinic acid quantification in callus culture of Satureja khuzistanica

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Jamzad (Lamiaceae). Iran J Pharm Res 2014, 13, 1447.

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54. Zgorka, G.; Głowniak, K., Variation of free phenolic acids in medicinal plants belonging to the

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Lamiaceae family. J Pharm Biomed Anal 2001, 26, 79-87.

647

55. Kintzios. S. E., In vitro rosmarinic acid production. Medicinal and Aromatic Plants-Industrial

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Approaches: The Genus Salvia, Second, Eds.; Publishers: Harwood, Amsterdam 2000, 14, 233-

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242.



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56. Kintzios, S.; Kollias, H.; Straitouris, E.; Makri, O., Scale-up micropropagation of sweet basil

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(Ocimum basilicum L.) in an airlift bioreactor and accumulation of rosmarinic acid. Biotechnol

652

Lett 2004, 26, 521-523.

653

57. Rady, M. R.; Nazif, N. M., Rosmarinic acid content and RAPD analysis of in vitro regenerated

654

basil (Ocimum americanum) plants. Fitoterapia 2005, 76, 525-533.

655

58. Bais, H. P.; Walker, T. S.; Schweizer, H. P.; Vivanco, J. M., Root specific elicitation and

656

antimicrobial activity of rosmarinic acid in hairy root cultures of Ocimum basilicum. Plant Physiol

657

Biochem 2002, 40, 983-995.

658

59. Sgherri, C.; Cecconami, S.; Pinzino, C.; Navari-Izzo, F.; Izzo, R., Levels of antioxidants and

659

nutraceuticals in basil grown in hydroponics and soil. Food Chem 2010, 123, 416-422.

660

60. Javanmardi, J.; Khalighi, A.; Kashi, A.; Bais, H.; Vivanco, J., Chemical characterization of

661

basil (Ocimum basilicum L.) found in local accessions and used in traditional medicines in Iran. J

662

Agric Food Chem 2002, 50, 5878-5883.

663

61. Hakkim, F. L.; Shankar, C.G.; Girija, S., Chemical composition and antioxidant property of

664

holy basil (Ocimum sanctum L.) leaves, stems, and inflorescence and their in vitro callus cultures.

665

J Agric Food Chem 2007, 55, 9109-9117.

666

62. Karam, N.S.; Jawad, F.M.; Arikat, N.A.; Shibl, R.A., Growth and rosmarinic acid accumulation

667

in callus, cell suspension, and root cultures of wild Salvia fruticosa. Plant Cell Tiss Organ

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Cult 2003, 73, 117–121.

669

63. Kintzios, S.; Makri, O.; Panagiotopoulos, E.; Scapeti, M., In vitro rosmarinic acid accumulation

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in sweet basil (Ocimum basilicum L.). Biotechnol Lett 2003, 25, 405-408.

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64. Srivastava, S.; Cahill, D. M.; Conlan, X. A.; Adholeya, A., A novel in vitro whole plant system

672

for analysis of polyphenolics and their antioxidant potential in cultivars of Ocimum basilicum.

673

J Agric Food Chem 2014, 62, 10064-10075.


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65. Petersen, M.; Simmonds, M. S., Rosmarinic acid. Phytochemistry 2003, 62, 121-125.

675

66. Petersen, M.; Abdullah, Y.; Benner, J.; Eberle, D.; Gehlen, K.; Hücherig, S.; Janiak, V.; Kim,

676

K. H.; Sander, M.; Weitzel, C., Evolution of rosmarinic acid biosynthesis. Phytochemistry 2009,70,

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1663-1679.

678

67. Phippen, W. B.; Simon, J. E., Anthocyanins in basil (Ocimum basilicum L.). J Agric Food

679

Chem 1998, 46, 1734-1738.

680

68. Prior, R. L.; Cao, G.; Martin, A.; Sofic, E.; McEwen, J.; Brien, C.; Lischner, N.; Ehlenfeldt,

681

M.; Kalt, W.; Krewer, G., Antioxidant capacity as influenced by total phenolic and anthocyanin

682

content, maturity, and variety of Vaccinium species. J Agric Food Chem 1998, 46, 2686-2693.

683

69. Grillo, M.; Colombatto, S., Advanced glycation end-products (AGEs): involvement in aging

684

and in neurodegenerative diseases. Amino acids 2008, 35, 29-36.

685

70. Bisquert, R.; Muniz-Calvo, S.; Guillamón, J. M., Protective Role of Intracellular Melatonin

686

Against Oxidative Stress and UV Radiation in Saccharomyces cerevisiae.

687

Front Microbiol 2018, 9, 318.

688 689 690 691 692 693 694 695 696 697 698 699



700

Figures Legends

701 702

Figure 1: Effect of PGRs on morphology and Calli biomass from leaf explant after 5 weeks of

703

culture (A) NAA 0.1 mg/l, (B) NAA 2.5mg/l, (C) NAA 5.0 mg/l, (D) TDZ 5.0 mg/l, (E) TDZ 10.0

704

mg/l, (F) BAP 0.1 mg/l, (G) NAA 1+ TDZ 01 mg/l, (H) NAA1+TDZ 2.5mg/l, (I) NAA 1 + BAP

705

5 mg/l.

706

Figure 2: (A) Fresh callus biomass and (B) dry callus biomass from leaf explant callus on MS

707

medium supplemented with TDZ, BAP, NAA (0.1, 1.0, 2.5, 5.0, 10.0 and 20.0 mg/l), NAA 1 +

708

BAP (0.1, 1.0, 2.5, 5.0, 10.0 and 20.0 mg/l) and NAA 1 + TDZ (0.1, 1.0, 2.5, 5.0, 10.0 and 20.0

709

mg/l).

710

(C) Total phenolic content (mg/g DW) from leaf explant callus culture.

711

(D) Total phenolic production (mg /l) from leaf explant callus culture

712

(E) Total flavonoid content (mg/g DW) from leaf explant callus.

713

(F) Total flavonoid production (mg GAE/l) from leaf explant callus culture on MS medium

714

supplemented with NAA, BAP, TDZ (0.1, 1.0, 2.5, 5.0, 10.0 and 20.0 mg/l) and NAA 1 + BAP

715

(0.1, 1.0, 2.5, 5.0, 10.0 and 20.0 mg/l) and NAA 1 + TDZ (0.1, 1.0, 2.5, 5.0, 10.0 and 20.0 mg/l).

716

Values are means of triplicates with the standard deviation.

717

Figure 3: (A). Chemical structure of the main phytochemicals accumulated in leaf-derived O.

718

basilicum callus (1) Caffeic acid, (2) Rosmarinic acid, (3) Chicoric acid, (4) Cyanidin and (5)

719

Peonidin).

720

(B). Typical HPLC chromatograms showing the separation of the main phytochemicals

721

accumulated in leaf-derived O basilicum callus extracts. HPLC chromatograms obtained for

722

“purple” (TDZ 5.0 mg/l) and “green” (NAA 1.0 mg/l) callus sample extracts are presented (here

723

detection was set at 320 nm).


Page 32 of 46

Page 33 of 46


724

Figure 4: (A). Reactive oxygen and nitrogen species (estimated with the DHR-123 fluorescence

725

signal) produced in yeast cells following UV-induced stress.

726

(B). Membrane lipid peroxidation (estimated by the TBARS methods) produced in yeast cells

727

following UV-induced stress.

728

UV (-): unstressed cells; UV (+): UV-induced oxidative stress; PBS (-): unwashed cells before UV

729

treatment; PBS (+): washed cells before UV treatment; values are means ± SD of 3 independent

730

experiments; superscript letters indicate significant differences (p< 0.05).

731 732



733

Supplementary Figures Legends

734 735

Supplementary Figure 1: DPPH radical scavenging activity (%) from leaf explant on MS medium

736

supplemented with NAA, BAP, TDZ (0.1, 1.0, 2.5, 5.0, 10.0 and 20.0 mg/l) and NAA 1 + BAP

737

(0.1, 1.0, 2.5, 5.0, 10.0 and 20.0 mg/l) and NAA 1 + TDZ (0.1, 1.0, 2.5, 5.0, 10.0 and 20.0 mg/l).

738

Values are means of triplicates with the standard deviation.

739 740

Supplementary Figure 2: Growth curves of yeast cells under control conditions and following

741

UV-induced oxidative stress monitored at λ = 630 nm. UV (-): unstressed cells; UV (+): UV-

742

induced oxidative stress; PBS (-): unwashed cells before UV treatment; PBS (+): washed cells

743

before UV treatment; values are means ± SD of 3 independent experiments; superscript letters

744

indicate significant differences (p< 0.05).

745 746 747 748 749 750 751 752 753 754 755 756 757


Page 34 of 46

Page 35 of 46

758 759


Table 1: Diﬀerent concentrations of PGRs, callus induction and callus morphology under coolwhite light (16/8-hrs, control) after 4weeks of inoculation.

761

PGRs Treatments (mg/l)

Callus initiation (day)

Callus color

Callus texture

762

NAA 0.1 NAA 1

7

PG

C

7

LG

C

NAA 2.5 NAA 5

7

PG

C

7

SLG

C

NAA 10

12

PG

C

765

NAA 20

12

FG

C

766

BAP 0.1 BAP 1

28 28

PB D

C C

BAP 2.5

28

D

C

760

763 764

767 768 769

BAP 5

28

D

C

BAP 10

28

D

C

BAP 20

28

D

C

TDZ 0.1

10

DP

C

TDZ 1

10

DP

C

TDZ 2.5

10

DP

C

771

TDZ 10

12

DP

C

772

TDZ 20

12

DP

C

NAA 1+TDZ 0.1

14

DG

C

NAA 1+TDZ 1

14

DG

C

NAA 1+TDZ 2.5

14

PG

C

770

773 774

NAA 1+TDZ 5

14

PG

C

NAA 1+TDZ 10

14

PG

C

NAA 1+TDZ 20

14

PG

C

776

NAA 1+BAP 0.1

21

PB

C

NAA 1+BAP 1

21

PB

C

777

NAA 1+BAP 2.5

21

PB

C

NAA 1+BAP 5

21

PB

C

NAA 1+BAP 10

21

PB

C

NAA 1+BAP 20

21

PB

C

775

778 779 780 781 782 783

C = Compact; F = Friable; D= Dead; DG = Dark green; FG = Fresh green; LG = Light green; SLG = Snowy light green; PG = Purplish green; PB= Purplish Brown; DP = Dark Purple

784 785 786 787 788



789

Page 36 of 46

Table 2: Validation parameters of the HPLC method.

Caffeic acid Rosmarinic acid Chicoric acid Cyanidin Peonidin

Precision (%RSD) Intraday Interday

Equation

R2

LOD (ng)

LOQ (ng)

y = 6.523x + 0.928

0.9991

3.1

10.2

0.63

y = 4.872x 0.123

0.9998

1.8

5.3

0.9997

2.3

0.9990 0.9988

y = 3.253 0.175 y = 5.321x + 1.254 y = 4.978x + 1.132

Repeatability (%RSD)

Recovery (%RSD)

1.25

1.44

102.3

0.21

0.81

0.97

100.4

8.8

0.55

1.12

1.23

99.8

3.0

9.8

0.71

1.35

1.56

96.7

2.9

9.7

0.82

1.54

0.73

95.1

790 791


Page 37 of 46

793 794


Table 3: Effect of different PGRs treatments on production of phenylpropanoid metabolites in leaf derived callus culture under white light (16/8-hrs, control) determined by HPLC. Hydroxycinnamic acid derivatives PGRs Treatments (mg/l) NAA (0.1) NAA (1.0) NAA (2.5) NAA (5.0) NAA (10.0) NAA (20.0) TDZ (0.1) TDZ (1.0) TDZ (2.5) TDZ (5.0) TDZ (10.0) TDZ (20.0) NAA (1.0) + TDZ (0.1) NAA (1.0) + TDZ (1.0) NAA (1.0) + TDZ (2.5) NAA (1.0) + TDZ (5.0) NAA (1.0) + TDZ (10.0) NAA (1.0) + TDZ (20.0)

795

Anthocyanins

Caffeic acid (mg/g DW)

Chicoric acid (mg/g DW)

Rosmarinic acid (mg/g DW)

Cyanidin (mg/g DW)

Peonidin (mg/g DW)

6.43 ± 1.31 9.44 ± 0.84 11.29 ± 1.14 10.48 ± 1.60 10.68 ± 0.71 8.92 ± 0.78 17.23 ± 1.39 29.62 ± 3.57 41.72 ± 3.24 44.67 ± 3.11 40.87 ± 2.91 20.93 ± 5.62 10.35 ± 1.22 14.98 ± 2.67 15.52 ± 3.98 17.68 ± 1.32 22.42 ± 1.14 23.03 ± 1.53

9.17 ± 1.91 11.34 ± 1.15 17.72 ± 0.34 22.77 ± 1.68 23.85 ± 1.13 24.72 ± 2.54 36.32 ± 2.18 40.76 ± 3.13 43.32 ± 0.88 43.89 ± 1.43 37.45 ± 1.05 33.41 ± 3.05 16.48 ± 2.57 17.85 ± 2.28 22.79 ± 1.59 21.83 ± 1.65 25.39 ± 0.77 25.92 ± 1.72

21.47 ± 2.51 23.08 ± 1.33 38.43 ± 1.27 35.46 ± 3.82 33.94 ± 2.61 35.14 ± 0.93 34.54 ± 0.91 44.17 ± 2.66 39.51 ± 0.78 52.22 ± 6.52 51.57 ± 0.90 50.87 ± 1.90 23.42 ± 1.18 25.20 ± 3.41 27.72 ± 1.34 33.18 ± 1.20 40.63 ± 0.73 40.15 ± 1.22

0.14 ± 0.06 0.37 ± 0.23 1.29 ± 0.18 1.48 ± 0.20 1.43 ± 0.17 1.51 ± 0.21 2.38 ± 0.37 6.84 ± 0.71 10.31 ± 1.11 16.39 ± 1.72 9.85 ± 1.89 9.72 ± 1.39 1.64 ± 0.29 0.48 ± 0.23 0.13 ± 0.23 0.13 ± 0.06 0.09 ± 0.02 0.12 ± 0.08

0.09 ± 0.04 2.15 ± 0.15 4.83 ± 0.35 4.31 ± 0.19 4.21 ± 0.10 4.31 ± 0.21 4.07 ± 0.26 5.41 ± 0.24 8.12 ± 0.56 10.77 ± 0.51 10.37 ± 0.15 10.38 ± 1.91 4.03 ± 0.14 4.18 ± 0.18 4.35 ± 0.24 2.15 ± 0.39 2.11 ± 0.47 1.75± 0.22

Values show means ± standard errors from triplicates

796



In vitro antioxidant activities PGRs Treatments (mg/l)

797 798 799

Page 38 of 46

In vitro anti-AGEs formation

ORAC (TEAC)

FRAP (TEAC)

vesperlysine-like AGEs (Inhibition %)

pentosidine-like AGEs (Inhibition %)

NAA (0.1)

418.89 ± 6.59

448.73 ± 31.45

26.87 ± 1.07

19.04 ± 2.32

NAA (1.0)

482.38 ± 8.70

464.66 ± 9.51

27.44 ± 2.48

22.86 ± 2.19

NAA (2.5)

583.54 ± 5.35

481.16 ± 25.58

28.90 ± 0.20

32.95 ± 1.42

NAA (5.0)

585.81 ± 6.38

492.67 ± 26.85

29.98 ± 1.08

34.03 ± 1.66

NAA (10.0)

589.23 ± 3.65

508.95 ± 4.30

29.24 ± 0.92

34.56 ± 2.27

NAA (20.0)

597.02 ± 11.46

502.91 ± 15.35

28.45 ± 1.83

34.74 ± 2.09

TDZ (0.1)

651.03 ± 6.91

526.74 ± 11.20

35.47 ± 1.43

40.38 ± 1.24

TDZ (1.0)

735.37 ± 20.51

569.96 ± 22.37

45.28 ± 3.46

48.89 ± 3.87

TDZ (2.5)

857.64 ± 14.90

615.68 ± 28.02

57.40 ± 3.63

53.27 ± 4.41

TDZ (5.0)

962.54 ± 12.76

656.04 ± 16.95

58.76 ± 4.81

57.99 ± 8.35

TDZ (10.0)

882.80 ± 10.23

644.04 ± 6.35

55.07 ± 3.44

53.97 ± 6.68

TDZ (20.0)

820.97 ± 5.41

623.97 ± 11.95

51.15 ± 3.07

50.04 ± 1.18

NAA (1.0) + TDZ (0.1)

518.44 ± 12.16

417.88 ± 3.22

29.32 ± 1.78

25.40 ± 2.20

NAA (1.0) + TDZ (1.0)

535.37 ± 12.16

450.66 ± 13.19

34.07 ± 2.63

27.94 ± 1.25

NAA (1.0) + TDZ (2.5)

559.03 ± 4.66

451.72 ± 36.05

38.67 ± 1.01

30.27 ± 1.18

NAA (1.0) + TDZ (5.0)

621.00 ± 17.66

450.60 ± 31.29

33.81 ± 2.28

33.03 ± 1.65

NAA (1.0) + TDZ (10.0)

694.62 ± 47.00

489.33 ± 12.63

36.27 ± 0.78

37.56 ± 0.76

NAA (1.0) + TDZ (20.0)

714.50 ± 27.41

479.77 ± 24.27

39.23 ± 2.67

39.15 ± 3.07

Table 4: Effect of different PGRs treatments on the in vitro antioxidant activities and anti-AGEs formation of leaf derived callus culture extracts Values are means of three replicates± SD; TEAC: Trolox equivalent antioxidant activity (in µM)

800 801 802 803 804 805


Page 39 of 46

806 807


Table 5: Correlation (PCCs) analysis linking the phytochemicals accumulated in leaf-derived callus of O. basilicum and the biological activities of the corresponding sample extracts

808

Caffeic acid Rosmarinic acid Chicoric acid Cyanidin Peonidin 809

ORAC

FRAP

Vesperlysine-like AGEs

Pentosidine-like AGEs

0.925*

0.830*

0.948**

0.895*

0.921*

0.868*

0.782*

0.926*

0.908*

0.868*

0.867*

0.958**

0.845* 0.821*

0.899* 0.883*

0.843* 0.833*

0.823* 0.833*

Pearson coefficient correlation; *p< 0.05, **p< 0.01, *** p< 0.001.

810 811 812



813 814

Page 40 of 46

Table 6: Growth index and viability of yeast cells following UV-induced oxidative stress in absence (CTL) or presence of the O. basilicum callus extracts Condition Treatment CTL

Ob_purple

Ob_green

UV (-) UV (+) – PBS (-) UV (+) – PBS (+) UV (-) UV (+) – PBS (-) UV (+) – PBS (+) UV (-) UV (+) – PBS (-) UV (+) – PBS (+)

Growth index

Cell viability

32.3 ± 1.7 11.6 ± 1.9

100 ± 2.1 25.0 ± 2.7

12.5 ± 0.5

24.3 ± 2.2

32.5 ± 1.0 16.1 ± 1.6*

106.0 ± 3.9 45.7 ± 3.9***

14.4 ± 1.1*

32.3 ± 1.8**

35.0 ± 1.8 17.1 ± 1.0*

108.3 ± 6.0 36.4 ± 2.1**

16.6 ± 0.6**

34.0 ± 2.3**

815 816 817 818

UV (-): unstressed cells; UV (+): UV-induced oxidative stress; PBS (-): unwashed cells before UV treatment; PBS (+): washed cells before UV treatment; values are means ± SD of 3 independent experiments; *p< 0.05, **p< 0.01, *** p< 0.001 (comparison with the corresponding control condition).

819 820 821 822 823 824 825 826 827 828 829 830 831 832 833


Page 41 of 46

834


Figure 1

835 836 837 838 839 840 841 842



843

Figure 2

844 845 846 847 848 849 850


Page 42 of 46

Page 43 of 46

851


Figure 3

852 853 854 855 856 857 858 859 860



861 862

Figure 4

863 864 865


Page 44 of 46

Page 45 of 46


866 867

Supplementary Figure 1

868 Supplementary Figure 2

Relative Growth (relative A630nm unit)

869

Time (hours)

870 871



872

Table of Contents

873

874 875 876


Page 46 of 46

Differential Production of Phenylpropanoid Metabolites in Callus

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