J Plant Mol Biol Biotechnol 2011 2: (2) 1-7

Plant Molecular Biology & Biotechnology

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Orjinal Article

Biochemical Role of Ascorbic acid during the Extraction of Nucleic Acids in Polyphenol Rich Medicinal Plant Tissues
Tushar BORSE1,2*, Prashant JOSHI 2 and Sushama CHAPHALKAR1,2
1 Vidya Pratishthans Arts Science & Commerce College 2 Vidya Pratishthans School of Biotechnology, Vidyanagari, Bhigwan Road, Baramati 413133 Received: 24.01.2011 Accepted: 13.02.2011 Published: 08.07.2011

Abstract Nucleic acid isolation from polyphenol rich plants fail to produce good quality DNA or RNA, as polyphenols adhere and interfere with DNA during isolation. To address this problem, we at our Institute are continuously trying to improve the method for genomic DNA isolation from these plants. Use of detergents and reducing agents has resulted in identification of ascorbate as the most potent reducing agent, avoiding oxidation of polyphenols and its interference with the genomic DNA. Ascorbic acid at 40 mM concentration decreased pH of extraction buffer thereby inhibiting activity of polyphenol oxidase which converts colourless polyphenols to coloured black-brown compounds tannins and melanins. Genomic DNA obtained is superior with respect to quality and quantity to be used for analytical applications. Concentration of ascorbate above 40 mM also decreased pH of extraction buffer in the range of 4-5 causing shearing of isolated DNA competing with polyphenols to adhere the DNA.
Key words: gallic acid; genomic DNA; melanin; polyphenoloxidase; tannins Corresponding Author: Tushar Borse, e-mail: thbvsbt@gmail.com, Phone: 91-2112-239388

INTRODUCTION Angiosperms containing pharmacological activities and therapeutic use has recently been overexploited as alternative medicine. This has resulted in domestication, cultivar development and in few cases the plants have become endangered species. Prior to envisaging the use of molecular tools for the proper identification and preservation of these plant species and to elucidate phenomena such as genetic variation, gene flow and hybridization, the establishment of a proper DNA extraction protocol remains the foremost step. These plant tissues contain high levels of polyphenols, polysaccharides and other secondary metabolites, which present a major contamination problem in the purification of plant DNA. The presence of these compounds renders the difficulty due to long and tedious extraction procedures and often does not result in good standards in terms of yield and quality. Also the biochemical composition of plant tissues and species varies considerably; therefore it is virtually impossible to supply a single isolation protocol which is optimally suited for each plant species. Thus different plant taxa often may not permit optimal DNA yields from one isolation protocol. Several protocols have been described for plants containing high amounts of polyphenols and polysaccharides including extraction of DNA from isolated nuclei (Hamilton et al. 1972; Katterman and Shattuck 1983; Pirttila et al. 2001) and purification by cesium chloride following the classic plant DNA protocol (Murray and Thompson 1980) using liquid nitrogen. Although these methods yield DNA of high purity, they are tedious and require expensive reagents and equipment such as high-speed or ultra centrifuges. Also, liquid nitrogen may be difficult to obtain in certain regions of the world (Honeycutt et al. 1972; Porebski et al. 1972).

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J Plant Mol Biol Biotechnol 2011 2: (2) 1-7 DNA isolation protocols generally use CTAB to avoid co-purification of polyphenols and polysaccharides from medicinally important plant tissues. Keeping in mind this fact of medicinal plants the modified method of Kulkarni et al. (2007) was tried in the experiments. Use of detergents like CTAB, SDS, SLS and antioxidants like - mercaptoethanol, ascorbic acid, bovine serum albumin (BSA), sodium azide, and polyvinylpyrrolidone-40 (PVPP-40) are commonly used to address problems related to phenolics (Clark 1997; Dawson and Magee 1995). CTAB, SDS/SLS are used to solubilize the membrane; break up the lipids associated with it and form complex with the DNA. Alterations in the concentration of these detergents are used to increase the efficiency of removing proteins from the extracted DNA (Sghaier and Mohammed 2005). Strong reducing agents like mercaptoethanol and DTT (Clelands Reagent) are introduced in the extraction buffer to denature proteins and the extraction of thiolated DNA by forming dimmers in the solution. PVPP40 has also been successfully used to remove polyphenols along with high molar concentration of NaCl to inhibit coprecipitation of polyphenols, polysaccharides and DNA. Ascorbic acid is a powerful antioxidant because it can donate a hydrogen atom to form a relatively stable ascorbyl free radical is also being used for isolation of nucleic acids in polyphenol rich plants (Seiob and Tolbert 1982). Also the use of BSA as a polyphenol absorbent at a concentration of 1-4 % found to be successful for these plants. Secondary metabolites synthesized in these plants are stored in the vacuoles of the leaf (Kulkarni et al. 2007), gets released along with genetic material by crushing the leaf sample using detergents like SDS or CTAB for the extraction procedure (Loomis 1974). Once released these polyphenols get oxidized with the atmospheric oxygen to tannins and melanins which have a high affinity for the nucleic acid. These oxidized polyphenols covalently bind to DNA and coprecipitate with it after alcohol addition giving it a brown color to form highly viscous solution (Guillemaut 1992). In the present study the modified CTAB method of Doyle and Doyle (1987, 1990) was used with an addition of ascorbic acid as an antioxidant which prevented the oxidation of polyphenols and change the pH of the extraction buffer to prevent the action of polyphenol oxidase, further helping in improving the quality and quantity of DNA obtained to be used for restriction digestion and molecular fingerprinting. Though the quantity of extracted DNA varied with respect to the type and amount of polyphenols present in different plant species. MATERIAL AND METHODS

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Collection of plant material: Fresh leaf samples were collected from 12 (angiosperms) - medicinally important) rich in polyphenols were obtained from the Nakshatra garden of the Institute. Estimation of Total Phenolic Content (TPC): Total phenolic content of the extracts was assessed by using Folin-Ciocalteau Phenol reagent according to the method previously reported by Folin and Denis (1912) and Mole and Waterman (1912) using standard curve generated with gallic acid and catechol. Isolation of genomic DNA: Protocol - The modified CTAB method (Kulkarni et al. 2007) was followed: 1. 2. Place the extraction buffer (10 ml; 2% CTAB; 100mM Tris; 20mM EDTA; 1.4M NaCl) in a water bath at 65C. Grind the fresh plant tissue (1g) into a fine powder in liquid nitrogen using a mortar and pestle. Without thawing immediately transfer the powder to the prewarmed isolation buffer. Vortex thoroughly and place the tube at 65C for 20 min. Add 20l of 2-mercaptoethanol and further incubate at 65C for 10mins. Cool the tubes to RT and centrifuge at 11,000 rpm for 10 min at room temperature. Transfer the supernatant into a new tube (discard the pellet) and extract with equal volume of chloroform-IAA (24:1). Centrifuge the tubes at 11,000 for 10 mins at 4C. Pipette the aqueous phase into a new tube and extract again with equal volume of chloroform-IAA (24:1). Centrifuge the tubes at 11,000 for 10 mins at 4C. Transfer the upper aqueous phase in fresh tube and add twice the volume of absolute alcohol (distilled) to precipitate the DNA. Incubate at -20oC, overnight (or at -80oC for 4 hours). Centrifuge the tubes at 11,000 for 10 mins at 4C. Discard the supernatant and dry the pellet either at 60C on a dry bath or air dry. Dissolve the pellet in 60-100 L of T10E1. Store at -20oC till further use.

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Analysis of the genomic DNA: The resultant genomic DNA was further electrophoresed for qualitative analysis on a 1% agarose gel containing 5l ethidium bromide at a concentration of 0.05 g/ml. The DNA yield per gram of leaf tissue was measured by using a UV-

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J Plant Mol Biol Biotechnol 2011 2: (2) 1-7 Visible spectrophotometer at 260nm. DNA purity was determined by calculating the absorbance A260/280 ratio. Pure DNA has a ratio of 1.8. Polyphenol contamination was assessed by calculating the absorbance ratio A260/230. A ratio >2.0 indicates no contamination in the isolated DNA sample. Effect of antioxidant on DNA quality: The genomic DNA isolation method was modified by addition of antioxidants such as ascorbic acid, PVP, sodium azide, -mercaptoethanol etc. Varying concentrations of these antioxidants was introduced in the extraction buffer to observe the effect on polyphenols and DNA quality obtained. Spectrophotometric experiments were also performed with pure phage DNA, gallic acid as polyphenol and antioxidants to verify the exact effect and mode of action. Applications of isolated genomic DNA: The isolated genomic DNA was further analysed for its analytical applications like restriction digestion by EcoRI according to Sambrook et al. (2001). Polymerase chain reaction amplification for RAPD analysis was performed with an initial denaturation at 950C for 5 min. followed by 35 cycles of 1 min denaturation at 950C, 1 min annealing at 450C and 1 min extension at 720C, with a final extension of 10 min at 720C in programmable thermal cycler (ABI). RESULTS AND DISCUSSION The need for a rapid and efficient procedure for obtaining high quality DNA is necessary for the advance studies like genome mapping and marker assisted selection (MAS) programs. We at our institute are continuously trying to improve upon the protocol for genomic DNA isolation from the polyphenol rich important plants. In our previous

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literature on genomic DNA isolation from Syzygium cumini (Kulkarni et al. 2007), we suggested the treatment of alcohol to the leaf sample to dehydrate it before the liquid nitrogen crushing and an ion exchange chromatography to purify the isolated DNA. But earlier protocol is not useful for plant species employed in this study; they require different treatments as discussed elsewhere. In the present study we have focused upon the introduction of ascorbic acid in the extraction buffer to improve the quality of DNA isolated by preventing the adherence of polyphenols to the DNA and avoiding the browning of sample. The extracted DNA was analyzed by electrophoresis in 1% agarose gels and quantified spectrophotometrically. The isolated DNA was further tested for molecular use by performing restriction digestion and molecular marker amplification (RAPDs). Figure 1 show the gel pattern of nucleic acid obtained by using different antioxidants in the genomic DNA isolation protocol. The figure clearly indicates that the DNA could be isolated with good quality and quantity in case of combination of ascorbic acid and -mercaptoethanol. While in case of only the extraction buffer with CTAB, the DNA cannot be isolated. Addition of PVP and -mercaptoethanol separately in the extraction buffer showed a very faint band with no RNA and protein contamination. But the DNA obtained was slightly brown in colour. The combination pair of PVP and -mercaptoethanol resulted in good quantity of DNA but showed shearing in repeated experiments along with RNA and protein impurities. Combination of mercaptoethanol and ascorbate gave the good results with respect to both quality and quantity of DNA with no protein, RNA and polyphenol contamination. The combination of PVP and ascorbate was also checked but resulted in smear of DNA.

Figure 1 Electropherogram showing the effect of different antioxidants on the on the quality and quantity of genomic DNA isolated (A - only CTAB; B - only PVPP; C- only -mercaptoethanol; D PVPP and -mercaptoethanol; E - -mercaptoethanol and ascorbate; F- PVPP and ascorbate)

The gel pattern of genomic DNA obtained by varying concentration of ascorbic acid in the extraction buffer keeping the concentration of -mercaptoethanol constant is depicted in Figure 2. Increasing the concentration of ascorbic acid resulted in high quality and quantity DNA, but after 60 mM concentration the quality remained same

and quantity decreased. Effective concentration of 40 mM resulted in good quality and quantity of the genomic DNA. Comparing the results with graph 1, depicts that the purity of DNA (A260/280 ratio) is near to the purity index of 1.8 and the concentration is also 1800 g/g approximately equal to the other known methods of DNA isolation. In case of 60-100mM

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Figure 2 Electropherogram showing the effect of varying concentration (mM) of ascorbic acid on the quality and quantity of genomic DNA isolated.

J Plant Mol Biol Biotechnol 2011 2: (2) 1-7 concentration the purity was maintained but the concentration dropped to 80% less as compared to 40 mM concentration The reason could be that the higher concentration of antioxidant may directly or indirectly causing damage to the DNA (Inoue et al. 2006). At high concentrations ascorbic acid may compete with the polyphenols to adhere and interact with the DNA. Accumulation of polyphenols in the leaf tissues is well known and we have also proved its presence by anatomical studies in Syzygium cumini (Kulkarni et al. 2007). The amount of total phenolic content of the leaf tissues was performed by the FC reagent and found to be 8001000g/g. The addition of an antioxidant like ascorbate in the extraction buffer is must, as the polyphenols also get released from the cells after crushing along with the nucleic acids and proteins, making the solution alkaline and activating the polyphenol oxidase to act upon the colourless

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polyphenols. To understand the biochemistry of these results obtained with ascorbic acid; we measured the change in pH during the DNA isolation protocol. The pH of extraction buffer after the addition of the ingredients was alkaline in the range of 7.5-8.5, addition of ascorbic acid (40mM) resulted in decrease in the pH of buffer towards acidic conditions in the range of 66.5. During the further procedure after the chloroform: IAA extraction step, the aqueous phase containing the DNA showed a pH range of 5.5-6.0. Comparing the results in graph 2, we can conclude that the pattern of pH change is same for all the concentrations of ascorbic acid used in the experiments. But above the 40 mM concentration the pH is too acidic and may be harmful to the pure DNA obtained as it is well known that ascorbic acid is beneficial and harmful depending on the sensitive balance of its concentration.

2.5 2.3
A (260/280) ratio

2.5 2.3 2.1 1.9 1.7 1.5 10mM 20mM 40mM 60mM 80mM 100mM
A (260/230) ratio

2.1 1.9 1.7 1.5

ASCO RBATE CO NCENTRATIO N (mM)

Graph 1 Effect of ascorbic acid concentration on A260/280 ratio and A260/230 ratio as an index of . DNA purity.

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J Plant Mol Biol Biotechnol 2011 2: (2) 1-7

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pH (units)

3 EB EB + AA CIA ext I CIA ext II

STEPS OF DNA ISOALTION

. Graph 2 pH profiles of solutions during steps of DNA isolation at different concentrations of ascorbic acid. (EB
Extraction buffer; AA Ascorbic acid; CIA chloroform: iso-amyl alcohol; ext extraction step I and II)

To confirm this fact we have performed experiments on pure phage DNA which showed a peak at 263 nm in a UVVIS spectrophotometer, which shifted to 400nm with the addition of gallic acid (a polyphenol with a peak at 258nm). This result indicated that the gallic acid binds to the phage DNA causing the shift. But the addition of ascorbate to the mixture showed a reversible nature of peak shift to around 268, whereas at higher concentration (80mM) of ascorbic acid the resultant was similar to that of mixture. This result is in accordance with the chemiluminescence studies of polyphenols by Inoue et al. (2006), describing the reversal of gallic acid oxidation by addition of ascorbate. It states that oxidation of gallic acid to quinone is the first step in a series of chemiluminescence reactions of gallic acid in alkaline conditions. But the addition of ascorbate quickly reduced the quinone produced from gallic acid to stop the chemiluminescence by a synergetic effect. These results clearly explain that ascorbic acid play an important role in protecting and reducing the oxidation of polyphenols to brown colour compounds which has high affinity for the nucleic acids. Browning of the cut surface of leaves is due the presence of a group of enzymes called polyphenoloxidases. These enzymes are released by the broken cells and they catalyze the reaction between colourless molecules called polyphenols and molecular oxygen to their quinone derivatives, which then spontaneously polymerize (Chaudhary 2008; Chunhua 2001; Mohammadi et al. 2009). This reaction creates colored compounds and these new compounds can spontaneously cross react with one another to form black-brown complexes called melanin and tannin.

The parameters standerdised for addition of ascorbate in the nucleic acid isolation protocol was extended to isolate genomic DNA from 12 different polyphenol rich plants (Table 1). The data in Table 1, shows the purity index and concentration of the DNA isolated from 12 different plant tissues. From Figure 3 it is clear that the quality and quantity of the DNA isolated was good, except for lane 1-3 where the concentration was low. Restriction endonuclease digestion requires fairly clean and large quantity of DNA. RAPD and related techniques require less DNA but purity is necessary to ensure repeatability and confidence. We successfully digested 2g of isolated DNA with 8U of EcoRI and 8U of Hind III (Figure 4). RAPD amplification of genomic DNA was performed for all the 12 polyphenol rich plants with a random primer 42 (5TTAACCCGGC-3). There was no interference with the PCR amplification reactions indicating the purity of DNA isolated.

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J Plant Mol Biol Biotechnol 2011 2: (2) 1-7 A B C D E F G H I J K L

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Figure 3 Electropherogram showing the isolated genomic DNA from 12 different medicinal plants using 40 mM ascorbic acid.

Figure 4 Electropherogram showing the restriction digestion by EcoRI of the genomic DNA isolated from the 12 polyphenol rich plants.

Table 1 Purity index of DNA in 12 polyphenol rich plant species and the concentration obtained by using 40 mM ascorbic acid in the extraction protocol. No. Botanical name A(260/A280) A(260/A230) DNA Conc. ratio ratio (g/g) A Strychnos nuxvomica 1.30 1.95 163 C Ficus racemosa 1.20 2.12 177 E Acacia catechu 1.71 2.01 1873 F Santalum album 1.76 2.10 1500 G Bambusa erandinasia 2.30 2.46 926 H Ficus religiosa 1.14 1.99 1072 I Messua ferrea 1.76 2.11 618 J Ficus benghalensis 1.33 1.88 1110 K Butea frondosa 2.40 1.20 537 The ratio is calculated by taking ratio of 260 and 280 absorbance for the isolations and amount of DNA calculated by considering 50 g concentration for dsDNA when A260 =1. The results are mean of three independent isolations. ACKNOWLEDGEMENT The authors wish to thank VSBT for granting the work to be done and the colleagues for their constant support. Abbreviations: DNA: Deoxy Nucleic Acid; RNA: Ribo Nucleic Acid; CTAB: Cetyl Trimethyl Ammonium Bromide; SDS: Sodium Dodecyl Sulphate; SLS: Sodium Lauryl Sulphate; BSA: Bovine Serum Albumin; NaCl: Sodium chloride; PVPP-40: Polyvinylpyrrolidone; DTT: Dithiothreitol; TPC: Total Phenolic Content; Tris: 2, amino,

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J Plant Mol Biol Biotechnol 2011 2: (2) 1-7 2, hydroxymethyl 1, 3 propanediol; EDTA: Ethylene Diamine Tetraacetic Acid; NaCl: Sodium Chloride; IAA: Isoamyl Alcohol; RAPD: Random Amplified Polymorphic DNA; PPO: Poly Phenol Oxidase

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Chunhua Y, Dai X, Xiaolong X, Xie Y, Liu Q. 2001. The Purification and Spectral Properties of Polyphenol Oxidase I from Nicotiana tabacum. Plant Molecular Biology Reporter 19: 381a381 Mohammadi A, Abdolmajid M, Dinarvand Z, 2009. Direct Electron Transfer of Polyphenol Oxidase on Carbon Nanotube Surfaces: Application in Biosensing, International Journal Electrochemical Science 495 905

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