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In vivo and in vitro Variation in Protein Profiling in Withania somnifera (L.) Dunal



Pallavi Sharma, Richa Bhardwaj, Ankita Yadav and R.A. Sharma
 
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ABSTRACT

Proteomics is a leading new technology for the high-throughput analysis of proteins on a genome-wide scale in living system. This technique was applied to investigate the protein changes under in vitro and in vivo conditions, since in vitro cultures is considered to be an alternative approach to traditional agriculture in the industrial production of the biomolecules. The overall goal of this project was to investigate the changes in protein expression under in vitro and in vivo leaves tissues of Withania somnifera. Of ~28 protein bands resolved in the two-dimensional gels, 21 protein bands were similarly expressed in both in vitro and in vivo root tissues 1 protein in in vivo condition and 2 protein bands were differentially expressed only in in vitro tissue. This is the first report on the comparison of in vitro and in vivo samples by establishment of a 2-D reference proteome map of Withania somnifera.

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Pallavi Sharma, Richa Bhardwaj, Ankita Yadav and R.A. Sharma, 2014. In vivo and in vitro Variation in Protein Profiling in Withania somnifera (L.) Dunal. Research Journal of Phytochemistry, 8: 25-34.

URL: https://scialert.net/abstract/?doi=rjphyto.2014.25.34
 
Received: December 18, 2013; Accepted: February 14, 2014; Published: March 29, 2014



INTRODUCTION

Withania somnifera Dunal (Solanaceae), known in India as ashwagandha or winter cherry, is one of the most valuable plants of the traditional Indian systems of medicines, is used in more than 1 00 formulations of Ayurveda, Unani and Sidha and is therapeutically equivalent to ginseng (Sangwan et al., 2004). Its ginseng like health-promoting effects has earned it the popular name of Indian ginseng. The biologically active chemical constituents are steroidal compounds, including ergostane type steroidal lactones, withaferine A, withanolides A-Y, withasomniferin-A, withanone, etc. (Ganzera et al., 2003) alkaloids (ashwagandhine, cuswhygrine, anahygrine, tropine, etc.). Many pharmacological studies have been carried out to describe multiple biological properties of W. somnifera (Mishra et al., 2000).

Different plant parts of various species of the genus W. somnifera have exhibited varied pharmacological activities, such as anti-inflammatory hypocholesterolemic (Asthana and Raina, 1989), cardiac and cerebral ischemia (Keller et al., 1998), immunomodulator and antitumor effect (Agarwal et al., 1999), neurodegenerative disorders (Perry et al., 2000), carcinogenesis (Kamat and Devasagayam, 2000), rheumatic disorders (Hanninen et al., 2000), anticancer and arthritis (Prakash et al., 2001), contributes a major role in the ageing process (Khodr and Khalil, 2001), anxiety and anti-depression (Al-Hindawi et al., 1989; Archana and Namasivayam, 1999; Bhattacharya et al., 2001; Singh et al., 2001), antioxidants (Ferguson, 2001; Tang et al., 2001), diabetes (Gorogawa et al., 2002), chronic stress (Archana and Namasivayam, 1999; Dhuley, 2000; Kaur et al., 2001; Bhattacharya and Muruganandam, 2003), anti-cancer chemotherapy and radio-sensitization (Devi et al., 1995; Devi, 1996; Chang et al., 2007; Ojha and Arya, 2009). As leaves contain a number of therapeutically applicable withanolides, mass cultivation of leaves in vitro will be an effective technique for the large scale production of these secondary metabolites.

Protein Profiling, an independent, emerging sub-specialty of proteomics, is poised to provide unprecedented insight into biological events. Protein profiling is defined here as the quantitative assessment of protein expression levels. As profiling evolves, the term will increasingly refer to the study of multiple proteins, protein forms or protein families, almost always comparing two different states (De Palma, 2006). Protein profiling in the high throughout mode is a most useful technique that allows formation of reference databases for cells and tissues and performance of comparative proteomics. The analysis of all proteins (proteome) and all metabolites (metabolome), however, continued to pose significant challenges. Proteins and metabolites are more divert and biochemically heterogenous, which precludes the application of a single standardized procedure for their analysis (Bino et al., 2004). In recent years SDS-proteins has been found wide application in resolving genetic diversity and for intra and interspecific studies.

These technologies will provide novel methods for early detection and diagnosis of cancer as well as classification and prognostic prediction. Proteomic profiling will also lead to better targeted therapies to enable the delivery of personalized medicine. Proteomic methods thus have the advantage of identifying the dynamic and transient interactions that are the sum of all molecular interactions impinging on a particular cellular pathway at the moment of analysis. Proteomics offers great potential for studying mechanisms of post translation regulation as well as biosynthetic pathways. However it has not been widely applied in plant biology. Currently, the complete genome of a number of plant species has been sequenced (Tabata, 2002; Frazier et al., 2003; Kav et al., 2007). However the functions of many of the identified genes remain unclear. Thus it is important to shift the focus towards the functional characterization of proteins that are encoded by the cellular genetic machinery (Kav et al., 2007; Senthil et al., 2011).

Global analysis of the system components (DNA, RNA, proteins and metabolites) is now possible, although, at different analytical depth at present. Protein profile system can be used as biochemical marker for selection superior genotype of the medicinally important plant. The protein banding profile system revealed the biochemical variation and evolutionary relationship. Protein profiling in high throughout mode is relatively simple and provides a snapshot of the major protein constituents of the cell (Yates, 2004). Each allele codes for the production of amino acids that string together to form protein. Thus differences in the nucleotide sequence of allele result in the production of slightly different strings of amino acids or variant forms of the proteins. These protein codes for the development of the anatomical and physiological characteristic of the organism, which are responsible for determining aspects of the behaviour of the organism (Shibata, 2005).With the advent of proteomics and mass spectrometry, systematic identification of proteins has become possible, as demonstrated in several studies in different organisms (Tyers and Mann, 2003). Proteins are major actors involved in many physiological processes, so profiling is used to predict those processes (Voelckel et al., 2010). Protein profiling of many species has been reviewed by few workers (Kumar and Kumari, 2009; Johnson, 2010; Shim et al., 2010; Hew and Gam, 2010). However the work on the protein profiling of Withania Species has been done by (Senthil et al., 2011). But no systematic efforts have been made to study protein expression of W. somnifera.

The present study was designed to investigate the protein changes under in vitro and in vivo conditions. Separation of proteins in in vitro and in vivo tissues of W. somnifera was done using 2-D gels.

MATERIALS AND METHODS

Proteome analysis
Sample preparation: The 21 to 28 days old calli was harvested for analysis kept at above 90°C for 3 to 5 min in a hot air oven to inactivate enzyme activity followed by continuous drying at 50°C to 60°C for 6 to 72 h (Jain et al., 2004). Dried callus was homogenized to fine powder and further exploited for protein extraction. Dried whole plant and powdered it with an electric homogenizer.

Protein extraction: The dried calli and dried plant were submitted to extraction with protein extraction buffer (Table 1).

The samples were sonicated and clarified by centrifugation at 1500 rpm for 10 min. The clear supernatants were collected and kept in boiling water for denaturation. These protein samples were stored at -20°C for long time. Supernatants were used as a sample for polyacrylamide gel electrophoresis.

Protein quantification: The protein content in the different fractions was performed according to Bradford (1976). A stock solution of Bovine Serum Albumin (BSA; Sigma Chemical Co., St. Louis, USA) was prepared (1 mg mL-1), out of which 0.2-1.0 mL of the standard was taken in separate test tube and volume in each case was raised to 2 mL by adding double distilled water. To each, 3 mL of Bradford reagent was added, mixed and kept at 37°C for 10 min and absorbance was measured at 595 nm. Similarly, 20 μL of extracted protein was diluted to 25 times in TBE buffer and in 0.5 mL of sample 1.5 mL of double distilled H2O and 3 mL of Bradford reagent was added to and the mixture was allowed to cool and absorbance was measured at 595 nm. Graph was drawn between concentration of standard protein and absorbance. The quantities of unknown samples were measured on the basis of standard curve and dilution factor.

Sample preparation for SDS-PAGE: Electrophoresis of equal volume of protein extracted from equal quantity (0.5 g) of sample was carried out on 12% polyacryamide gel. Since amount of sample and buffer were same in all samples, it was assumed that quantity of protein in mg mL-1 is as per expression level of proteins. In order to load protein samples properly for SDS-PAGE the extracted protein samples were mixed with 10 X bromophenol blue dye. The sample so prepared contained, 34 μL of extracted protein as per procedure described earlier and 2 μL 10X Bromophenol blue dye.

Preparation of polyacrylamide gel (Sambrook et al., 1989): In order to resolve various polypeptides SDS-PAGE was conducted using 12% resolving gels. A 12% separating gel (25 mL) was used for resolving the polypeptides whereas 5% stacking gel (5 mL) was used to stack the polypeptides. Glass plates and spacers were properly cleaned with double distilled water followed by spirit. Plates are set in the gel caster and sealed with the help of sealing agar. Whole cassette of glass plate and spacers were assembled properly. Vaseline or tape was used at bottom of cassette to prevent leakage of gel solution. Separating gel solution for 12% gel (Table 2) was prepared in conical flask and degassed using vacuum pump for 4-5 min until bubbles stopped forming at the surface.

Separating gel solution was then poured in the chamber between the glass plates leaving the space for stacking gel. Distilled water was overlaid to form about a 2-5 mm layer and left to set for 30 min. On polymerization of the separating gel, water overlay was carefully removed by inverting the whole cassette.

Table 1: Composition of protein extraction buffer
Image for - In vivo and  in vitro Variation in Protein Profiling in Withania somnifera (L.) Dunal

Table 2: Solution components for resolving and stacking gel
Image for - In vivo and  in vitro Variation in Protein Profiling in Withania somnifera (L.) Dunal

Immediately after overlaying water from cassette, APS and TEMED was in stacking gel and poured on the surface of the separating gel. The teflon comb was then placed in stacking gel. The comb was removed carefully after 10 min and washed the wells with dd H2O to remove air bubbles. The gel was allowed to polymerize for at least 2 h at cool temperature. The gel assembly was removed from the casting stand and snapped it into the cooling core. Cooling core with clamped gel plates and one dummy plate was placed in vertical electrophoresis tank. Prepared samples were then loaded in the wells with protein molecular weight marker at one end. Upper and lower tanks were then filled with Tris-glycine buffer (1X) to complete the circuit. The power supply was turned to 100 V till the samples were in stacking gel then turned to 200 V until the bromophenol blue reached the bottom of the resolving gel. After completion the power supply was turned off and the cooling core unit was pulled out from electrophoresis tank, the unit was disassembled and gel was detached from glass plates. The gel was placed in coomassie brilliant blue stain for overnight. After complete staining, gel was destained in destaining solution on a slowly rocking platform and changed the destaining solution 3-4 times, till the background became quite clear. After destaining the gel was analysed on densitometer (Bio RAD Multi-Analyst). The multi-analyst software of densitometer gave quantity and intensity of each band of every lane. The gel was scanned and photographed.

The identification/comparison of the components of the storage proteins in the extract was made using a protein marker (Banglore Genei). The molecular weights of the marker corresponded to 20.1, 29, 43, 66 and 97.4 kDa.

RESULTS AND DISCUSSION

Two-dimensional polyacrylamide gel electrophoresis (2-D) is an established and powerful technique for analyzing the complex mixtures of protein. Proteomic analysis offers a new approach to identify a broad spectrum of genes that are expressed in living system. We applied this technique to investigate the protein changes under in vivo and in vitro conditions, since in vitro cultures is considered to be an alternative approach to traditional agriculture in the industrial production of the biomolecules. To better understand the proteins and enzymes involved in biosynthetic pathway, detailed two-dimensional gel electrophoresis (2-D) of in vivo leaves samples and in vitro grown callus of W. somnifera were conducted. Total soluble proteins were extracted and then total amount of proteins were estimated using Bradford method. The concentration of proteins in estimated sample was found to be from 3.8 to 7.9 mg 120 mg-1). Finally the equal amount of extracted and estimated proteins subjected to SDS-PAGE to generate banding pattern with a protein molecular weight marker (PMW-M from G Brand). After electrophoretic separation of proteins, 28 band positions were observed and finally scored for analysis. 23 protein spots were similarly expressed in both in vivo and in vitro tissues 1 protein in in vivo condition and 2 protein spots were differentially expressed only in in vitro tissue. Total proteins were analyzed by Bradford assay method. Unlike Lowry’s method, metal ions such as NH4+, Na+, K+ phenols and carbohydrates such as sucrose do not interfere in this assay(Kumar et al.,2010). The standard curve was prepared on the basis of absorbance at 595nm from the standard curve, the concentration of protein was found to be 6.8 and 4.3 mg g-1 in in vivo plant and in vitro (callus) (Table 3, 4). The report of Cormack et al. (2001) suggested that leaves record high amount of proteins compared to roots and seeds. Generally, 18% proteins were profiled in leaves whereas roots have less than 0.25% protein in Arabidopsis thaliana root. The quantity of the protein content in the leaves of W. somnifera was analyzed and it was observed that leaves contain high amount of protein compared to roots and seeds (Khanna et al., 2006).

Comparative analysis of 2-D gels revealed high level of similarity in the protein pattern of both in vivo and in vitro leaves sample. The 23 spots were commonly present in the in vivo and in vitro leaves samples. One for presence and zero for absence was considered and tabulated in electrophoretic banding pattern. Twenty three bands with molecular weights 99.2, 96.4, 94.1, 92.6, 88.2, 64.2, 60.2, 55.2, 52.3, 50.2, 48.8, 40.2, 38.2, 37.1, 36.9, 36.2, 34.2, 29.5, 28.1, 27.2, 26.2, 22.3 and 19.2 kDa were expressed in both conditions (Fig. 1; Table 5). The intensity of bands was high in in vivo sample as compared to in vitro sample (Table 6).

Interestingly, two (in vitro specific protein) differential spots were present in the in vitro (66.2 and 39.6 kDa) that were not found in the in vivo leaves. These proteins accumulated in immature cells grown in vitro conditions that permits callus formations. These may be callus associated protein for proliferative growth and cellular differentiation (Table 5).

The protein profiles indicate that most of the proteins present in leaves and callus was of a same molecular weight. But in callus protein bands of (39.6 kDa) molecular weight region were not detected.

Table 3: Observation table for known protein solution
Image for - In vivo and  in vitro Variation in Protein Profiling in Withania somnifera (L.) Dunal

Table 4: Observation table for unknown leaf and callus protein solution
Image for - In vivo and  in vitro Variation in Protein Profiling in Withania somnifera (L.) Dunal

Image for - In vivo and  in vitro Variation in Protein Profiling in Withania somnifera (L.) Dunal
Fig. 1: In vivo and in vitro protein profiling of isolated protein from Withania somnifera L.

Table 5: Electrophoretic banding pattern of in vitro and in vivo samples derived from SDS-PAGE of leaf and callus proteins
Image for - In vivo and  in vitro Variation in Protein Profiling in Withania somnifera (L.) Dunal
1: Presence, 0: Absence

Table 6: Electrophoretic densitogram of in vitro and in vivo samples of leaf and callus proteins
Image for - In vivo and  in vitro Variation in Protein Profiling in Withania somnifera (L.) Dunal

The protein bands of molecular weight indicate that the number of protein identified in in vitro condition was more compared to in vivo condition and for development of callus it required the same protein compared to in vivo condition.

CONCLUSION

Until now no reports are available on the comparison of in vivo and in vitro leaves in plants of commercial importance. Hence pioneering attempt has been made to study the profile of proteins expressed in in vivo and in vitro samples of W. somnifera.

REFERENCES

  1. Sangwan, R.S., N.D. Chaurasiya, L.N. Misra, P. Lal and G.C. Uniyal et al., 2004. Phytochemical variability in commercial herbal products and preparations of Withania somnifera (Ashwagandha). Curr. Sci., 86: 461-465.
    Direct Link  |  


  2. Agarwal, R., S. Diwanay, P. Patki and B. Patwardhan, 1999. Studies on immunomodulatory activity of Withania somnifera (ashwagandha) extracts in experimental immune inflammation. J. Ethnopharmacol., 67: 27-35.
    CrossRef  |  PubMed  |  Direct Link  |  


  3. Archana, R. and A. Namasivayam, 1999. Antistressor effect of Withania somnifera. J. Ethnopharmacol., 64: 91-93.
    PubMed  |  Direct Link  |  


  4. Asthana, R. and M.K. Raina, 1989. Pharmacology of Withania somnifera (L.) dunal: A review. Indian Drugs, 26: 199-205.


  5. Bhattacharya, A., S. Ghosal and S.K. Bhattacharya, 2001. Anti-oxidant effect of Withania somnifera glycowithanolides in chronic footshock stress-induced perturbations of oxidative free radical scavenging enzymes and lipid peroxidation in rat frontal cortex and striatum. J. Ethnopharmacol., 74: 1-6.
    PubMed  |  Direct Link  |  


  6. Chang, H.C., F.R. Chang, Y.C. Wu, M.R. Pan, W.C. Hung and Y.C. Wu, 2007. A bioactive withanolide tubocapsanolide A inhibits proliferation of human lung cancer cells via repressing Skp2 expression. Mol. Cancer Ther., 6: 1572-1578.
    CrossRef  |  PubMed  |  


  7. Devi, P.U., A.C. Sharada and F.E. Solomon, 1995. In vivo growth inhibitory and radiosensitizing effects of withaferin a on mouse Ehrlich ascites carcinoma. Cancer Lett., 16: 189-193.
    PubMed  |  Direct Link  |  


  8. Devi, P.U., 1996. Withania somnifera Dunal (Ashwagandha): potential plant source of a promising drug for cancer chemotherapy and radiosensitization. Indian J. Exp. Biol., 34: 927-932.
    PubMed  |  


  9. Ferguson, L.R., 2001. Role of plant polyphenols in genomic stability. Mutat. Res./Fundam. Mol. Mech. Mutagen., 475: 89-111.
    CrossRef  |  PubMed  |  Direct Link  |  


  10. Ganzera, M., M.I. Choudhary and I.A. Khan, 2003. Quantitative HPLC analysis of withanolides in Withania somnifera. Fitoterapia, 74: 68-76.
    CrossRef  |  PubMed  |  Direct Link  |  


  11. Gorogawa, S.I., Y. Kajimoto, Y. Umayahara, H. Kaneto and H. Watada et al., 2002. Probucol preserves pancreatic β-cell function through reduction of oxidative stress in type 2 diabetes. Diabetes Res. Clin. Pract., 57: 1-10.
    PubMed  |  Direct Link  |  


  12. Hanninen, O., K. Kaartinen, A.L. Rauma, M. Nenonen and R. Torronen et al., 2000. Antioxidants in vegan diet and rheumatic disorders. Toxicology, 155: 45-53.
    PubMed  |  


  13. Al-Hindawi, M.K., I.H. Al-Deen, M.H. Nabi and M.H. Ismail, 1989. Anti-inflammatory activity of some Iraqi plants using intact rats. J. Ethnopharmacol., 26: 163-168.
    PubMed  |  Direct Link  |  


  14. Keller, J.N., M.S. Kindy, F.W. Holtsberg, D.K.S. Clair and H.C. Yen et al., 1998. Mitochondrial manganese superoxide dismutase prevents neural apoptosis and reduces ischemic brain injury: Suppression of peroxynitrite production, lipid peroxidation and mitochondrial dysfunction. J. Neurosci., 18: 687-697.
    Direct Link  |  


  15. Kamat, J.P. and T.P.A. Devasagayam, 2000. Oxidative damage to mitochondria in normal and cancer tissues and its modulation. Toxicology, 155: 73-82.
    PubMed  |  Direct Link  |  


  16. Khodr, B. and Z. Khalil, 2001. Modulation of inflammation by reactive oxygen species: Implications for aging and tissue repair. Free Rad. Biol. Med., 30: 1-8.
    PubMed  |  Direct Link  |  


  17. Perry, G., A.K. Raina, A. Nunomura, T. Wataya, L.M. Sayre and M.A. Smith, 2000. How important is oxidative damage? Lessons from Alzheimer's disease. Free Radic. Biol. Med., 28: 831-834.
    PubMed  |  Direct Link  |  


  18. Prakash, J., S.K. Gupta, V. Kochupillai, N. Singh, Y.K. Gupta and S. Joshi, 2001. Chemopreventive activity of Withania somnifera in experimentally induced fibrosarcoma tumours in Swiss albino mice. Phytother. Res., 15: 240-244.
    CrossRef  |  PubMed  |  Direct Link  |  


  19. Mishra, L.C., B.B. Singh and S. Dagenais, 2000. Scientific basis for the therapeutic use of Withania somnifera (ashwagandha): A review. Altern. Med. Rev., 5: 334-346.
    PubMed  |  Direct Link  |  


  20. Singh, B., A.K. Saxena, B.K. Chandan, D.K. Gupta, K.K. Bhutani and K.K. Anand, 2001. Adaptogenic activity of a novel, withanolide-free aqueous fraction from the roots of Withania somnifera Dun. Phytother. Res., 15: 311-318.
    CrossRef  |  


  21. Tang, S., J.P. Kerry, D. Sheehan, D.J. Buckley and P.A. Morrissey, 2001. Antioxidative effect of added tea catechins on susceptibility of cooked red meat, poultry and fish patties to lipid oxidation. Food Res. Int., 34: 651-657.
    CrossRef  |  Direct Link  |  


  22. Ojha, S.K. and D.S. Arya, 2009. Withania somnifera Dunal (Ashwagandha): A promising remedy for cardiovascular diseases. World J. Med. Sci., 4: 156-158.
    Direct Link  |  


  23. De Palma, A., 2006. Protein profiling poised to make its mark. Feature Articles, Vol. 26.
    Direct Link  |  


  24. Bino, R.J., R.D. Hall, O. Fiehn, J. Kopka and K. Saito et al., 2004. Potential of metabolomics as a functional genomics tool. Trends Plant Sci., 9: 418-425.
    CrossRef  |  PubMed  |  


  25. Yates, J.R., 2004. Mass spectral analysis in proteomics. Annu. Rev. Biophys. Biomol. Struct., 33: 297-316.
    CrossRef  |  PubMed  |  


  26. Tabata, S., 2002. Impact of genomics approaches on plant genetics and physiology. J. Plant Res., 115: 271--275.
    CrossRef  |  


  27. Senthil, K., N. Karunanithi, G.S. Kim, A. Nagappan, S. Sundareswaran, S. Natesan and R. Muthurajan, 2011. Proteome analysis of in vitro and in vivo root tissue of Withania somnifera. Afr. J. Biotechnol., 10: 16875-16883.
    Direct Link  |  


  28. Frazier, M.E., G.M. Johnson, D.G. Thomassen, C.E. Oliver and A. Patrino, 2003. Realizing the potential of the genome revolution: The genomes to life program. Science, 300: 290-293.
    CrossRef  |  


  29. Kav, N.N., S. Srivastava, W. Yajima and N. Sharma, 2007. Application of proteomics to investigate plant-microbe interactions. Curr. Proteomics, 4: 28-43.
    CrossRef  |  


  30. Shibata, D., 2005. Genome sequencing and functional genomics approaches in tomato. J. Gen. Plant Pathol., 71: 1-7.
    CrossRef  |  


  31. Tyers, M. and M. Mann, 2003. From genomics to proteomics. Nature, 422: 193-197.
    PubMed  |  


  32. Voelckel, C., M. Mirzaei, M. Reichelt, Z. Luo and D. Pascovici et al., 2010. Transcript and protein profiling identify candidate gene sets of potential adaptive significance in New Zealand pachycladon. BMC Evolutionary Biol., Vol. 10.
    CrossRef  |  


  33. Shim, S.Y., R.M. Ali and L.H. Gam, 2010. Protein profiling of Piper sarmentosum. Asia-Pacific J. Mol. Biol. Biotechol., 18: 303-313.
    Direct Link  |  


  34. Khanna, P.K., A. Kumar, A. Ahuja and M.K. Kaul, 2006. Biochemical composition of roots of Withania somnifera (L.) dunal. Asian J. Plant Sci., 5: 1061-1063.
    CrossRef  |  Direct Link  |  


  35. Kumar, S.P. and B.D.R. Kumari, 2009. In vitro and in vitro identification of variation of protein expression in Artemisia vulgaris L. Adv. Biol. Res., 3: 237-241.
    Direct Link  |  


  36. Kaur, P., S. Mathur, M. Sharma, M. Tiwari, K.K. Srivastava and R. Chandra, 2001. A biologically active constituent of Withania somnifera (ashwagandha) with antistress activity. Ind. J. Clin. Biochem., 16: 195-198.
    CrossRef  |  


  37. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72: 248-254.
    CrossRef  |  PubMed  |  Direct Link  |  


  38. Hew, C.S. and L.H. Gam, 2010. The identification of high abundant proteins in the leaves of Gynura procumbens. Biotechnol. Biotechnol. Equip., 24: 2132-2136.
    CrossRef  |  Direct Link  |  


  39. Johnson, M., 2010. Biochemical variation studies in Aegle marmelos (L) Corr-A medicinally important plant. J. Chem. Res., 2: 454-462.
    Direct Link  |  


  40. Jain, S.C., R. Jain and A.J. Vlietinck, 2004. In vivo and in vitro antimicrobial efficacy of Mimosa hamata. Indian J. Biotechnol., 3: 271-273.
    Direct Link  |  


  41. Kumar, A., B.R. Garg, G. Rajput, D. Chandel, A. Muwalia, I. Bala and S. Singh, 2010. Antibacterial activity and quantitative determination of protein from leaf of Datura stramonium and Piper betle plants. Pharmacophore, 1: 184-195.
    Direct Link  |  


  42. Dhuley, J.N., 2000. RETRACTED: Adaptogenic and cardioprotective action of ashwagandha in rats and frogs. J. Ethnopharmacol., 70: 57-63.
    CrossRef  |  Direct Link  |  


  43. Bhattacharya, S.K. and A.V. Muruganandam, 2003. Adaptogenic activity of Withania somnifera: An experimental study using a rat model of chronic stress. Pharmacol. Biochem. Behav., 75: 547-555.
    CrossRef  |  Direct Link  |  


  44. Sambrook, J., E.F. Fritsch and T. Maniatis, 1989. Gel Electrophoresis of DNA. In: Molecular Cloning a Laboratory Manual, Sambrook, J., E.F. Fritsch and T. Maniatis (Eds.). 2nd Edn., Cold Spring Harbor Laboratory Press, New York, pp: 6.3-6.17


  45. Cormack, Y.W., S.M. Basin, Y. Sahir and J.Y. Liu, 2001. Comparative proteomic analysis provides new insights into the fiber elongating process in leaves of Arabidopsis thaliana. J. Proteome Res., 7: 4623-4637.


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