Protection of Beta Boswellic Acid against Streptozotocin-induced Alzheimerʼs Model by Reduction of Tau Phosphorylation Level and Enhancement of Reelin Expression

 

 Buy Article Permissions and Reprints

Abstract

Alzheimerʼs disease is a growing general health concern with huge implications for individuals and society. Beta boswellic acid, a major compound of the Boswellia serrata plant, has long been used for the treatment of various inflammatory diseases. The exact mechanism of beta boswellic acid action in Alzheimerʼs disease pathogenesis remains unclear. In the current study, the protective effect of beta boswellic acid on streptozotocin-induced sporadic Alzheimerʼs disease was surveyed. Alzheimerʼs disease model was induced using streptozotocin followed by an assessment of the treatment effects of beta boswellic acid in the presence of streptozotocin. The prevention effect of beta boswellic acid on Alzheimerʼs disease induction by streptozotocin was evaluated. Behavioral activities in the treated rats were evaluated. Histological analysis was performed. Phosphorylation of tau protein at residues Ser396 and Ser404 and the expression of reelin protein were determined. Glial fibrillary acidic protein immunofluorescence staining was applied in the hippocampus regions. Our findings indicated that beta boswellic acid decreased traveled distance and escape latency in the prevention (beta boswellic acid + streptozotocin) and treatment (streptozotocin + beta boswellic acid) groups compared to control during the acquisition test. It increased “time spent” (%) in the target quadrant. Reelin level was enhanced in rats treated with beta boswellic acid. Tau hyperphosphorylation (p-tau404) and glial fibrillary acidic protein were decreased in the prevention group while the expression of reelin protein in both groups was increased. We could suggest that the anti-inflammatory property of beta boswellic acid is one of the main factors involving in the improvement of learning and memory in rats. Therefore the antineurodegenerative effect of beta boswellic acid may be due to its ability to reactivate reelin protein.

Publication History

Received: 07 February 2020

Accepted after revision: 04 May 2021

Article published online:
11 June 2021

© 2021. Thieme. All rights reserved.

Georg Thieme Verlag KG
Rüdigerstraße 14, 70469 Stuttgart, Germany

• References

• 1 Hooper C, Killick R, Lovestone S. The GSK3 hypothesis of Alzheimerʼs disease. J Neurochem 2008; 104: 1433-1439
  CrossrefPubMedGoogle Scholar
• 2 Jaul E, Barron J. Age-related diseases and clinical and public health implications for the 85 years old and over population. Front Public Health 2017; 5: 335
  CrossrefPubMedGoogle Scholar
• 3 Mayeux R, Stern Y. Epidemiology of Alzheimer disease. Cold Spring Harb Perspect Med 2012; 2: a006239
  CrossrefPubMedGoogle Scholar
• 4 Rizzi L, Rosset I, Roriz-Cruz M. Global epidemiology of dementia: Alzheimerʼs and vascular types. BioMed Res Int 2014; 2014: 908915-908923
  CrossrefPubMedGoogle Scholar
• 5 Fang F, Lue LF, Yan S, Xu H, Luddy JS, Chen D, Walker DG, Stern DM, Yan S, Schmidt AM. RAGE-dependent signaling in microglia contributes to neuroinflammation, Aβ accumulation, and impaired learning/memory in a mouse model of Alzheimerʼs disease. FASEB J 2010; 24: 1043-1055
  CrossrefPubMedGoogle Scholar
• 6 Kumar A, Singh A. A review on Alzheimerʼs disease pathophysiology and its management: an update. Pharmacol Rep 2015; 67: 195-203
  CrossrefPubMedGoogle Scholar
• 7 Brunden KR, Trojanowski JQ, Lee VMY. Advances in tau-focused drug discovery for Alzheimerʼs disease and related tauopathies. Nat Rev Drug Discov 2009; 8: 783-793
  CrossrefPubMedGoogle Scholar
• 8 Iqbal K, Liu F, Gong CX, Grundke-Iqbal I. Tau in Alzheimer disease and related tauopathies. Curr Alzheimer Res 2010; 7: 656-664
  CrossrefPubMedGoogle Scholar
• 9 Liu F, Iqbal K, Grundke-Iqbal I, Gong CX. Involvement of aberrant glycosylation in phosphorylation of tau by cdk5 and GSK-3β . FEBS Lett 2002; 530: 209-214
  CrossrefPubMedGoogle Scholar
• 10 Baumann K, Mandelkow EM, Biernat J, Piwnica-Worms H, Mandelkow E. Abnormal Alzheimer-like phosphorylation of tau-protein by cyclin-dependent kinases cdk2 and cdk5. FEBS Lett 1993; 336: 417-424
  CrossrefPubMedGoogle Scholar
• 11 Cai Z, Li B, Li K, Zhao B. Down-regulation of amyloid-β through AMPK activation by inhibitors of GSK-3β in SH-SY5Y and SH-SY5Y-AβPP695 cells. J Alzheimerʼs Dis 2012; 29: 89-98
  CrossrefPubMedGoogle Scholar
• 12 Lindwall G, Cole RD. Phosphorylation affects the ability of tau protein to promote microtubule assembly. J Biol Chem 1984; 259: 5301-5305
  CrossrefPubMedGoogle Scholar
• 13 Moreira-Silva D, Carrettiero DC, Oliveira ASA, Rodrigues S, Canas P, Cunha RA, Köfalvi A, Almeida MC, Ferreira TL. Anandamide effects in a streptozotocin-induced alzheimerʼs disease-like sporadic dementia in rats. Front Neurosci 2018; 12: 653-667
  CrossrefPubMedGoogle Scholar
• 14 El Halawany AM, Sayed NSE, Abdallah HM, El Dine RS. Protective effects of gingerol on streptozotocin-induced sporadic Alzheimerʼs disease: emphasis on inhibition of β-amyloid, COX-2, alpha-, beta-secretases and APH1a. Sci Rep 2017; 7: 2902-2913
  CrossrefPubMedGoogle Scholar
• 15 Xu ZP, Li L, Bao J, Wang ZH, Zeng J, Liu EJ, Li XG, Huang RX, Gao D, Li MZ. Magnesium protects cognitive functions and synaptic plasticity in streptozotocin-induced sporadic Alzheimerʼs model. PLoS One 2014; 9: 1-11
  CrossrefPubMedGoogle Scholar
• 16 Su F, Bai F, Zhang Z. Inflammatory cytokines and Alzheimerʼs disease: a review from the perspective of genetic polymorphisms. Neurosci Bull 2016; 32: 469-480
  CrossrefPubMedGoogle Scholar
• 17 González-Reyes RE, Nava-Mesa MO, Vargas-Sánchez K, Ariza-Salamanca D, Mora-Muñoz L. Involvement of astrocytes in Alzheimerʼs disease from a neuroinflammatory and oxidative stress perspective. Front Mol Neurosci 2017; 10: 427-447
  CrossrefPubMedGoogle Scholar
• 18 Yang Y, Ge W, Chen Y, Zhang Z, Shen W, Wu C, Poo M, Duan S. Contribution of astrocytes to hippocampal long-term potentiation through release of D-serine. Proc Natl Acad Sci 2003; 100: 15194-15199
  CrossrefPubMedGoogle Scholar
• 19 Pascual O, Casper KB, Kubera C, Zhang J, Revilla-Sanchez R, Sul JY, Takano H, Moss SJ, McCarthy K, Haydon PG. Astrocytic purinergic signaling coordinates synaptic networks. Science 2005; 310: 113-116
  CrossrefPubMedGoogle Scholar
• 20 Ding S, Fellin T, Zhu Y, Lee SY, Auberson YP, Meaney DF, Coulter DA, Carmignoto G, Haydon PG. Enhanced astrocytic Ca2+ signals contribute to neuronal excitotoxicity after status epilepticus. J Neurosci 2007; 27: 10674-10684
  CrossrefPubMedGoogle Scholar
• 21 Jourdain P, Bergersen LH, Bhaukaurally K, Bezzi P, Santello M, Domercq M, Matute C, Tonello F, Gundersen V, Volterra A. Glutamate exocytosis from astrocytes controls synaptic strength. Nat Neurosci 2007; 10: 331-339
  CrossrefPubMedGoogle Scholar
• 22 Rajkowska G, Miguel-Hidalgo JJ, Wei J, Dilley G, Pittman SD, Meltzer HY, Overholser JC, Roth BL, Stockmeier CA. Morphometric evidence for neuronal and glial prefrontal cell pathology in major depression. Biol Psychiatry 1999; 45: 1085-1098
  CrossrefPubMedGoogle Scholar
• 23 Webster M, OʼGrady J, Kleinman J, Weickert C. Glial fibrillary acidic protein mRNA levels in the cingulate cortex of individuals with depression, bipolar disorder and schizophrenia. Neuroscience 2005; 133: 453-461
  CrossrefPubMedGoogle Scholar
• 24 Forman MS, Lal D, Zhang B, Dabir DV, Swanson E, Lee VMY, Trojanowski JQ. Transgenic mouse model of tau pathology in astrocytes leading to nervous system degeneration. J Neurosci 2005; 25: 3539-3550
  CrossrefPubMedGoogle Scholar
• 25 Halassa MM, Fellin T, Haydon PG. The tripartite synapse: roles for gliotransmission in health and disease. Trends Mol Med 2007; 13: 54-63
  CrossrefPubMedGoogle Scholar
• 26 Kamphuis W, Middeldorp J, Kooijman L, Sluijs JA, Kooi EJ, Moeton M, Freriks M, Mizee MR, Hol EM. Glial fibrillary acidic protein isoform expression in plaque related astrogliosis in Alzheimerʼs disease. Neurobiol Aging 2014; 35: 492-510
  CrossrefPubMedGoogle Scholar
• 27 Yiannopoulou KG, Papageorgiou SG. Current and future treatments for Alzheimerʼs disease. Ther Adv Neurol Disord 2013; 6: 19-33
  CrossrefPubMedGoogle Scholar
• 28 Roy NK, Parama D, Banik K, Bordoloi D, Devi AK, Thakur KK, Padmavathi G, Shakibaei M, Fan L, Sethi G. An update on pharmacological potential of boswellic acids against chronic diseases. Int J Mol Sci 2019; 20: 4101-4128
  CrossrefPubMedGoogle Scholar
• 29 Ebrahimpour S, Fazeli M, Mehri S, Taherianfard M, Hosseinzadeh H. Boswellic acid improves cognitive function in a rat model through its antioxidant activity:-neuroprotective effect of boswellic acid. J Pharmacopuncture 2017; 20: 10-17
  CrossrefPubMedGoogle Scholar
• 30 Khalaj-Kondori M, Sadeghi F, Hosseinpourfeizi MA, Shaikhzadeh-Hesari F, Nakhlband A, Rahmati-Yamchi M. Boswellia serrata gum resin aqueous extract upregulatesBDNF but not CREB expression in adult male rat hippocampus. Turk J Med Sci 2016; 46: 1573-1578
  CrossrefPubMedGoogle Scholar
• 31 Arora RB, Kapoor V, Basu N, Jain AP, Jain AP. Anti-inflammatory studies on Curcuma longa (turmeric). Indian J Med Res 1971; 59: 1289-1295
  PubMedGoogle Scholar
• 32 Ammon HP. Boswellic acids in chronic inflammatory diseases. Planta Med 2006; 72: 1100-1116
  Article in Thieme ConnectPubMedGoogle Scholar
• 33 Karima O, Riazi G, Khodadadi S, Yousefi R, Mahnam K, Mokhtari F, Cheraghi T, Hoveizi E, Moosavi-Movahedi AA. An in vitro study of the role of β-boswellic acid in the microtubule assembly dynamics. FEBS Lett 2012; 586: 4132-4138
  CrossrefPubMedGoogle Scholar
• 34 Jalili C, Salahshoor M, Pourmotabbed A, Moradi S, Roshankhah S, Darehdori AS, Motaghi M. The effects of aqueous extract of Boswellia serrata on hippocampal region CA1 and learning deficit in kindled rats. Res Pharmaceutical Sci 2014; 9: 351-358
  PubMedGoogle Scholar
• 35 Kamat PK, Kalani A, Rai S, Swarnkar S, Tota S, Nath C, Tyagi N. Mechanism of oxidative stress and synapse dysfunction in the pathogenesis of Alzheimerʼs disease: understanding the therapeutics strategies. Mol Neurobiol 2016; 53: 648-661
  CrossrefPubMedGoogle Scholar
• 36 Ammon H, Safayhi H, Mack T, Sabieraj J. Mechanism of antiinflammatory actions of curcumine and boswellic acids. J Ethnopharmacol 1993; 38: 105-112
  CrossrefPubMedGoogle Scholar
• 37 Banno N, Akihisa T, Yasukawa K, Tokuda H, Tabata K, Nakamura Y, Nishimura R, Kimura Y, Suzuki T. Anti-inflammatory activities of the triterpene acids from the resin of Boswellia carteri. J Ethnopharmacol 2006; 107: 249-253
  CrossrefPubMedGoogle Scholar
• 38 Alkon DL, Sun MK, Nelson TJ. PKC signaling deficits: a mechanistic hypothesis for the origins of Alzheimerʼs disease. Trends Pharmacol Sci 2007; 28: 51-60
  CrossrefPubMedGoogle Scholar
• 39 Khan TK, Alkon DL. An internally controlled peripheral biomarker for Alzheimerʼs disease: Erk1 and Erk2 responses to the inflammatory signal bradykinin. Proc Natl Acad Sci 2006; 103: 13203-13207
  CrossrefPubMedGoogle Scholar
• 40 Sailer ER, Subramanian LR, Rall B, Hoernlein RF, Ammon HP, Safayhi H. Acetyl-11-keto-beta-boswellic acid (AKBA): structure requirements for binding and 5-lipoxygenase inhibitory activity. Br J Pharmacol 1996; 117: 615-618
  CrossrefPubMedGoogle Scholar
• 41 Rankin CA, Sun Q, Gamblin TC. Tau phosphorylation by GSK-3β promotes tangle-like filament morphology. Mol Neurodegener 2007; 2: 12
  CrossrefPubMedGoogle Scholar
• 42 Altmann A, Fischer L, Schubert-Zsilavecz M, Steinhilber D, Werz O. Boswellic acids activate p42(MAPK) and p38 MAPK and stimulate Ca(2+) mobilization. Biochem Biophys Res Commun 2002; 290: 185-190
  CrossrefPubMedGoogle Scholar
• 43 Altmann A, Poeckel D, Fischer L, Schubert-Zsilavecz M, Steinhilber D, Werz O. Coupling of boswellic acid-induced Ca2+ mobilisation and MAPK activation to lipid metabolism and peroxide formation in human leucocytes. Br J Pharmacol 2004; 141: 223-232
  CrossrefPubMedGoogle Scholar
• 44 Taghizadeh M, Maghaminejad F, Aghajani M, Rahmani M. The effect of tablet containing Boswellia serrata and Melisa officinalis extract on older adultsʼ memory: a randomized controlled trial. Arch Gerontol Geriatr 2018; 75: 146-150
  CrossrefPubMedGoogle Scholar
• 45 Mahboubi M, Taghizadeh M, Talaei SA, Firozeh SMT, Rashidi AA, Tamtaji OR. Combined administration of Melissa officinalis and Boswellia serrata extracts in an animal model of memory. Iranian J Psychiatry Behav Sci 2016; 10: e681-e687
  PubMedGoogle Scholar
• 46 Siddiqui M. Boswellia serrata, a potential antiinflammatory agent: an overview. Ind J Pharm Sci 2011; 73: 255
  PubMedGoogle Scholar
• 47 Grünblatt E, Salkovic-Petrisic M, Osmanovic J, Riederer P, Hoyer S. Brain insulin system dysfunction in streptozotocin intracerebroventricularly treated rats generates hyperphosphorylated tau protein. J Neurochem 2007; 101: 757-770
  CrossrefPubMedGoogle Scholar
• 48 Neddens J, Temmel M, Flunkert S, Kerschbaumer B, Hoeller C, Loeffler T, Niederkofler V, Daum G, Attems J, Hutter-Paier B. Phosphorylation of different tau sites during progression of Alzheimerʼs disease. Acta Neuropathol Commun 2018; 6: 52-67
  CrossrefPubMedGoogle Scholar
• 49 Regan P, Piers T, Yi JH, Kim DH, Huh S, Park SJ, Ryu JH, Whitcomb DJ, Cho K. Tau phosphorylation at serine 396 residue is required for hippocampal LTD. J Neurosci 2015; 35: 4804-4812
  CrossrefPubMedGoogle Scholar
• 50 Peineau S, Bradley C, Taghibiglou C, Doherty A, Bortolotto ZA, Wang YT, Collingridge GL. The role of GSK-3 in synaptic plasticity. Br J Pharmacol 2008; 153: S428-S437
  CrossrefPubMedGoogle Scholar
• 51 Mondragón-Rodríguez S, Trillaud-Doppia E, Dudilot A, Bourgeois C, Lauzon M, Leclerc N, Boehm J. Interaction of endogenous tau protein with synaptic proteins is regulated by N-methyl-D-aspartate receptor-dependent tau phosphorylation. J Biol Chem 2012; 287: 32040-32053
  CrossrefPubMedGoogle Scholar
• 52 Hanger DP, Hughes K, Woodgett JR, Brion JP, Anderton BH. Glycogen synthase kinase-3 induces Alzheimerʼs disease-like phosphorylation of tau: generation of paired helical filament epitopes and neuronal localisation of the kinase. Neurosci Lett 1992; 147: 58-62
  CrossrefPubMedGoogle Scholar
• 53 Liu SJ, Zhang JY, Li HL, Fang ZY, Wang Q, Deng HM, Gong CX, Grundke-Iqbal I, Iqbal K, Wang JZ. Tau becomes a more favorable substrate for GSK-3 when it is prephosphorylated by PKA in rat brain. J Biol Chem 2004; 279: 50078-50088
  CrossrefPubMedGoogle Scholar
• 54 Peineau S, Taghibiglou C, Bradley C, Wong TP, Liu L, Lu J, Lo E, Wu D, Saule E, Bouschet T. LTP inhibits LTD in the hippocampus via regulation of GSK3β . Neuron 2007; 53: 703-717
  CrossrefPubMedGoogle Scholar
• 55 Kimura T, Whitcomb DJ, Jo J, Regan P, Piers T, Heo S, Brown C, Hashikawa T, Murayama M, Seok H. Microtubule-associated protein tau is essential for long-term depression in the hippocampus. Philos Trans R Soc Lond B Biol Sci 2014; 369: 20130144-20130152
  CrossrefPubMedGoogle Scholar
• 56 Yassin N, El-Shenawy S, Mahdy KA, Gouda N, Marrie A, Farrag A, Ibrahim BM. Effect of Boswellia serrata on Alzheimerʼs disease induced in rats. J Arab Soc Med Res 2013; 8: 1-11
  PubMedGoogle Scholar
• 57 Heneka MT, Carson MJ, El Khoury J, Landreth GE, Brosseron F, Feinstein DL, Jacobs AH, Wyss-Coray T, Vitorica J, Ransohoff RM. Neuroinflammation in Alzheimerʼs disease. Lancet Neurol 2015; 14: 388-405
  CrossrefPubMedGoogle Scholar
• 58 Osborn LM, Kamphuis W, Wadman WJ, Hol EM. Astrogliosis: an integral player in the pathogenesis of Alzheimerʼs disease. Prog Neurobiol 2016; 144: 121-141
  CrossrefPubMedGoogle Scholar
• 59 Verkhratsky A, Parpura V, Pekna M, Pekny M, Sofroniew M. Glia in the pathogenesis of neurodegenerative diseases. Biochem Soc Trans 2014; 42: 1291-1301
  CrossrefPubMedGoogle Scholar
• 60 Masgrau R, Guaza C, Ransohoff RM, Galea E. Should we stop saying ‘glia’ and ‘neuroinflammation’?. Trends Mol Med 2017; 23: 486-500
  CrossrefPubMedGoogle Scholar
• 61 Cheon SY, Cho KJ, Song J, Kim GW. Knockdown of apoptosis signal-regulating kinase 1 affects ischaemia-induced astrocyte activation and glial scar formation. Eur J Neurosci 2016; 43: 912-922
  CrossrefPubMedGoogle Scholar
• 62 Yan L, Gu H, Li J, Xu M, Liu T, Shen Y, Chen B, Zhang G. RKIP and 14-3-3ε exert an opposite effect on human gastric cancer cells SGC7901 by regulating the ERK/MAPK pathway differently. Dig Dis Sci 2013; 58: 389-396
  CrossrefPubMedGoogle Scholar
• 63 Nishino T, Matsunaga R, Konishi H. Functional relationship between CABIT, SAM and 14-3-3 binding domains of GAREM1 that play a role in its subcellular localization. Biochem Biophys Res Communications 2015; 464: 616-621
  CrossrefPubMedGoogle Scholar
• 64 Xiao Q, Yan P, Ma X, Liu H, Perez R, Zhu A, Gonzales E, Burchett JM, Schuler DR, Cirrito JR. Enhancing astrocytic lysosome biogenesis facilitates Aβ clearance and attenuates amyloid plaque pathogenesis. J Neurosci 2014; 34: 9607-9620
  CrossrefPubMedGoogle Scholar
• 65 Förster E, Bock HH, Herz J, Chai X, Frotscher M, Zhao S. Emerging topics in Reelin function. Eur J Neurosci 2010; 31: 1511-1518
  CrossrefPubMedGoogle Scholar
• 66 Beffert U, Morfini G, Bock HH, Reyna H, Brady ST, Herz J. Reelin-mediated signaling locally regulates protein kinase B/Akt and glycogen synthase kinase 3β . J Biol Chem 2002; 277: 49958-49964
  CrossrefPubMedGoogle Scholar
• 67 Pujadas L, Rossi D, Andrés R, Teixeira CM, Serra-Vidal B, Parcerisas A, Maldonado R, Giralt E, Carulla N, Soriano E. Reelin delays amyloid-beta fibril formation and rescues cognitive deficits in a model of Alzheimerʼs disease. Nature Commun 2014; 5: 34433454
  CrossrefPubMedGoogle Scholar
• 68 Kocherhans S, Madhusudan A, Doehner J, Breu KS, Nitsch RM, Fritschy JM, Knuesel I. Reduced Reelin expression accelerates amyloid-β plaque formation and tau pathology in transgenic Alzheimerʼs disease mice. J Neurosci 2010; 30: 9228-9240
  CrossrefPubMedGoogle Scholar
• 69 Lopez LP. Brain maps: structure of the rat brain (2nd edn) by LW Swanson. Trends Neurosci 2000; 23: 88-89
  CrossrefPubMedGoogle Scholar
• 70 Swanson L. Brain Maps: Structure of the Rat Brain. London: Gulf Professional Publishing; 2004
  Google Scholar
• 71 Dehghan-Shasaltaneh M, Naghdi N, Choopani S, Alizadeh L, Bolouri B, Masoudi-Nejad A, Riazi GH. Determination of the best concentration of streptozotocin to create a diabetic brain using histological techniques. J Mol Neurosci 2016; 59: 24-35
  CrossrefPubMedGoogle Scholar
• 72 Brandeis R, Brandys Y, Yehuda S. The use of the Morris water maze in the study of memory and learning. Int J Neurosci 1989; 48: 29-69
  CrossrefPubMedGoogle Scholar
• 73 Zhou S, Yu G, Chi L, Zhu J, Zhang W, Zhang Y, Zhang L. Neuroprotective effects of edaravone on cognitive deficit, oxidative stress and tau hyperphosphorylation induced by intracerebroventricular streptozotocin in rats. Neurotoxicology 2013; 38: 136-145
  CrossrefPubMedGoogle Scholar
• 74 Gould TD, Dao DT, Kovacsics CE. The open Field Test. In: Gould TD. ed. Mood and Anxiety related Phenotypes in Mice. Saskatoon: Springer; 2009: 1-20
  Google Scholar
• 75 Gittins R, Harrison PJ. Neuronal density, size and shape in the human anterior cingulate cortex: a comparison of Nissl and NeuN staining. Brain Res Bull 2004; 63: 155-160
  CrossrefPubMedGoogle Scholar
• 76 Alvarez-Buylla A, Ling CY, Kirn JR. Cresyl violet: a red fluorescent Nissl stain. J Neurosci Methods 1990; 33: 129-133
  CrossrefPubMedGoogle Scholar
• 77 Ooigawa H, Nawashiro H, Fukui S, Otani N, Osumi A, Toyooka T, Shima K. The fate of Nissl-stained dark neurons following traumatic brain injury in rats: difference between neocortex and hippocampus regarding survival rate. Acta Neuropathol 2006; 112: 471-481
  CrossrefPubMedGoogle Scholar
• 78 Clodfelder-Miller BJ, Zmijewska AA, Johnson GV, Jope RS. Tau is hyperphosphorylated at multiple sites in mouse brain in vivo after streptozotocin-induced insulin deficiency. Diabetes 2006; 55: 3320-3325
  CrossrefPubMedGoogle Scholar
• 79 Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976; 72: 248-254
  CrossrefPubMedGoogle Scholar