Pharmacological perspectives and phytochemical reservoir of Withania somnifera: A review

[1] Woods, D., Epistemic Things in Charles and Ray Eames’s Powers of Ten. In Scale in Literature and Culture, Springer: 2017; pp 61-92.

[2] Hayat, S.; Ali, B.; Ahmad, A., Salicylic acid: biosynthesis, metabolism and physiological role in plants. In Salicylic acid: A plant hormone, Springer: 2007; pp 1-14.

[3] Koutsospyros, A. D.; Koutsospyros, D. A.; Strigul, N.; Braida, W.; Christodoulatos, C., Tungsten: Environmental pollution and health effects. In Encyclopedia of environmental health, Elsevier: 2019; pp 161-169.

[4] Kong, J.-M.; Goh, N.-K.; Chia, L.-S.; Chia, T.-F., Recent advances in traditional plant drugs and orchids. Acta Pharmacologica Sinica 2003, 24 (1), 7-21.

[5] Petrovska, B. B., Historical review of medicinal plants’ usage. Pharmacognosy reviews 2012, 6 (11), 1.

[6] Sangwan, N. S.; Tripathi, S.; Srivastava, Y.; Mishra, B.; Pandey, N., Phytochemical genomics of Ashwagandha. In Science of Ashwagandha: Preventive and Therapeutic Potentials, Springer: 2017; pp 3-36

[7] Rates, S. M. K., Plants as source of drugs. Toxicon 2001, 39 (5), 603-613.

[8] Mukherjee, P. K.; Harwansh, R. K.; Bahadur, S.; Banerjee, S.; Kar, A.; Chanda, J.; Biswas, S.; Ahmmed, S. M.; Katiyar, C., Development of Ayurveda–tradition to trend. Journal of ethnopharmacology 2017, 197, 10-24.

[9] Patwardhan, B.; Warude, D.; Pushpangadan, P.; Bhatt, N., Ayurveda and traditional Chinese medicine: a comparative overview. Evidence-Based Complementary and Alternative Medicine 2005, 2.

[10] Tiwari, S., Plants: A rich source of herbal medicine. Journal of natural products 2008, 1 (0), 27-35.

[11] Yesilada, E., Past and future contributions to traditional medicine in the health care system of the Middle-East. Journal of Ethnopharmacology 2005, 100 (1-2), 135-137.

[12] Ackerknecht, E. H.; Haushofer, L., A short history of medicine. JHU Press: 2016.

[13] Rai, A.; Rai, A. C., Microbial endophytes and their intellectual property rights. In Microbial Endophytes, Elsevier: 2020; pp 349-387.

[14] Dutfield, G.; Suthersanen, U., Traditional Knowledge and Genetic Resources: Observing Legal Protection through the Lens of Historical Geography and Human Rights. Washburn LJ 2019, 58, 399.

[15] Alamgir, A., Secondary Metabolites: Secondary Metabolic Products Consisting of C and H; C, H, and O; N, S, and P Elements; and O/N Heterocycles. In Therapeutic Use of Medicinal Plants and their Extracts: Volume 2, Springer: 2018; pp 165-309

[16] Fischer, R.; Stoger, E.; Schillberg, S.; Christou, P.; Twyman, R. M., Plant-based production of biopharmaceuticals. Current opinion in plant biology 2004, 7 (2), 152-158.

[17] Twyman, R. M.; Stoger, E.; Schillberg, S.; Christou, P.; Fischer, R., Molecular farming in plants: host systems and expression technology. TRENDS in Biotechnology 2003, 21 (12), 570-578.

[18] Verpoorte, R., Exploration of nature's chemodiversity: the role of secondary metabolites as leads in drug development. Drug Discovery Today 1998, 3 (5), 232-238.

[19] Tripathi, S.; Srivastava, Y.; Sangwan, R. S.; Sangwan, N. S., In silico mining and functional analysis of AP2/ERF gene in Withania somnifera. Scientific reports 2020, 10 (1), 1-1

[20] Tripathi, S.; Sangwan, R. S.; Narnoliya, L. K.; Srivastava, Y.; Mishra, B.; Sangwan, N. S., Transcription factor repertoire in Ashwagandha (Withania somnifera) through analytics of transcriptomic resources: Insights into regulation of development and withanolide metabolism. Scientific reports 2017, 7 (1), 1-17.

[21] Edreva, A.; Velikova, V.; Tsonev, T.; Dagnon, S.; Gürel, A.; Aktaş, L.; Gesheva, E., Stress-protective role of secondary metabolites: diversity of functions and mechanisms. Gen Appl Plant Physiol 2008, 34 (1-2), 67-78.

[22] Srivastava, Y.; Sangwan, N. S., Chapter 17 - Improving medicinal crops through phytochemical perspective: Withania somnifera (Ashwagandha). In Advancement in Crop Improvement Techniques, Tuteja, N.; Tuteja, R.; Passricha, N.; Saifi, S. K., Eds. Woodhead Publishing: 2020; pp 275-295.

[23] Aharoni, A.; Galili, G., Metabolic engineering of the plant primary–secondary metabolism interface. Current opinion in biotechnology 2011, 22 (2), 239-244.

[24] Pichersky, E.; Gang, D. R., Genetics and biochemistry of secondary metabolites in plants: an evolutionary perspective. Trends in plant science 2000, 5 (10), 439-445.

[25] Phillipson, J. D., Phytochemistry and medicinal plants. Phytochemistry 2001, 56 (3), 237-243.

[26] Bourgaud, F.; Gravot, A.; Milesi, S.; Gontier, E., Production of plant secondary metabolites: a historical perspective. Plant science 2001, 161 (5), 839-851.

[27] Achar, G. P. K.; Prabhakar, B.; Rao, S.; George, T.; Abraham, S.; Sequeira, N.; Baliga, M. S., Scientific Validation of the Usefulness of Withania somnifera Dunal in the Prevention and Treatment of Cancer. In Anticancer Plants: Properties and Application, Springer: 2018; pp 285-301.

[28] Gurib-Fakim, A., Medicinal plants: traditions of yesterday and drugs of tomorrow. Molecular aspects of Medicine 2006, 27 (1), 1-93.

[29] Almoulah, N. F. Phytochemical study and biological evaluation of five plants belonging to the family Solanaceae. Université de Lorraine, 2017.

[30] Kumar, A.; Kaul, M.; Bhan, M.; Khanna, P. K.; Suri, K., Morphological and chemical variation in 25 collections of the Indian medicinal plant, Withania somnifera (L.) Dunal (Solanaceae). Genetic Resources and Crop Evolution 2007, 54 (3), 655-660.

[31] Rajeswara Rao, B., Opportunities and challenges in the cultivation of Ashwagandha {Withania somnifera (L.) Du-nal}. Journal of Pharmacognosy, ISSN 2012.

[32] Singh, N.; Bhalla, M.; de Jager, P.; Gilca, M., An overview on ashwagandha: A Rasayana (Rejuvenator) of Ayurveda. African Journal of Traditional, Complementary and Alternative Medicines 2011, 8 (5S).

[33] Mirjalili, M. H.; Moyano, E.; Bonfill, M.; Cusido, R. M.; Palazón, J., Steroidal lactones from Withania somnifera, an ancient plant for novel medicine. Molecules 2009, 14 (7), 2373-2393.

[34] Wasnik, N. G.; Muthusamy, M.; Chellappan, S.; Vaidhyanathan, V.; Pulla, R.; Senthil, K.; Yang, D. C., Establishment of in vitro root cultures and analysis of secondary metabolites in Indian Ginseng-Withania somnifera. Korean J Plant Res 2009, 22, 584-91.

[35] Khan, J.; Srivastava, P.; Singh, S., Sensitive detection of a phytoplasma associated with little leaf symptoms in Withania somnifera. European journal of plant pathology 2006, 115 (4), 401-408.

[36] Martín-Herrera, D.; Abdala, S.; Benjumea, D.; Pérez-Paz, P., Diuretic activity of Withania aristata: An endemic Canary Island species. Journal of ethnopharmacology 2007, 113 (3), 487-491.

[37] Mir, B. A.; Khazir, J.; Mir, N. A.; Hasan, T.-u.; Koul, S., Botanical, chemical and pharmacological review of Withania somnifera (Indian ginseng): an ayurvedic medicinal plant. Indian J Drugs Dis 2012, 1, 147-60.

[38] Shah, S.; Saravanan, R.; Gajbhiye, N., Leaf gas exchange, chlorophyll fluorescence, growth and root yield of Ashwagandha (Withania somnifera Dunal.) under soil moisture stress. Indian Journal of Plant Physiology 2010, 15 (2), 117-124.

[39] Singh, P. K.; Sharma, Y. K., Withania somnifera (Ashwagandha): A wonder herb with multiple medicinal properties. Asian Journal of Pharmacy and Pharmacology 2018, 4 (2), 123-130.

[40] Kumar, V.; Dey, A.; Hadimani, M. B.; Emeraldjavascript, M., Chemistry and pharmacology of Withania somnifera: An update. Tang 2015, 5 (1), 1.1-1.13.

[41] Sharada, M.; Ahuja, A.; Suri, K.; Vij, S.; Khajuria, R.; Verma, V.; Kumar, A., Withanolide production by in vitro cultures of Withania somnifera and its association with differentiation. Biologia Plantarum 2007, 51 (1), 161-164.

[42] Nagella, P.; Murthy, H. N., Establishment of cell suspension cultures of Withania somnifera for the production of withanolide A. Bioresource Technology 2010, 101 (17), 6735-6739.

[43] Gupta, P.; Goel, R.; Pathak, S.; Srivastava, A.; Singh, S. P.; Sangwan, R. S.; Asif, M. H.; Trivedi, P. K., De novo assembly, functional annotation and comparative analysis of Withania somnifera leaf and root transcriptomes to identify putative genes involved in the withanolides biosynthesis. PLoS One 2013, 8 (5), e62714.

[44] Dhar, N.; Razdan, S.; Rana, S.; Bhat, W. W.; Vishwakarma, R.; Lattoo, S. K., A decade of molecular understanding of withanolide biosynthesis and in vitro studies in Withania somnifera (L.) Dunal: prospects and perspectives for pathway engineering. Frontiers in plant science 2015, 6, 1031.

[45] Singh, A. K.; Varshney, R.; Sharma, M.; Agarwal, S. S.; Bansal, K. C., Regeneration of plants from alginate-encapsulated shoot tips of Withania somnifera (L.) Dunal, a medicinally important plant species. Journal of plant physiology 2006, 163 (2), 220-223.

[46] Sivamani, S.; Joseph, B.; Kar, B., Anti-inflammatory activity of Withania somnifera leaf extract in stainless steel implant induced inflammation in adult zebrafish. Journal of Genetic Engineering and Biotechnology 2014, 12 (1), 1-6.

[47] Pawar, P.; Gilda, S.; Sharma, S.; Jagtap, S.; Paradkar, A.; Mahadik, K.; Ranjekar, P.; Harsulkar, A., Rectal gel application of Withania somnifera root extract expounds anti-inflammatory and muco-restorative activity in TNBS-induced Inflammatory Bowel Disease. BMC complementary and alternative medicine 2011, 11 (1), 34.

[48] Gupta, A.; Singh, S., Evaluation of anti-inflammatory effect of Withania somnifera root on collagen-induced arthritis in rats. Pharmaceutical biology 2014, 52 (3), 308-320.

[49] Sudhir, S.; Budhiraja, R.; Miglani, G.; Arora, B.; Gupta, L.; Garg, K., Pharmacological studies on leaves of Withania somnifera. Planta medica 1986, 52 (01), 61-63.

[50] Rasool, M.; Varalakshmi, P., Immunomodulatory role of Withania somnifera root powder on experimental induced inflammation: An in vivo and in vitro study. Vascular pharmacology 2006, 44 (6), 406-410.

[51] Gupta, M.; Kaur, G., Withania somnifera as a potential anxiolytic and anti-inflammatory candidate against systemic lipopolysaccharide-induced Neuroinflammation. Neuromolecular medicine 2018, 20 (3), 343-362.

[52] Naidoo, D. B.; Chuturgoon, A. A.; Phulukdaree, A.; Guruprasad, K. P.; Satyamoorthy, K.; Sewram, V., Withania somnifera modulates cancer cachexia associated inflammatory cytokines and cell death in leukaemic THP-1 cells and peripheral blood mononuclear cells (PBMC’s). BMC complementary and alternative medicine 2018, 18 (1), 126.

[53] Manchanda, S.; Kaur, G., Withania somnifera leaf alleviates cognitive dysfunction by enhancing hippocampal plasticity in high fat diet induced obesity model. BMC complementary and alternative medicine 2017, 17 (1), 136.

[54] Hahm, E.-R.; Lee, J.; Kim, S.-H.; Sehrawat, A.; Arlotti, J. A.; Shiva, S. S.; Bhargava, R.; Singh, S. V., Metabolic alterations in mammary cancer prevention by withaferin A in a clinically relevant mouse model. Journal of the National Cancer Institute 2013, 105 (15), 1111-1122.

[55] Winters, M., Ancient medicine, modern use: Withania somnifera and its potential role in integrative oncology. Alternative Medicine Review 2006, 11 (4).

[56] Vyas, A. R.; Singh, S. V., Molecular targets and mechanisms of cancer prevention and treatment by withaferin a, a naturally occurring steroidal lactone. The AAPS journal 2014, 16 (1), 1-10.

[57] Rai, M.; Jogee, P. S.; Agarkar, G.; Santos, C. A. d., Anticancer activities of Withania somnifera: Current research, formulations, and future perspectives. Pharmaceutical biology 2016, 54 (2), 189-197.

[58] Nema, R.; Khare, S.; Jain, P.; Pradhan, A., Anticancer activity of Withania somnifera (leaves) flavonoids compound. Int J Pharm Sci Rev Res 2013, 19 (1), 103-106.

[59] Manoharan, S.; Kaur, J., Anticancer, antiviral, antidiabetic, antifungal and phytochemical constituents of medicinal plants. Am J PharmTech Res 2013, 3, 149-69.

[60] Malik, F.; Kumar, A.; Bhushan, S.; Mondhe, D. M.; Pal, H. C.; Sharma, R.; Khajuria, A.; Singh, S.; Singh, G.; Saxena, A. K., Immune modulation and apoptosis induction: Two sides of antitumoural activity of a standardised herbal formulation of Withania somnifera. European Journal of Cancer 2009, 45 (8), 1494-1509.

[61] Srinivasan, S.; Ranga, R. S.; Burikhanov, R.; Han, S.-S.; Chendil, D., Par-4-dependent apoptosis by the dietary compound withaferin A in prostate cancer cells. Cancer research 2007, 67 (1), 246-253.

[62] Sachdeva, H.; Sehgal, R.; Kaur, S., Studies on the protective and immunomodulatory efficacy of Withania somnifera along with cisplatin against experimental visceral leishmaniasis. Parasitology research 2013, 112 (6), 2269-2280.

[63] Davis, L.; Kuttan, G., Immunomodulatory activity of Withania somnifera. Journal of Ethnopharmacology 2000, 71 (1-2), 193-200.

[64] Agarwal, R.; Diwanay, S.; Patki, P.; Patwardhan, B., Studies on immunomodulatory activity of Withania somnifera (Ashwagandha) extracts in experimental immune inflammation. Journal of Ethnopharmacology 1999, 67 (1), 27-35.

[65] Rasool, M.; Varalakshmi, P., Protective effect of Withania somnifera root powder in relation to lipid peroxidation, antioxidant status, glycoproteins and bone collagen on adjuvant‐induced arthritis in rats. Fundamental & clinical pharmacology 2007, 21 (2), 157-164.

[66] Ali, K.; Shuaib, M.; Ilyas, M.; Hussain, F.; Hussain, F., Medicinal uses of chemical extracts from Withania somnifera and its antimicrobial activity: a mini-review. PSM Microbiology 2017, 2 (1), 20-23.

[67] Siddiqui, N. A.; Singh, S.; Siddiquei, M. M.; Khan, T. H., Immunomodulatory effect of Withania somnifera, Asparagus racemosus and Picrorhiza kurroa roots. International Journal of Pharmacology 2012, 8 (2), 108-114.

[68] Zahran, E.; Abd El-Gawad, E. A.; Risha, E., Dietary Withania sominefera root confers protective and immunotherapeutic effects against Aeromonas hydrophila infection in Nile tilapia (Oreochromis niloticus). Fish & shellfish immunology 2018, 80, 641-650.

[69] Jain, S.; Shukla, S. D.; Sharma, K.; Bhatnagar, M., Neuroprotective effects of Withania somnifera Dunn. in hippocampal sub‐regions of female albino rat. Phytotherapy research 2001, 15 (6), 544-548.

[70] Srivastava, A. N.; Ahmad, R.; Khan, M. A., Evaluation and Comparison of the In Vitro Cytotoxic Activity of Withania somnifera Methanolic and Ethanolic Extracts against MDA-MB-231 and Vero Cell Lines. Sci Pharm 2016, 84 (1), 41-59.

[71] Bano, A.; Sharma, N.; Dhaliwal, H. S.; Sharma, V., A systematic and comprehensive review on Withania somnifera (L.) Dunal-an Indian ginseng. Journal of Pharmaceutical Research International 2015, 63-75.

[72] Sankar, S. R.; Manivasagam, T.; Krishnamurti, A.; Ramanathan, M., The neuroprotective effect of Withania somnifera root extract in MPTP-intoxicated mice: an analysis of behavioral and biochemical variables. Cell Mol Biol Lett 2007, 12 (4), 473-81.

[73] Bhatnagar, M.; Sharma, D.; Salvi, M., Neuroprotective effects of Withania somnifera dunal.: A possible mechanism. Neurochem Res 2009, 34 (11), 1975-83.

[74] Kumar, P.; Kumar, A., Possible neuroprotective effect of Withania somnifera root extract against 3-nitropropionic acid-induced behavioral, biochemical, and mitochondrial dysfunction in an animal model of Huntington's disease. J Med Food 2009, 12 (3), 591-600.

[75] Durg, S.; Dhadde, S. B.; Vandal, R.; Shivakumar, B. S.; Charan, C. S., Withania somnifera (Ashwagandha) in neurobehavioural disorders induced by brain oxidative stress in rodents: a systematic review and meta-analysis. J Pharm Pharmacol 2015, 67 (7), 879-99.

[76] Chatterjee, S.; Srivastava, S.; Khalid, A.; Singh, N.; Sangwan, R. S.; Sidhu, O. P.; Roy, R.; Khetrapal, C. L.; Tuli, R., Comprehensive metabolic fingerprinting of Withania somnifera leaf and root extracts. Phytochemistry 2010, 71 (10), 1085-94.

[77] Minhas, U.; Minz, R.; Das, P.; Bhatnagar, A., Therapeutic effect of Withania somnifera on pristane-induced model of SLE. Inflammopharmacology 2012, 20 (4), 195-205.

[78] Prakash, J.; Yadav, S. K.; Chouhan, S.; Singh, S. P., Neuroprotective role of Withania somnifera root extract in maneb-paraquat induced mouse model of parkinsonism. Neurochem Res 2013, 38 (5), 972-80.

[79] Kataria, H.; Wadhwa, R.; Kaul, S. C.; Kaur, G., Water extract from the leaves of Withania somnifera protect RA differentiated C6 and IMR-32 cells against glutamate-induced excitotoxicity. PLoS One 2012, 7 (5), e37080.

[80] Kumar, S.; Harris, R. J.; Seal, C. J.; Okello, E. J., An aqueous extract of Withania somnifera root inhibits amyloid β fibril formation in vitro. Phytotherapy research 2012, 26 (1), 113-117.

[81] Mohanty, I.; Gupta, S. K.; Talwar, K. K.; Dinda, A.; Joshi, S.; Bansal, P.; Saxena, A.; Arya, D. S., Cardioprotection from ischemia and reperfusion injury by Withania somnifera: a hemodynamic, biochemical and histopathological assessment. Molecular and cellular biochemistry 2004, 260 (1), 39-47.

[82] Reuland, D. J.; Khademi, S.; Castle, C. J.; Irwin, D. C.; McCord, J. M.; Miller, B. F.; Hamilton, K. L., Upregulation of phase II enzymes through phytochemical activation of Nrf2 protects cardiomyocytes against oxidant stress. Free Radical Biology and Medicine 2013, 56, 102-111.

[83] Mohanty, I.; Arya, D. S.; Dinda, A.; Talwar, K. K.; Joshi, S.; Gupta, S. K., Mechanisms of cardioprotective effect of Withania somnifera in experimentally induced myocardial infarction. Basic & clinical pharmacology & toxicology 2004, 94 (4), 184-190.

[84] Hina, S.; Rehman, K.; Dogar, Z.-u.-H.; Jahan, N.; Hameed, M.; Khan, Z. I.; Ahmad, K.; Mukhtar, K.; Valeem, E. E., Cardioprotective effect of gemmotherapeutically treated Withania somnifera against chemically induced myocardial injury. Pak J Bot 2010, 42 (2), 1487-1499.

[85] Hamza, A.; Amin, A.; Daoud, S., The protective effect of a purified extract of Withania somnifera against doxorubicin-induced cardiac toxicity in rats. Cell biology and toxicology 2008, 24 (1), 63-73.

[86] Mohanty, I. R.; Arya, D. S.; Gupta, S. K., Withania somnifera provides cardioprotection and attenuates ischemia–reperfusion induced apoptosis. Clinical Nutrition 2008, 27 (4), 635-642.

[87] Gorelick, J.; Rosenberg, R.; Smotrich, A.; Hanuš, L.; Bernstein, N., Hypoglycemic activity of withanolides and elicitated Withania somnifera. Phytochemistry 2015, 116, 283-289.

[88] Andallu, B.; Radhika, B., Hypoglycemic, diuretic and hypocholesterolemic effect of winter cherry (Withania somnifera, Dunal) root. 2000.

[89] Anwer, T.; Sharma, M.; Pillai, K. K.; Iqbal, M., Effect of Withania somnifera on insulin sensitivity in non‐insulin‐dependent diabetes mellitus rats. Basic & clinical pharmacology & toxicology 2008, 102 (6), 498-503.

[90] Udayakumar, R.; Kasthurirengan, S.; Mariashibu, T. S.; Rajesh, M.; Anbazhagan, V. R.; Kim, S. C.; Ganapathi, A.; Choi, C. W., Hypoglycaemic and hypolipidaemic effects of Withania somnifera root and leaf extracts on alloxan-induced diabetic rats. International journal of molecular sciences 2009, 10 (5), 2367-2382.

[91] Anup, K.; Mohan, K.; Suraj, S.; Sandip, F.; Bhushan, F.; Prashant, W., Antimicrobial Activity of Some Important Medicinal Plants of India against Some Plant and Human Pathogens. Research Journal of Pharmacy and Technology 2010, 3 (3), 924-926.

[92] Bisht, P.; Rawat, V., Antibacterial activity of Withania somnifera against Gram-positive isolates from pus samples. Ayu 2014, 35 (3), 330.

[93] Al-Ani, N.; Hadi, S. A.; Nazar, R., Antimicrobial activities of Withania somnifera Crude extract. Sci Agri 2013, 4, 74-76.

[94] Mwitari, P. G.; Ayeka, P. A.; Ondicho, J.; Matu, E. N.; Bii, C. C., Antimicrobial activity and probable mechanisms of action of medicinal plants of Kenya: Withania somnifera, Warbugia ugandensis, Prunus africana and Plectrunthus barbatus. PloS one 2013, 8 (6), e65619.

[95] Owais, M.; Sharad, K.; Shehbaz, A.; Saleemuddin, M., Antibacterial efficacy of Withania somnifera (ashwagandha) an indigenous medicinal plant against experimental murine salmonellosis. Phytomedicine 2005, 12 (3), 229-235.

[96] Singh, N.; Bhalla, M.; de Jager, P.; Gilca, M., An overview on ashwagandha: A Rasayana (Rejuvenator) of Ayurveda. African Journal of Traditional, Complementary and Alternative Medicines 2011, 8 (5S).

[97] Kaur, K.; Rani, G.; Widodo, N.; Nagpal, A.; Taira, K.; Kaul, S.; Wadhwa, R., Evaluation of the anti-proliferative and anti-oxidative activities of leaf extract from in vivo and in vitro raised Ashwagandha. Food and chemical toxicology 2004, 42 (12), 2015-2020.

[98] Mahdi, A. A.; Shukla, K. K.; Ahmad, M. K.; Rajender, S.; Shankhwar, S. N.; Singh, V.; Dalela, D., Withania somnifera improves semen quality in stress-related male fertility. Evidence-Based Complementary and Alternative Medicine 2011, 2011.

[99] Baitharu, I.; Jain, V.; Deep, S. N.; Hota, K. B.; Hota, S. K.; Prasad, D.; Ilavazhagan, G., Withania somnifera root extract ameliorates hypobaric hypoxia induced memory impairment in rats. Journal of ethnopharmacology 2013, 145 (2), 431-441.