Morphometric and biochemical screening of old mulberry trees (Morus alba L.) in the former sericulture region of Slovenia

Andreja Urbanek Krajnc, Tina Ugulin, Andrej Paušič, Johannes Rabensteiner, Vesna Bukovac, Maja Mikulič Petkovšek, Franc Janžekovič, Tamás Bakonyi, Rebeka Lucijana Berčič, Mateja Felicijan


Over centuries, in many European countries, the white mulberry trees (Morus alba L.) became an integral part of the cultural landscape, bearing witness to past sericulture activities. The distribution records of white mulberry trees in the cultural landscape are incomplete and in general poorly documented. The aim of the presented research was to collect data regarding geographical locations of mulberry trees and to define their morphological and biochemical variability in Goriška region, one of the historical sericulture regions in Slovenia. Principal component analysis of all morphometrical leaf traits allowed us to characterize two separate groups of morphotypes. Recordings of the tree pruning management revealed that annual base cutting is traditionally used in Goriška region. Significant correlations between pruning management and leaf morphology traits showed that frequently pruned trees form larger leaves. Biochemically, mulberry leaves are shown to be rich in proteins containing threonine, arginine, asparagine, serine, and glutamine as the most prominent free amino acids. The main phenolic compounds were identified as caffeoylquinic acid derivatives, quercetin malonyl-hexoside, rutin, kaempferol acetyl-hexoside, quercetin-3-glucoside, and p-coumaric acid derivatives. The difference in concentrations of the investigated metabolites is correlated either with the pruning management or the morphotype. Pruning significantly affected the levels of asparagine, alanine, and serine, which were higher in the annually pruned trees regardless of the morphotype. Furthermore, we were able to confirm a significant effect of pruning on total phenolics as well as on the levels of rutin, quercetin malonyl-hexoside, and quercetin-3-glucoside contents. Multivariate analysis allowed us to determine seven chemotypes with distinctive biochemical traits. Our results are the basis for defining superior high-yielding genotypes with optimum metabolic composition for both silkworm feeding as well as for innovative usage in food processing and pharmaceutical industries.


free amino acids; crop intraspecific diversity; leaf traits; morphological variability; Morus alba L.; phenolics; proteins; sericulture

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Vijayan K, Jayarama Raju P, Tikader A, Saratchnadra B. Biotechnology of mulberry (Morus L.) – a review. Emir J Food Agric. 2014;26(6):472–496.

von Bretton CW. Practische Anleitung zur Seidenzucht. Ein auf eigene Erfahrung gestützter Vorschlag zur Verbreitung und Hebung des Seidenbaues in den ausser-italienischen Ländern der Österreichischen Monarchie. Wien: In Cummission bei Carl Gerold & Sohn; 1852.

Grünberg K. Die Entwicklung der Seidenindustrie in Österreich 1660–1840. Wien: Verlagsbuchhandlung Carl Konegen; 1909.

Ipavec VM. Murve in kavalirji: Svilogojstvo na Goriškem. Ljubljana: Inštitut za slovensko narodopisje; 2008

Žontar J. Svilogojstvo in svilarstvo na Slovenskem od 16. do 20. stoletja. Ljubljana: Inštitut za zgodovino SAZU; 1957.

Khyade VB. Antioxidant activity and phenolic compounds of mulberry, Morus alba (L.) (variety: Baramatiwali). Journal of Medicinal Plants Studies. 2016;4(1):4–7.

Kandylis K, Hadjigeorgiou I, Harizanis P. The nutritive value of mulberry leaves (Morus alba) as a feed supplement for sheep. Trop Anim Health Prod. 2009;41:17–24.

Kumar V, Chauhan S, Kumar D. Nutritional composition in leaves of some mulberry varieties: a comparative study. In: Mahadevan V, Zhou J, editors. 2010 International Conference of Bioinformatics and Biomedical Technology; 2010 Apr 16–18; Chengdu, China. Piscataway, NJ: IEEE; 2010. p. 438–442.

Radojković MM, Zeković ZP, Vidović SS, Kočar DD, Mašković PZ. Free radical scavenging activity and total phenolic and flavonoid contents of mulberry (Morus spp. L., Moraceae) extracts. Hem Ind. 2012;66:547–552.

Memon AA, Memon N, Luthria DL, Bhanger MI, Pitafi AA. Phenolic acids profiling and antioxidant potential of mulberry (Morus laevigata W., Morus nigra L., Morus alba L.) Leaves and fruits grown in Pakistan. Polish Journal of Food and Nutrition Sciences. 2010;60(1):25–32.

Kim DS, Kang YM, Jin WM, Sung YY, Choi G, Kim HK. Antioxidant activities and polyphenol content of Morus alba leaf extracts collected from varying regions. Biomed Rep. 2014;2:675–680.

Flaczyk E, Kobus-Cisowska J, Przeor M, Korczak J, Remiszewski M, Korbas E, et al. Chemical characterization and antioxidative properties of Polish variety of Morus alba L. leaf aqueous extracts from the laboratory and pilot-scale processes. Agricultural Sciences. 2013;4(5):141–147.

Butt MS, Nazir A, Sultan MT, Schroen K. Morus alba L. nature’s functional tonic. Trends Food Sci Technol. 2008;19:505–512.

Zafar MS, Muhammad F, Javed I, Akhtar M, Khaliq T, Aslam B, et al. White mulberry (Morus alba): a brief phytochemical and pharmacological evaluations account. Int J Agric Biol. 2013;15:612–620.

Gryn-Rynkoa A, Bazylaka G, Olszewska-Slonina D. New potential phytotherapeutics obtained from white mulberry (Morus alba L.) leaves. Biomed Pharmacother. 2016;84:628–636.

Brahmachari G. Bio-flavonoids with promising antidiabetic potentials: a critical survey. In: Tiwari VK, Mishra BB, editors. Opportunity, challenge and scope of natural products in medicinal chemistry. Kerala: Research Signpost; 2011. p. 187–212.

Al-kirschi RA, Alimon AR, Zulkifli I. Zahari MW, Sazili AQ. The chemical composition and nutritive value of mulberry leaf as a protein source in poultry diets. In: “Feed and nutrition”: the 1st International Seminar on Animal Industry; 2009 Nov 23–24; Bogor, Indonesia. Bogor: Bogor Agricultural University; 2009. p. 98–102.

Zou Y, Liao S, Shen W, Liu F, Tang C, Chen CYO, et al. Phenolics and antioxidant activity of mulberry leaves depend on cultivar and harvest month in Southern China. Int J Mol Sci. 2012;13:16544–16553.

Katsube T, Imavaka N, Kawano Y, Yamazaki Y, Shiwaku K, Yamane Y. Antioxidant flavonol glycosides in mulberry (Morus alba L.) leaves isolated based on LDL antioxidant activity. Food Chem. 2006;97:25–31.

Ju WT, Kwon OC, Lee MK, Kim HB, Sung GB, Kim YS. Quali-quantitative analysis of flavonoids for mulberry leaf and fruit of ‘Suhyang’. Korean Journal of Environmental Agriculture. 2007;36(4):249–255.

Ju WT, Kwon OC, Kim HB, Sung GB, Kim HW, Kim QS. Qualitative and quantitative analysis of flavonoids from 12 species of Korean mulberry leaves. J Food Sci Technol. 2018;55(5):1789–1796.

Enkhmaa B, Shiwaku K, Katsube T, Kitajima K, Anuurad E, Yamasaki M, et al. Mulberry (Morus alba L.) leaves and their major flavonol quercetin-3-(6-malonylglucoside) attenuate atherosclerotic lesion development in LDL receptor-deficient mice. J Nutr. 2005;135:729–734.

Choi SW, Lee YJ, Ha SB, Jeon YH, Lee DH. Evaluation of biological activity and analysis of functional constituents from different parts of mulberry (Morus alba L.) tree. Journal of the Korean Society of Food Science and Nutrition. 2015;44(6):823–831.

Dugo P, Donato P, Cacciola F. Characterization of the polyphenolic fraction of Morus alba leaves extracts by HPLC coupled to a hybrid IT-TOF MS system. J Sep Sci. 2009;32:3627–3634.

Thabti I, Elfalleh W, Hannachi H, Ferchichi A, Campos MG. Identification and quantification of phenolic acids and flavonol glycosides in Tunisian Morus species by HPLC-DAD and HPLC-MS. J Funct Foods. 2012;4:367–374.

Lee WJ, Choi SW. Quantitative changes of polyphenolic compounds in mulberry (Morus alba L.) leaves in relation to varieties, harvest period and heat processing. Prev Nutr Food Sci. 2012;17:280–285.

Iqbal S, Younas U, Sirajuddin KWC, Sarfraz RA, Uddin MK. Proximate composition and antioxidant potential of leaves from three varieties of mulberry (Morus sp.): a comparative study. Int J Mol Sci. 2012;13:6651–6664.

WorldClim – Global Climate Data [Internet]. 2016 [cited 2018 Aug 25]. Available from:

Perko D, Orožen Adamič M, editors. Slovenija – pokrajine in ljudje. Ljubljana: Mladinska knjiga; 1998.

Čarni A, Košir P, Karadžić B, Matevski V, Redžić S, Škvorc Ž. Thermophilous deciduous forests in Southeastern Europe. Plant Biosyst. 2009;143(1):1–13.

Thangamalar A. Influence of pruning techniques and improved integrated nutrient management on productivity and quality of mulberry (Morus alba L.) and their impact on silkworm (Bombyx mori L.) bioassay [PhD thesis]. Coimbatore: Department of Sericulture Center for Plant Protection Studies, Tamil Nadu Agricultural University; 2014.

Lowry OH, Rosenbrough NJ, Farr AL, Randall RJ. Protein measurement with folin phenol reagent. J Biol Chem. 1951;193:265–275.

Kumar K, Mohan M, Tiwari N, Kumar S. Production potential and leaf quality evaluation of selected mulberry (Morus alba) clones. J Pharmacogn Phytochem. 2018;7(2):482–486.

Noctor G, Bergot G, Mauve C, Thominet D, Lelarge-Trouverie C, Prioul JL. A comparative study of amino acid measurement in leaf extracts by gas chromatography-time of flight-mass spectrometry and high performance liquid chromatography with fluorescence detection. Metabolomics. 2007;3(2):161–174.

Perucho J, Gonzalo-Gobernado R, Bazan E, Casarejos MJ, Jiménez-Escrig A, Asensio MJ, et al. Optimal excitation and emission wavelengths to analyze amino acids and optimize neurotransmitters quantification using precolumn OPA-derivatization by HPLC. Amino Acids. 2015;47:963–973.

Ainsworth EA, Gillespie KM. Estimation of total phenolic content and other oxidation substrates in plant tissues using Folin–Ciocalteu reagent. Nat Protoc. 2007;2:875–877.

Bukovac V, Strlič M, Kočar D. Comparison of methods for determination of polyphenols in wine by HPLC-UV/VIS, LC/MS/MS and spectrophotometry. Acta Chim Slov. 2009;56(3):698–703.

Hammer O, Harper DAT. PAST: paleotological statistics software package for education and data analysis. Palaeontologia Electronica. 2001;4(1):[9 p.].

Evans JD. Straightforward statistics for the behavioral sciences. Pacific Grove, CA: Brooks/Cole Publishing. 1996.

Peris NW, Gacheri KM, Theophillus MM, Lucas N. Morphological characterization of mulberry (Morus spp.) accessions grown in Kenya. Sustainable Agriculture Research. 2014;3(1):10–17.

Pawan Sharma JR, Baloda S, Singh S, Kumar M. Effect of the time and severity of pruning on growth, yield and quality in mulberry (Morus alba L.). International Journal of Agriculture Sciences. 2017;9(50):4861–4863.

Eltayb MTA, Warag EEI, Elhiuri A. Effect of pruning height on growth on five Morus species. Journal of Forest Products and Industries. 2013;2(2):27–30.

Krishnaswami S. Mulberry cultivation in South India. Bongalore: Central Silk Board; 1986.

Radjabi R. Effect of mulberry leaves enrichment with amino acid supplementary nutrients on silkworm, Bombyx mori L. at north of Iran. Academic Journal of Entomology. 2010;3(1):45–51.

Kumar VR, Chauhan S. Biochemical constituents of different parts of mulberry genotypes. International Journal of Agriculture Sciences. 2011;3(2):90–96.

Thirumalaisamy R, Gowrishankar J, Suganthapriya S, Prakash B, Ashok Kumar L, Arunachalam G. Genetic variability in Morus alba L. by biochemical and bioassay methods for increased silk productivity. Journal of Biomedical Sciences and Research. 2009;1(1):11–18.

Horie Y. Quantitative requirement of nutrients for growth of the silkworm, Bombyx mori L. JARQ. 1978;12(4):211–217.

Kozlowski RM, editor. Handbook of natural fibers. Volume 2. Processing and applications. Cambridge: Woodhead Publishing Limited; 2012.

Fedič R, Žurovec M, Sehnal F. Correlation between fibroin amino acid sequence and physical silk properties. J Biol Chem. 2003;278(37):35255–35264.

Hara S, Yamakawa M. Moricin, a novel type of antibacterial peptide isolated from the silkworm, Bombyx mori. J Biol Chem. 1995;270(50):29923–29927.

Inokuchi T, Horie Y, Ito T. Urea cycle in the silkworm, Bombyx mori. Biochem Biophys Res Commun. 1969;35(6):783–787.

Krishnaswami S, Roy D, Mikherjee SK. Yield and nutritive value of mulberry leaves as influenced by planting season, spacing and frequency of pruning. Indian Journal of Sericulture. 1970;9(1):38–42.

Chaluvachari, Bongale UD. Leaf quality evaluation of selected mulberry genotypes by biochemical and bioassay studies. In: Manna GK, Sinha U, editors. Proceedings of the Fourth All India Congress of Cytology and Genetics “Perspectives in cytology and genetics”. Vol. 4; 1981 Oct 17–21; Bhagalpur, India. Kalyani: Department of Zoology, Kalyani University; 1984. p. 121–124.

Suzuki T. Free amino acids in upper leaves of Morus species and their exudates after pruning during growing season. The Journal of Sericultural Science of Japan. 1983;53:216–221.

Yamashita T. Changes in sugar and free amino acid contents in mulberry plant after top pruning and defoliation. The Journal of Sericultural Science of Japan. 1983;53(1):1–8.

Hunyadi A, Liktor-Busa E, Márki A, Martins A, Jedlinszki N, Hsieh TJ, et al. Metabolic effects of mulberry leaves: exploring potential benefits in type 2 diabetes and hyperuricemia. Evid Based Complementary Altern Med. 2013;2013:948627.

Jeszka-Skowron M, Flaczyk E, Jeszka J, Krejpcio Z, Król E, Buchowski MS. Mulberry leaf extract intake reduces hyperglycaemia in streptozotocin (STZ)-induced diabetic rats fed high-fat diet. J Funct Foods. 2014;8:9–17.

Sánchez-Salcedo EM, Mena P, García-Viguera C, Hernández F, Martínez JJ. (Poly)phenolic compounds and antioxidant activity of white (Morus alba) and black (Morus nigra) mulberry leaves: their potential for new products rich in phytochemicals. J Funct Foods. 2015;18:1039–1046.

Urbanek Krajnc A, Novak M, Felicijan M, Kraševec N, Lešnik M, Zupanec N, et al. Antioxidative response patterns of Norway spruce bark to low-density Ceratocystis polonica inoculation. Trees. 2014;28(4),1145–1160.

Germano MP, D’Angelo V, Catania S, Miano TC, Perna V, Farago S, Cappellozza L, Cappellozza S. Phenolic content of leaf of different mulberry cultivars affect growth in the silkworm. In: Proceedings of the 21st International Sericultural Congress of ISC; 2008 Nov 3–6; Athens, Greece. Athens: International Sericultural Commission; 2008. p. 25–29.

Hirayama C, Ono H, Tamura Y, Konno K, Nakamura M. Regioselective formation of quercetin 5-O-glucoside from orally administered quercetin in the silkworm, Bombyx mori. Phytochemistry. 2008;69:1141–1149.

Vihakas M. Flavonoid and other phenolic compounds: characterization and interactions with Lepidopteran and sawfly larvae [PhD thesis]. Turku; Department of Chemistry, Faculty of Mathematics and Natural Sciences, University of Turku; 2014.

Hunyadi A, Martins A, Hsieh TJ, Seres A, Zupkó I. Chlorogenic acid and rutin play a major role in the in vivo anti-diabetic activity of Morus alba leaf extract on type II diabetic rats. PloS One. 2012;7(11):e50619.

Maudu M, Mudau FN, Mariga IK. The effect of pruning on growth and chemical composition of cultivated bush tea (Athrixia phylicoides D. C.). J Med Plant Res. 2010;4(22):2353–2358.

Sugiyama M, Katsube T, Koyama A, Itamura H. Seasonal changes in functional component contents in mulberry (Morus alba L.) leaves. Hort J. 2017;86(4):534–542.