MORUS SPP. Material Conservation and Characterization and Its Importance for Romanian Sericulture and GCEARS-PSP Development- a Review

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MORUS SPP. Material Conservation and Characterization and its Importance for Romanian Sericulture and GCEARS-PSP Development- A Review

and 1 * 1 2 3 Adela Ramona MOISE , Liviu1 Alexandru MARGHITAS , Otilia BOBIS , Florina Maria COPACIU Daniel Severus DEZMIREAN 1 Department of Apiculture and Sericulture, University of Agricultural Science and Veterinary Medicine Cluj-Napoca2 Life Sciences Institute “King Michael I” of Romania, University of Agricultural Sciences and Veterinary Medicine3* Cluj-Napoca Department of Biochemistry, University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca corresponding author: [email protected]

Bulletin UASVM Animal Science and Biotechnologies 75(2)/ 2018 Print ISSN 1843-5262; Electronic ISSN 1843-536X DOI:10.15835/buasvmcn-asb: 2018.0001

Abstract Morus spp. Moraceae Morus

is a perennial and woody plant, belongingMorus to spp. family, genus and is essential for sericulture. One of the main aims of Global Centre of Excellence for Advance Research in Sericulture and Promotion of Silk Production (GCEARS-PSP) is to create a reserve of , to multiply the vegetal material and to provide it to farmers and also to create an important biotechnological support for further scientifically works. As in Romania the intensive plantations are almost non-existent, one of the main objective of the centre is to create a real base for sericulture revival (the existence of nutritional base for silkworms) and to develop the biotechnological scientific component of the centre. The aims of this review are to create a clear overview of moriculture for sericulture and to reveal the biotechnological potentialMulberry and spp. pharmacological uses of different mulberry parts. In this review the major findings regarding description of Kokuso 21 variety, mulberry micropropagation and bioactive compounds of different vegetalKeywords: parts from GCEARS-PSP, mulberry, are presented, conservation, and represent bioactive only the first stage of the GCEARS-PSP activity.

INTRODUCTION Global Centre of Excellence for Advance Research in Sericulture and Promotion of Silk Morus spp Production (GCEARS-PSP) areresearches Morus regardsalba and the Morus biotechnological nigra aspects of . The most popular species of mulberry (www.usamvcluj. , and Yongkang ro/crcas-ppm) is located in UASVM CN and it (2000) states that the intensive selection and is recognised by the International Sericulture mutation breedings have resulted in thousands Commission (http://www.inserco.org/) since of cultivars, hybrids and polyploids. Mulberry 2014. The centre was created in order to achieve trees are valuable species not only for silkworm development of advanced research in sericulture rearing, but also for gardening and landscaping, and moriculture, to offer diagnostic services using street shade and reduction of pollution level in the modern techniques, to maintain and reproduce environment and soil (Rafati et al., 2011). the gene pool of Romanian and foreign silkworm`s The purpose of the present review is to breeds and to promote the silk production. thecreate moriculture. a realistic image of the main GCEARS-PSP Working in close collaboration with Laboratory activities in the past and in the future regarding of Bee’s Products Control – APHIS, the GCEARS-PSP 58 et al.

MOISE et al.

As it was stated by literature, the components Papanastasiset al. (1995) was found very effective of mulberry trees exhibiting their biological in increasing the production of this shrub. activity in the various pathological and health Dolis (2016) reported in leaves a general human ailments. Our research team want to draw chemical composition, containing proteins the attention that mulberry is a source of potential 21.16%, fat 3.54%, crude fibre 17.88%, nitrogen indigenousDescription nutraceuticals of Kokuso and 21 functional variety food. free extract 43.42% and ash content 13.96%. The biological and productive parameters of Kokuso 21, gived by Tănase (2007) are: number One of the main achievements of GCEARS- of branches/shrub – 24.6, branches length 115.2 PSP in 2017 was to set up a mulberry plantation cm, branches thickness 15 cm,2 internodes length in UASVM CN. This plantation was necessary for 3.8 cm, leaf surface 292.3 m , leaf weight 6.2 g, providing the vegetal material as food for the leaf production 3.0 kg/shrub/ series, leaves water silkworms that the Department of Apiculture content 75.73% and 25.45% protein content. inand vitroSericulture owns and material for the further Even if the scientifically reports regarding biotechnological researches in moriculture (i.e., Kokuso 21 variety are pretty poor, the existing micropropagation, the extraction of studies presents very encouraging data, which biological active compounds, etc.). The mulberry made the decision of planting Kokuso 21 variety from Kokuso 21 variety was purchased from in ourMulberry University, micropropagation very easily. Sericulture and Agriculture Experiment Station Vratza (Bulgaria) (http://ses-vratza.bacsa-silk. Morus org), accompanied by phytosanitary certificate. spp. As we state in the introduction one of the goal The Sericulture Experiment Station (SES) in of GCEARS-PSP is to create a reserve of Vratza collects, characterizes and evaluates , and this aim willMorus be spp reached by mulberry both indigenous and exotic mulberry varieties. micropropagation. Currently more than 140 mulberry accessions The mulberry ( ) is anBombyx important mori. are maintained in the germplasm at SES-Vratza tree in the sericulture industry because its leaves (Tzenov, 2002, citedMorus by Weiguo, latifolia 2011). A number represent the only source of food for of 570 saplings were planted on 0.12 ha. Qualitative and quantitative improvements in Kokuso 21 ( ) has Japanese mulberry varieties play a vital role in industrial origins; it derives from the crossing between advances. Unfortunately, the perennial nature of Naganua, Gariin and Shiso varieties and it is a the plants, together with the species prolonged bisexual variety. juvenile period, slows this process. Plant tissue- In intensive type plantations it can produce culture techniques have been used extensively from the first years a high quantity of leaves for the stock improvement. During the last fortyet with high protein content. Kokuso 21 has a good alyears, several researchers (Chattopadhyay and development with long branches, large unlobed Datta, 1990; Chitra and Padmaja, 2001; Hossain (23 cm x 17 cm), thick and juicy nutritive leaves ., 1990; Thomas, 2002) have reported success in with slow withering. Leaves yield under rain fed plant regeneration from different explant types. conditions is higher than 15 000 kg/ha (http://ses- Mulberry has been micropropagated from vratza.bacsa-silk.org/en/chernichevi-fidanki). many explants sources, such as sooth tips and FAO Electronic Conference on Mulberry axillary, isolated, or winter buds. The earliest for Animal Production (Morus1-L) states that reports, from Japan, include one from Ohyama “Ichinose” and “Kokuso 21” varieties have been (1970), who obtained the first complete plantlets used as parents for other varieties. One of the from axillary-bud tissue. The majority of the major reasons why “Ichinose” and “Kokuso 21” research has involved in the use of MS (Murashige have been selected as parents is that “Ichinose” and Skoog, 1962) media for nodal cuttings, and for is female and “KokusoM. alba 21” is male. They have culture of axillary and apical buds. Other authors desirable traits, and crossing is easy and simple. (Sharma and Thope, 1990) observedin vitro that an MS Fertilization of Kokuso 21 with 120 medium supplementedMorus alba with 2.5 µM BSA (bovine kg N/ha applied in a large study described by serum albumin) was optimum for -raised Bulletin UASVM Animal Science and Biotechnologies 75(2) / 2018 seedlings of . Their explants exhibited MORUS SPP. 59

Material Conservation and Characterization and its Importance for Romanian Sericulture and GCEARS-PSP

significant differences in shoot-multiplication0 An important progress has been made in rates, and could be stored at 4 C for at least six mulberry improvement through Morus tissue-culture months without any decrease in vigor. Rooting techniques. Micropropagation procedures are was enhanced by activated charcoal (0.05-0.10%). already standardized in various species, Transplantation was 100% successful when the fact that provides good chances to GCEARS-PSP to shoots were grown in a 1:1 sand-vermiculite mix realize new mulberry plants. in the greenhouse (Pattnaik and Chand, 1997). Anyway, more researches are still needed in Thomas (2002) in a review presents the main the area of haploid and triploid plant production, researched on tissue culture in mulberry. somatic embryogenesis, etc. The attention should The success of shoot-culture experiments be focused on genetic engineering in mulberry. is highly influenced by the source and age of the Judicious choice of the explant source, coupled explant, as well as the conditions underet al which it with some refinements in media composition, is cultured. Positioning of the explant also plays a should increase the success rate for recalcitrant crucial role in bud induction (Hossain ., 1991). genotypes. Improvements in regeneration ability The same authors showed that, in the 10 studied and control of somaclonal variation are crucial genotypes, the et maximum al response of multiple- for maximum exploitation of this genus. When shoot induction was obtained from subterminal more emphasis is paid to these research areas, the buds (Hossain Morus., 1992). leavigata They were tree. able Shoot to futureBioactive of sericulture compounds industry of will different be assured. vegetal produce multiple shoots from nodal explants matrices from Mulberry spp. of a 10 year old proliferation was higher when the BA level was raised to as much as 11 µM, but further increases Mulberry is exclusively used for rearing suppressed development. The highest percentage silkworm due to the presence of unique chemo- of explants giving rise to shoots, and the maximum factors like morin, β-sitosterol in leaves. Plant is length of those shoots, was observed on MS a potential source for curing debilitating diseases. media supplemented with 4.4 µM BA, whereas Flavonoids, anthocyanin and alkaloids present the greatestet al. number of shoots per explantsin vitro was in the leaves, bark, root and fruits of mulberry obtained from 11 µM BA. play a pivotal role in containing free radicalset al Paul (2001) described a rapid - and prooxidants generated in the body due to propagation procedure, using for culture young metabolismMorus alba and phagocytosis (Ramesh ., axillaries buds, which were multiplied, rooted and 2014). transplanted to soil within 3 months. tree bark, fruits, and leaves have When micropropagated plants are comparedet been used in conventional and natural medicine alwith those generated through cutting, the (China and India) for the treatment of diabetes, former have significantly higher vigor. Zaman atherosclerosis, hyperlipidemia,et al hypertension, and . (1997) have assessed the field performance more recently, some cancer and neurogenerative and biochemical analyses of both tissue culture- diseases (Butt ., 2008). Mulberry leaves derived plants and plants raised according to have been usedet foral years to treat hyperglycemia, conventional methods. Their studies confirmed inflammation, cough, hypertension, cancerMorus alba and that micropropagated plants had significantly fever (Kang ., 2006). Because of its good greater morphogenic vigor. The biochemical therapeutic activity andMorus low alba toxicity, is reported to analysis of the leaves revealed no significant has been extensively used in conventional Chinese nutritional differenceMorus nigra between theet twoal types. medicine (Li, 1998). SurvivalMorus rates nigra for acclimatizedet al plants varied have neuroprotective, skin tonic, antioxidant,et anti- al Morusfrom 75% ihou in Morus cathayana (Yadav and Morus., 1990) serrata up to hyperglycemic, antibacterial, antihypertensive 90-95% in (Jain ., Morus1992a, australis 1992b); and anti-hyperlipidemic activities (Nomura ., et, al 1980). Tables 1-3 present the general chemical transplanted(Pattnaik and Morus Chand, alba 1997) and composition of mulberry leaves. (Pattnaik ., 1996). Sharma and Thrope (1990) plants with 100% success. Bulletin UASVM Animal Science and Biotechnologies 75(2) / 2018 60 et al.

MOISE Table 1. et al The average values of the main constituents in mulberry leaves (According to Flaczyk ., 2013) Concentration Phenolic Concentration Concentration Compound Flavonols acids Water g/100g g/100g DW g/100g DW 5.2 Gallic 0.02 Rutin 0.91 Quercetin 3-β-D- Protein 14.70 Protocatechuic 0.16 0.54 glucoside Kaempferol 3-β-D- Ash 22.37 p-hydroxybenzoic 0.11 0.32 glucopyranoside Succharose Fat 0.11 Vanillic 0.71 Total Flavonols 1.76 18.62 Chlorogenic 4.64 Glucose 4.90 Caffeic 2.54 Fructose 1.61 p-coumaric 0.27 Sinapic Xylose 0.85 Ferulic 0.39 Galactose 0.27 0.21 Other 17.04 Total phenolic acids 9.09 carbohydrates DW = dry weigh

Table 2 et al . Antioxidant compounds and activity of mulberry leaves (according to Flaczyk ., 2013)

Compound Unit Value Total phenolics g gallic acid equivalents/100g DW 14.42

Ascorbic acid mg/100g of DW 1.76

DPPH (μMol Trolox /g DW) 214.08 ABTS (μMol Trolox /g DW 51.76 Antioxidant activity 29.88 Chelating activity (%) for 0.002g DW = dry weight Table 3. et al.

Bioactive compounds in mulberry fruits, according to Zhang (2008) Concentration Concentration

Phenolic compound Flavonoid µg/g FW µg/g FW Protocatechiuc acid 14.66 Taxifolin 6.53 Chlorogenic acid 24.72 hexosideRutin 111.38 Kamepferon- 4-CQA (CAE) 7.32 Quercetin 20.80

3,5-diCQA (CAE) 7.99 3.29 Total 54.68 Total 142 Total nonantocyanins phenolic (GAE) 653.2 FW = fresh weight Bulletin UASVM Animal Science and Biotechnologies 75(2) / 2018 MORUS SPP. 61

Material Conservation and Characterization and its Importance for Romanian Sericulture and GCEARS-PSP

In the last period, wild food plants have become The high phenolic content and antioxidant very attractive to the food industry, promoting activity of mulberry underline the nutritiveMorus spp and. their use as replacements for synthetic chemicals phyto-medicinal potential of this fruit. The results and nutraceuticals, in this way mulberry became presented in this review indicate that properties.a very important resource for its phytochemical has the potential to be further developed into a composition, nutritional value andet antioxidant al. nutritionally and biotechnological valuable raw material for food and beverage application. As other authors done, Donno (2014) CONCLUSIONS identified mulberry as a rich source of antioxidant compounds; the observed fingerprint demonstra ted that the species represent a rich source of phy- The recovery of Romanian sericulture is tochemicals, like organic acids, monoterpenes and possible through the GCEARS-PSP actions. Its polyphenolic compounds, especially flavonols and success depends on different factors,Bombyx such as mori the anthocyanins, which led to reasonably good over- production performances of mulberry plantation, all fruit quality. obtaining high quality leaves for Among the bioactive compounds, one of the et al., feeding and rich mulberry leaves and fruitsMorus for most important constituents of mulberry fruits latifoliaproduction diversification. is represented by anthocyanins (Lee 2005). A number of 570 mulberry trees ( Several studies have investigated the content ) were planted in USAMV CN. of phenolics as flavonoids and anthocyanins in The literature study presented in this review, mulberry extracts, as well as carotenoids content contains the characterisation of this plant, its (Arabshdi-Delousee and Urooi, 2007). leaves and fruits composition, bioactivity and Due to this health-promoting compounds their therapeutically effects on human are shortly mulberry fruits are traditionally used as a laxative, presented here, but it demonstrates that the odontalgic, anthelmintic, expectorant, hypogly- GCEARS-PSP has the ability to reach its economical caemic and emetic agent; the traditional Chinese and scientifically objectives. medicine used mulberry fruit as et folk al remedy to in orderFurther to provide studies to willthe local be pharmaceutical done on the treat oral and dental diseases, diabetes, hyperten- components of Kokuso 21 variety leafs and fruits sion, arthritis and anaemiaMorus spp (Liang ., 2012). Several researches investigated the nutraceu- industry a very valuable vegetal raw material and tical properties of . fruits, studying their to provideREFERENCES to the silkworms a quality fodder base. nutritional potentials, but a complete profile with quality traits, phytochemical composition and an- Morus tioxidant activity evaluation is still lacking. Pre- 1. indicaArabshahi-Delouee S., Urooj A. (2007). Antioxidant vious studies have examined the total content of properties of various solvent extracts of mulberry ( Morus spp L.) leaves. Food Chemistry 102: 1233-1240. phenols, flavonoids, anthocyanins and antioxidant et al et al., 2. Bobis, O., Dezmirean, D. S., Marghitas, L. A., Bonta, activity of . grown in different regions V., Urcan, A., Pasca, C., Moise, A.R. (2018) - Morus spp. (Chen ., 2012; Ozgen 2009; Uzun and for revigoratingth silkworm breeding in Romania and Bayir, 2012), but total polyphenol content, anti- promoting health benefits of leaves and fruits. Book of Abstracts 9 CASEE Conference, 6-9 June, Bucharest p. 7. oxidant activity and most of the potential health- Morus alba promoting agents of mulberry fruits still remain 3. Butt, M.S., Nazir A., Tauseef Sultan M., Schroen K. (2008) under scribed. - L. nature’s functional tonic. Trends in Food Morus spp varieties. Science & Technology, 19,505-512. Our research team has already started a Morus alba 4. Chattopadhyay th S., Datta S.K., (1990). A rapid clonal chemical characterisationMorus nigra of propagation of mulberry tree ( ) through tissue We started to analyze mulberry leafs and fruits, culture. Proc 76 Ind Sci Cong Part III (Abstr): 208-209. belonging to specie - Ukraina variety; 5. Chen, S.K., Zhao P., Shao Y. X., Li Z., Zhang C., He X. using modern techniques for sugar spectrum, (2012). Moracin M from Morus alba L. is a natural lipid, protein and mineral content, free aminoacid phosphodiesterase-4 inhibitor. Bioorganic & Medicinal profile, polyphenolic and flavones contents and Chemistry Letters 22(9): 3261-3264. the methods are presented by Bobiş et al., 2018. Bulletin UASVM Animal Science and Biotechnologies 75(2) / 2018 62 et al.

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Material Conservation and Characterization and its Importance for Romanian Sericulture and GCEARS-PSP Morus alba Morus nigra 37. Yadav U., Lal M., Jaiswal V.S., (1990). Micropropagation of Mulberry ( L.). Journal of Food Science 73(6): L from shoot tip and nodal explants of mature 512-518. trees. Sc Hort 44:61-67. 41. Weiguo, Z., Venugopalan N., Chirakkara, Ercisli, S., 38. Yongkang H., (2000). Mulberry cultivation and utilization Tsou, Chi-Hua, (2011). Morus. C. Kole (ed.), Wild Crop in China. Electronic Conference on Mulberry for Animal Relatives: Genomic and Breeding Resources, Tropical and Production (Morus 1-L), Rome, Italy, 11-43. Subtropical Fruits, DOI 10.1007/978-3-642-20447-0_5, 39. Zaman A., Islam R., Joarder O.I., (1997). Field performance Springer-Verlag Berlin Heidelberg. and biochemical evaluation of micropropagated mulberry 42. www.usamvcluj.ro/crcas-ppm plants. Plant Cell Tiss Organ Cult 51: 61:64. 43. http://www.inserco.org/ 40. Zhang, W., (2008). HPLC-DAD_ESI-MS/MS Analysis and 44. http://ses-vratza.bacsa-silk.org Antioxidant Activities of Nonanthocyanin Phenolics in 45. http://ses-vratza.bacsa-silk.org/en/chernichevi-fidanki

Bulletin UASVM Animal Science and Biotechnologies 75(2) / 2018