Mulberry is a common crop rich in flavonoids, and its leaves (ML), fruits (M), and branches (Ramulus Mori, RM) have medicinal value. In the present study, a total of 118 flavonoid metabolites (47 flavone, 23 flavonol, 16 flavonoid, 8 anthocyanins, 8 isoflavone, 14 flavanone, and 2 proanthocyanidins) and 12 polyphenols were identified by ultra-performance liquid chromatography-electrospray ionization-tandem mass spectrometry. The most abundant in ML were 8-C-hexosyl-hesperetin O-hexoside and astragalin, the most abundant in M were 8-C-hexosyl-hesperetin O-hexoside and naringenin, and the most abundant in RM were cyanidin 3-O-galactoside and gallocatechin-gallocatechin. The total flavonoid compositions of ML and RM were essentially the same, but the contents of flavonoid metabolite in more than half of them were higher than those in M. Compared with ML, the contents of flavone and flavonoid in RM and M were generally down-regulated. Each tissue part had a unique flavonoid, which could be used as a marker to distinguish different tissue parts. In this study, the differences between flavonoid metabolite among RM, ML, and M were studied, which provided a theoretical basis for making full use of mulberry resources.

Mulberry (Morus alba L) fruit is traditionally used in Chinese medicine and has several beneficial effects, such as hypoglycemic, hypolipidemic, and anti-oxidative effects. We previously developed the synbiotic mulberry (SM) containing probiotic Lactobacilli, prebiotic inulin, and mulberry powder. In food supplement development, toxicity is the most important criterion in food and drug regulations before commercialization. Thus, this study aimed to investigate the subchronic toxicity of SM in male and female Wistar rats to evaluate its biosafety. The subchronic toxicity study was conducted by daily oral administration of SM at doses of 250, 500, and 1000 mg/kgBW for 90 days. Male and female rats were evaluated for body weight, organ coefficients, biochemical and hematological parameters, and vital organ histology. The results showed no mortality or toxic changes in the subchronic toxicity study. These results suggested that no observed adverse effect level (NOAEL) of SM in male and female rats has been considered at 1000 mg/kgBW for subchronic toxicity study.

Glutamine synthetase (GS) is a key enzyme involved in nitrogen metabolism. GS can be divided into cytosolic and plastidic subtypes and has been reported to respond to various biotic and abiotic stresses. However, little research has been reported on the function of GS in mulberry. In this study, the full length of MaGS2 was cloned, resulting in 1302 bp encoding 433 amino acid residues. MaGS2 carried the typical GS2 motifs and clustered with plastidic-subtype GSs in the phylogenetic analysis. MaGS2 localized in chloroplasts, demonstrating that MaGS2 is a plastidic GS. The expression profile showed that MaGS2 is highly expressed in sclerotiniose pathogen-infected fruit and sclerotiniose-resistant fruit, demonstrating that MaGS2 is associated with the response to sclerotiniose in mulberry. Furthermore, the overexpression of MaGS2 in tobacco decreased the resistance against Ciboria shiraiana, and the knockdown of MaGS2 in mulberry by VIGS increased the resistance against C. shiraiana, demonstrating the role of MaGS2 as a negative regulator of mulberry resistance to C. shiraiana infection.

Mulberry has also been regarded as a valuable source of forage for ruminants. This study was developed to investigate the impact of four additives and combinations thereof on fermentation quality and bacterial communities associated with whole-plant mulberry silage. Control fresh material (FM) was left untreated, while other groups were treated with glucose (G, 20 g/kg FM), a mixture of Lactobacillus plantarum and L. buchneri (L, 106 CFU/g FM), formic acid (A, 5 mL/kg FM), salts including sodium benzoate and potassium sorbate (S, 1.5 g/kg FM), a combination of G and L (GL), a combination of G and A (GA), or a combination of G and S (GS), followed by ensiling for 90 days. Dry matter content in the A, S, GA, and GS groups was elevated relative to the other groups (p < 0.01). Relative to the C group, all additives and combinations thereof were associated with reductions in pH and NH3-N content (p < 0.01). The A groups exhibited the lowest pH and NH3-N content at 4.23 and 3.27 g/kg DM, respectively (p < 0.01), whereas the C groups demonstrated the highest values at 4.43 and 4.44 g/kg DM, respectively (p < 0.01). The highest levels of lactic acid were observed in the GA and A groups (70.99 and 69.14 g/kg DM, respectively; p < 0.01), followed by the GL, L, and GS groups (66.88, 64.17 and 63.68 g/kg DM, respectively), with all of these values being higher than those for the C group (53.27 g/kg DM; p < 0.01). Lactobacillus were the predominant bacteria associated with each of these samples, but the overall composition of the bacterial community was significantly impacted by different additives. For example, Lactobacillus levels were higher in the G, A, and GA groups (p < 0.01), while those of Weissella levels were raised in the L, GL, and GS groups (p < 0.01), Pediococcus levels were higher in the A and GA groups (p < 0.01), Enterococcus levels were higher in the G and S groups (p < 0.01), and Lactococcus levels were raised in the S group (p < 0.01). Relative to the C group, a reduction in the levels of undesirable Enterobacter was evident in all groups treated with additives (p < 0.01), with the greatest reductions being evident in the A, S, GA, and GS groups. The additives utilized in this study can thus improve the quality of whole-plant mulberry silage to varying extents through the modification of the associated bacterial community, with A and GA addition achieving the most efficient reductions in pH together with increases in lactic acid content and the suppression of undesirable bacterial growth.

UNASSIGNED: Manganese (Mn) plays a pivotal role in plant growth and development. Aside aiding in plant growth and development, Mn as heavy metal (HM) can be toxic in soil when applied in excess. Morus alba is an economically significant plant, capable of adapting to a range of environmental conditions and possessing the potential for phytoremediation of contaminated soil by HMs. The mechanism by which M. alba tolerates Mn stresses remains obscure.
UNASSIGNED: In this study, Mn concentrations comprising sufficiency (0.15 mM), higher regimes (1.5 mM and 3 mM), and deficiency (0 mM and 0.03 mM), were applied to M. alba in pot treatment for 21 days to understand M. alba Mn tolerance. Mn stress effects on the net photosynthetic rate (Pn), stomatal conductance (Gs), transpiration rate (Tr), intercellular CO2 concentration (Ci), chlorophyll content, plant morphological traits, enzymatic and non-enzymatic parameters were analyzed as well as metabolome signatures via non-targeted LC-MS technique.
UNASSIGNED: Mn deficiency and toxicity decrease plant biomass, Pn, Ci, Gs, Tr, and chlorophyll content. Mn stresses induced a decline in the activities of catalase (CAT) and superoxide dismutase (SOD), while peroxidase (POD) activity, and leaf Mn content, increased. Soluble sugars, soluble proteins, malondialdehyde (MDA) and proline exhibited an elevation in Mn deficiency and toxicity concentrations. Metabolomic analysis indicates that Mn concentrations induced 1031 differentially expressed metabolites (DEMs), particularly amino acids, lipids, carbohydrates, benzene and derivatives and secondary metabolites. The DEMs are significantly enriched in alpha-linolenic acid metabolism, biosynthesis of unsaturated fatty acids, galactose metabolism, pantothenate and CoA biosynthesis, pentose phosphate pathway, carbon metabolism, etc.
UNASSIGNED: The upregulation of Galactinol, Myo-inositol, Jasmonic acid, L-aspartic acid, Coproporphyrin I, Trigonelline, Pantothenol, and Pantothenate and their significance in the metabolic pathways makes them Mn stress tolerance metabolites in M. alba. Our findings reveal the fundamental understanding of DEMs in M. alba\'s response to Mn nutrition and the metabolic mechanisms involved, which may hold potential significance for the advancement of M. alba genetic improvement initiatives and phytoremediation programs.

Morus sp. (mulberry) has a long tradition of use as a medicinal treatment, including for cardiovascular disease and type 2 diabetes, being shown to have antioxidant properties and to promote wound healing. Extracellular vesicles (EVs) are sub-micron, membrane-enclosed particles that were first identified in mammalian bodily fluids. EV-like particles have been described in plants (PDVs) and shown to have similar characteristics to mammalian EVs. We hypothesised that some of the health benefits previously attributed to the fruit of Morus sp. could be due to the release of PDVs. We isolated PDVs from Morus nigra and Morus alba via ultracentrifugation and incubated THP-1 monocytes, differentiated THP-1 macrophages, or HMEC-1 endothelial cells with pro-oxidant compounds DMNQ (THP-1) and glucose oxidase (HMEC-1) or lipopolysaccharide (LPS) in the presence of different fractions of mulberry EVs. Mulberry EVs augmented ROS production with DMNQ in THP-1 and caused the downregulation of ROS in HMEC-1. Mulberry EVs increased LPS-induced IL-1β secretion but reduced CCL2 and TGF-β secretion in THP-1 macrophages. In scratch wound assays, mulberry EVs inhibited HMEC-1 migration but increased proliferation in both low and high serum conditions, suggesting that they have opposing effects in these two important aspects of wound healing. One of the limitations of plant-derived therapeutics has been overcoming the low bioavailability of isolated compounds. We propose that PDVs could provide the link between physiological dose and therapeutic benefit by protecting plant active compounds in the GIT as well as potentially delivering genetic material or proteins that contribute to previously observed health benefits.

Mulberry is a rapidly growing plant that thrives in diverse climatic, topographical, and soil types, spanning temperature and temperate countries. Mulberry plants are valued as functional foods for their abundant chemical composition, serving as a significant reservoir of bioactive compounds like proteins, polysaccharides, phenolics, and flavonoids. Moreover, these compounds displayed potent antioxidant activity by scavenging free radicals, inhibiting reactive oxygen species generation, and restoring elevated nitric oxide production induced by LPS stimulation through the downregulation of inducible NO synthase expression. Active components like oxyresveratrol found in Morus demonstrated anti-inflammatory effects by inhibiting leukocyte migration through the MEK/ERK signaling pathway. Gallic and chlorogenic acids in mulberry leaves (ML) powder-modulated TNF, IL-6, and IRS1 proteins, improving various inflammatory conditions by immune system modulation. As we delve deeper into understanding its anti-inflammatory potential and how it works therapeutically, it is crucial to refine the extraction process to enhance the effectiveness of its bioactive elements. Recent advancements in extraction techniques, such as solid-liquid extraction, pressurized liquid extraction, superficial fluid extraction, microwave-assisted extraction, and ultrasonic-assisted extraction, are being explored. Among the extraction methods tested, including Soxhlet extraction, maceration, and ultrasound-assisted extraction (UAE), UAE demonstrated superior efficiency in extracting bioactive compounds from mulberry leaves. Overall, this comprehensive review sheds light on the potential of mulberry as a natural immunomodulatory agent and provides insights into its mechanisms of action for future research and therapeutic applications.

In this study, we investigated the anti-hypertensive properties of mulberry products by modulating the renin-angiotensin system (RAS). Comparative analysis showed that the ethyl acetate fractions, particularly from the Cheongil and Daeshim cultivars, contained the highest levels of polyphenols and flavonoids, with concentrations reaching 110 mg gallic acid equivalent (GE)/g and 471 mg catechin equivalent (CE)/g of extract, respectively. The ethyl acetate fraction showed superior angiotensin-converting enzyme (ACE) inhibitory activity, mainly because of the presence of the prenylated flavonoids kuwanon G and H. UPLC/Q-TOF-MS analysis identified kuwanon G and H as the primary active components, which significantly contributed to the pharmacological efficacy of the extract. In vivo testing of mice fed a high-salt diet showed that the ethyl acetate fraction substantially reduced the heart weight and lowered the serum renin and angiotensinogen levels by 34% and 25%, respectively, highlighting its potential to modulate the RAS. These results suggested that the ethyl acetate fraction of mulberry root bark is a promising candidate for the development of natural ACE inhibitors. This finding has significant implications for the management of hypertension through RAS regulation and the promotion of cardiovascular health in the functional food industry.

Mulberries (genus Morus), belonging to the order Rosales, family Moraceae, are important woody plants due to their economic values in sericulture, as well as for nutritional benefits and medicinal values. However, the taxonomy and phylogeny of Morus, especially for the Asian species, remains challenging due to its wide geographical distribution, morphological plasticity, and interspecific hybridization. To better understand the evolutionary history of Morus, we combined plastomes and a large-scale nuclear gene analyses to investigate their phylogenetic relationships. We assembled the plastomes and screened 211 single-copy nuclear genes from 13 Morus species and related taxa. The plastomes of Morus species were relatively conserved in terms of genome size, gene content, synteny, IR boundary and codon usage. Using nuclear data, our results elucidated identical topologies based on coalescent and concatenation methods. The genus Morus was supported as monophyletic, with M. notabilis as the first diverging lineage and the two North American Morus species, M. microphylla and M. rubra, as sister to the other Asian species. In the Asian Morus species, interspecific relationships were completely resolved. However, cyto-nuclear discordances and gene tree-species tree conflicts were detected in the phylogenies of Morus, with multiple evidences supporting hybridization/introgression as the main cause of discordances between nuclear and plastid phylogenies, while gene tree-species tree conflicts were mainly caused by ILS.

Hop stunt viroid (HSVd), a small, single stranded, circular, non-coding infectious RNA known to cause infection in various economically important crop plants. In the present investigation, a study was conducted in the southern part of Karnataka districts of India to detect the possible association of HSVd infection in mulberry plants. A total of 41 mulberry plants showing typical viroid-like symptoms along with asymptomatic samples were collected and screened using conventional Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) using a specific set of HSVd-Fw/ HSVd-Re primers. Out of 41 samples, the study confirmed the presence of HSVd in six samples of mulberry collected from Ramanagara (1 sample), Chikkaballapur (3 samples) and Doddaballapura (2 samples) regions with an expected HSVd amplicon size of ∼ 290-300 nucleotides. The mechanical transmission of HSVd was also confirmed on cucumber (cv. Suyo) seedlings through bioassay, which was reconfirmed by RT-PCR. The amplicons were cloned, sequenced, and the representative nucleotide sequences were deposited in the NCBI GenBank. Subsequently, molecular phylogenetic analysis showed that HSVd mulberry isolates from this study were most closely related to grapevine isolates, indicating a common origin. On the other hand, it was shown to belong to a different group from mulberry isolates so far reported from Iran, Italy, Lebanon, and China. The secondary structure analysis of HSVd mulberry Indian isolates exhibited substitutions in the terminal left, pathogenicity, and variable regions compared to those of the Indian grapevine isolates. As far as this study is concerned, HSVd was detected exclusively in some mulberry plants with viral-like symptoms, but the pathogenesis and symptom expression needs to be further investigated to establish the relationship between HSVd and the disease symptoms in the mulberry plants.