1. Mulberry Extracts and Their Natural Products Studied for Therapeutic Potential in Diabetes

Before 2010, the effect of black mulberry leaf extracts on diabetes was studied by two in vitro studies and two in vivo settings. However, more studies were published after 2010. As seen in Table 1, the extracts are accounted for reducing diabetic symptoms, neuropathy, hyperglycemia, and inhibiting the carbohydrate metabolism enzyme activities.

Diabetes mellitus is a chronic disease defined by the overproduction of ROS, which affects the lysosomal membrane integrity, and, as a metabolic disorder, diabetes is also described by enzymatic alterations ^[14]. In 2015, a study investigated the catalytic characters and gene expression of hepatic arylsulfatase B (ASB) in streptozotocin-induced diabetic rats treated with black mulberry leaf extract. The results indicated that the black mulberry leaf and fruit extract administered as a powder managed to suppress the blood sugar levels in diabetic rats and substantially diminished hepatic ASB activity. The study’s published data revealed that the daily supplementation of M. nigra foliage has a beneficial action in the correction of diabetes-stimulated ASB alteration ^[4].

Moreover, about four years before the above study, an aqueous black mulberry leaf extract (Botucatu, São Paulo State, Brazil) was evaluated for its effects on maternal reproductive results, lipid and oxidative stress outline, and fetal anomaly prevalence when orally administered to streptozotocin-induced diabetic and non-diabetic WR. The study concluded that administering a dose of 400 mg/kg/day of black mulberry leaf extract, by gavage, from day 0 until day 20 of pregnancy resulted in an antioxidant outcome contributive to a reduced occurrence of skeletal and visceral abnormalities in progenies from diabetic dams. However, it could not regulate maternal hyperglycemia, pregnancy rate, and placental–fetal development in diabetic rats ^[5].

Another study evaluated an ethanolic mulberry leaf extract’s properties in diabetes models induced by a high-fat diet and injection of 35 mg/kg STZ in male WR. The extract was administered to one of the four groups over the curse of four weeks. Kidney and blood samples were subjected to biochemical analysis such as fasting blood glucose level, albumin, creatinine, urea and uric acid concentrations, white blood cells, hemoglobin, hematocrit, and histological assessment. The results showed that fasting blood glucose, creatinine, urea, and uric acid displayed considerably lower levels in the extract-treated group than the group lacking mulberry leaf treatment. Moreover, histology evaluation proved that glycogen accumulation, fatty degeneration, and lymphocyte infiltration in the extract-treated group were only mild while they were moderate in the non-treated group ^[6]. These results were associated with the contents of mulberry leaves in 1-deoxynojirimycin (1- DNJ), quercetin-3-O-βD-glucopyranoside, phytoalexins moracin C, moracin N, and chalcomoracin.

It was also indicated that mulberry leaf extracts, such as mulberry leaves tea, can be addressed to hyperuricemia and nephropathy in diabetes. Diabetic nephropathy results from overly increased oxidative stress and mulberry leaves contain antioxidant components such as 1-DNJ, flavonoids, polyphenols, and polysaccharides with beneficial therapeutic actions ^[7].

To explain the regulatory mechanism of mulberry in treating diabetes, in 2011, a study evaluated 1-DNJ and polysaccharides extracted from mulberry leaves on alloxan-induced diabetic mice. Administering a daily oral treatment with polysaccharides (150 mg/kg body weight) to diabetic mice for three months resulted in postprandial blood glucose reduction and lessened toxicity caused by prolonged supra-physiological glucose to pancreatic cells. Moreover, it was concluded that polysaccharides might control hepatic glucose metabolism and gluconeogenesis by up/down-regulating the expression of rate-limiting enzymes (glucokinase, phosphoenolpyruvate carboxykinase, and glucose-6-phosphatase) in the liver and up-regulating the pancreatic and duodenal homeobox factor-1 (PDX-1), insulin-1, and insulin-2 expressions in the pancreas ^[8].

Mulberry foliage extract enriched with deoxynojirimycin (DNJ) was studied on postprandial hyperglycemia in patients with impaired glucose metabolism. After three months of supplementation with M. nigra extract (6 mg DNJ, t.i.d.) to 76 subjects with fasting plasma glucose varying between 110–140 mg/dL, it was determined that prolonged ingestion of M. nigra leaf extract with high DNJ contents can result in enhanced postprandial glycemic control, especially in cases of impaired glucose metabolism ^[2].

Another research investigated the coincubation of 0.1–10 μmol/L DNJ with mature 3T3-L1 adipocytes and established that the genes/proteins expression of the insulin receptor, phosphatidylinositol-3-kinase, protein kinase B, and adiponectin were amplified in a dose-dependent manner. However, it was concluded that further studies are needed to clarify the molecular mechanism of action ^[15].

In 2020, there was another attempt to assess the mechanism of action and bioactivity of mulberry foliage polyphenols in preventing T2D by inhibiting disaccharidase and glucose transport in Caco-2 cells. The mulberry leaves extract’s main composition consisted of chlorogenic acid, rutin, benzoic acid, and hyperoside. The results indicated that mulberry leaf polyphenols hindered glucose absorption by curbing the sodium-dependent glucose cotransporter-1–glucose transporter 2 pathway by downregulating phospholipase mRNA expression, protein kinase A, and protein kinase C ^[13].

The leaves of black mulberry were studied compared to the leaves of Bauhinia variegate L., which is a species of flowering plant in the legume family Fabaceae. The study evaluated the impact of administering these plants’ extracts orally on hyperglycemia, insulin activity, and renal and hepatic function in an STZ-induced diabetic murine model. The HPLC with a diode-array detection analysis of the extracts highlighted major flavonoids being rutin, quercitrin, quercetin, and hesperidin, and phenolic acids as chlorogenic and p-coumaric acids. Quercitrin was found as the key flavonoid in M. nigra foliage extract (2.75 mg/g), and rutin was the main one in B. variegate extract (4.38 mg/g). Given the elevated insulin serum levels measured in the rats treated, it was suggested that the black mulberry leaf extract might be boosting the endogenous insulin secretion. Moreover, it was advised that the amount of elevated polyphenols identified in M. nigra foliage extract might add to its antioxidant action in protecting the liver and kidneys from tissue injury linked to hyperglycemia ^[9].

In January 2020, a group of researchers explored a hexane fraction of black mulberry Brazilian foliage (Hex-Mn) and their action on carbohydrate digestion and absorption in diabetic mice. HPLC analysis revealed the presence of flavonoids isoquercitrin and kaempferol-3-O-rhamnoside. It was shown that administering Hex-Mn through an oral route to diabetic mice led to the delay of carbohydrate digestion but not to the glucose transport through a brush border membrane of the intestine, which contributed to the reduction of postprandial hyperglycemia. Moreover, it was suggested that Hex-Mn was more effective in suppressing the α-glucosidase enzyme than the a-amylase activity in vitro ^[10]. Similarly, a later study concluded that the black mulberry leaf extract obtained through maceration acts as an α-glucosidase inhibitor. Their M. nigra extract inhibited the α-glucosidase enzyme with an IC₅₀ value of 549.7 μg/mL ^[16].

Figure 1 shows some of the mechanisms of actions investigated regarding M. nigra’s effect in managing diabetes.

Figure 1. Mechanisms of action regarding mulberry’s effect in managing diabetes.

2. Mulberry Extracts and Their Natural Products Studied for Therapeutic Potential in Hepatic Disorders

After checking the literature published before 2010, it was found that, before 2010, there are no relevant studies regarding black mulberry extracts and their potential benefits in treating or preventing hepatic diseases. However, in Table 2, there are summaries of the later studies conducted to assess mulberry’s action in managing hepatic disorders.

As in the case of dandelion leaf extracts, black mulberry leaves have a potentially beneficial and partly proven impact in cases of hepatic lesions induced by excessive or chronic drug administration. In 2014, a MeOH- H₂O extract of M. nigra foliage was dispensed in doses of 250 mg/kg and 500 mg/kg through an oral route to mice suffering from paracetamol-induced hepatic damage, and it resulted in lower hepatic enzymes and total bilirubin levels. These hepatoprotective properties were attributed to the high contents of quercetin, luteolin, and isorhamnetin ^[17].

The antioxidant actions of Morus leaf ethanolic extracts were also examined in other cases of drug-induced hepatotoxicity. In vivo and in vitro studies were performed on methotrexate-induced hepatotoxicity models of male rats and on human HepG2 cells. The results indicated that the administration of M. nigra leaves extract is advised simultaneously with the anti-rheumatic drug therapy given the extract’s antioxidant and cytoprotective characteristics, which likely resulted from the plant’s flavonoid content ^[18].

Black mulberry leaves and pulp were also evaluated in what concerns their impact on the glycemic response and redox profile on a hepatic level in alloxan-induced diabetic rats. The findings indicated that the hydroalcoholic foliage extracts diminished the SOD-CAT ratio and carbonylated protein levels by decreasing oxidative stress. Finally, it was suggested that black mulberry leaves could be used to control hyperglycemia as they increased the serum insulin level and present hepatoprotective action given the attenuated liver damage markers ^[19]. The authors concluded as well that the black mulberry leaves extract contained more phenolic compounds than the fruit extract, and that the therapeutic results are most likely triggered by caffeoylquinic acid contents, which were identified in the leaves but not in the fruits.

The effect of lyophilized black mulberry leaf extracts on CCI₄-induced hepatic injury in a rat model was investigated. The leaf extracts at doses ranging from 150 to 300 mg/kg were administered to rats by i.p. injection for eight weeks. Black mulberry extracts counteracted protein oxidation caused by CCI₄ and exhibited the capability to control the activity of SOD and GPx. Moreover, the extracts counteracted the CCI₄-induced rise in AST and gamma-glutamyl transferase levels. Finally, leaf extracts offered substantial protection from CCl₄-induced hepatic injury, and it was suggested that they might also represent a novel approach for treating hepatic diseases ^[20].

In what concerns the M. nigra’s toxicity, a study published in 2018 aimed to identify the chemical profile of EtOH leaves extract and perform a toxicological study in rats of both genders. In the acute exposure group, 2000 mg/kg of the extract was orally administered, and signs of toxicity and mortality were observed. The extract was orally dispensed for 28 days in doses of 500, 750, and 1000 mg/kg in the sub-acute exposure group. The extract’s chemical profile was determined through HPLC/DAD analysis and the findings indicated that quercetin and caffeic acid were the main compounds. After evaluating its action following acute administration, the EtOH M. nigra leaf extract was graded as a safe product (category 5), according to the protocol. Moreover, in the subacute exposure group, the researchers noted a reduction in AST in males (750 and 1000 mg/kg) and females (1000 mg/kg) and a decrease in TC in females (750 and 1000 mg/kg). Finally, the ethanolic M. nigra leaf extracts were marked as safe when administered orally. They exhibited protective action of organs along with cholesterol-lowering actions, likely due to the extract’s content of quercetin and caffeic acid, which are known as the mulberry leaf’s major compounds ^[21].

A recent study attempted to assess the mechanism through which the MeOH extract of black mulberry foliage curbs the damage caused by APAP in various murine tissues, including hepatic tissues. APAP (500 mg/kg) was administered orally with or without black mulberry leaf extract (150, 300, and 500 mg/kg) over the course of four days. It was, thus, indicated that the crude extract had powerful antioxidant activity (EC50 = 42.97 µg extract/mL) given the significant contents of polyphenols and flavonoids, such as gallic acid, chlorogenic acid, catechin, and rutin. Furthermore, the initially modified levels of glutathione S transferase activity, lipid peroxidation, and liver and kidney functions were radically overturned when black mulberry MeOH extract was administered to the APAP group. Finally, it was indicated that M. nigra leaves’ natural products including potential candidates for genetic protection and the treatment of organotoxicity was caused by heightened oxidative stress ^[22].

Figure 2 shows some of the mechanisms of actions investigated regarding M. nigra’s effect in managing hepatic disorders.

Figure 2. Mechanisms of action regarding mulberry’s effect in managing hepatic disorders.

3. Mulberry Extracts and Their Natural Products Evaluated for Therapeutic Potential in Cardiovascular Disease

After checking the literature published before 2010, it was found that there are no relevant studies regarding M. nigra leaf extracts and their potential benefits in treating or preventing cardiovascular disorders. However, in Table 3, there are some summaries of the later studies conducted to assess mulberry’s action in managing cardiovascular disease.

Black mulberry leaves were traditionally used to remedy ailments connected with various hepatic disorders and heart diseases. Currently, their extracts are studied for pharmacological and nutraceutical applications due to the reported bioactive compounds. Over the last 10 years, a few in vivo and in vitro studies were published on the M. nigra’s leaves' therapeutic properties on a cardiovascular level, attributed mainly to its antioxidant properties.

In 2016, a black mulberry leaf extract at a dose of 200 mg/kg combined with binahong (Anredera cordifolia [Ten.] Stennis) leaf extract was administered to hyperlipidemic-induced rats for 21 days. Powdered crude herb of binahong and mulberry leaves were extracted by ethanol 70% and ethanol 95%, following the reflux method for 3 h with three repetitions. The combination of extracts showed superior effectiveness in a dose-dependent manner than that of the reference drug simvastatin in reducing TC and LDL serum levels ^[23].

Two black mulberry leaves’ aqueous solutions obtained through infusion and decoction and a hydromethanolic extract of black mulberry leaves was compared to fenofibrate-based therapy to highlight their hypolipidemic abilities. Fenofibrate treatment is usually recommended for lowering cholesterol and for preventing cardiovascular complications. However, in what concerns the extracts, it was determined that the infusion extract of black mulberry leaves had superior contents of antioxidant polyphenols like chlorogenic acid and quercetin, while decoction extract offered a greater ascorbic acid content. The hyperlipidemic rats were treated with 100, 200, or 400 mg/kg of black mulberry extracts and showed diminished serum cholesterol, triglycerides, and regulated lipoproteins. Moreover, black mulberry leaves infusion reduced lipid peroxidation in the liver, kidney, and brain. It also resulted in a stronger hypolipidemic effect where high-density lipoprotein (HDL) improved, and LDL dropped. Finally, the potential therapeutic effect attributed to the high contents of quercetin and chlorogenic acid of the black mulberry leaf infusion was indicated as beneficial in dyslipidemia and related oxidative stress ^[24].

Although some studies are exploring the action of white mulberry leaves and black mulberry fruits over triggering conditions of cardiovascular diseases, obesity, and diabetes ^[26][27], there is very little preclinical information given over the last 20 years on the therapeutic effects of black mulberry leaves on cardiovascular disease, with the fruits being mainly studied for their content of anthocyanins, proven to be effective in managing cardiovascular disease and triggering factors such as hyperlipidemia ^[28][29].

As mentioned in the previous chapters, cardiovascular ailments are often triggered by diabetes and obesity. In these regards, a study published in 2020 investigated the impact and the action mechanism of dietary black mulberry leaf powder on fat deposition in fattening pigs. The animals were randomly allotted to a normal diet or a 5% (w/w) mulberry leaf supplemented diet. Changes in backfat thickness were assessed with blood triglycerides, cholesterol, serum hormones, and leptin-related signaling activity. The mulberry leaf supplemented diet reduced serum triglycerides and free cholesterol concentrations while it increased the ratio of HDL to LDL. Finally, it was indicated that administering black mulberry leaves as diet supplements in fattening pigs have potent obesity preventive action, offering a promising start for developing mulberry leaves-based products as anti-obesity agents ^[25].

M. nigra leaf extract’s therapeutic action was also studied on symptoms and quality of life among climacteric women. Women go through various hormonal modifications during the menopausal transition that heightens the probability of developing certain diseases and deteriorate women’s quality of life. This study is included in this chapter given that the most frequently met symptoms of hormonal changes also comprise cardiovascular affections among palpitations, headaches, depression, irritability, and fatigue. Furthermore, 250 mg of black mulberry leaf powder, 1 mg of estradiol, or placebo were administered for 60 days to 62 climacteric women. The conclusion was that climacteric symptoms and the women’s quality of life improved after administrating M. nigra leaf powder, similarly to the estradiol outcome ^[30].

In Figure 3, there are some mechanisms of actions investigated regarding M. nigra’s effect in managing cardiovascular disease.

Figure 3. Mechanisms of action regarding mulberry’s effect in managing cardiovascular disease.

4. Conclusions

In what concerns the Morus species, it seems like black mulberry leaves and some of their natural products are mainly proven to be effective in managing diabetes due to their antioxidant action and reported the capacity of increasing a serum insulin level. These therapeutic properties in managing diabetes are thought to be attributed to mulberry leaf compounds such as 1-deoxynojirimycin, chlorogenic acid, quercetin, and polysaccharides. In hepatic ailments, black mulberry leaves present antioxidant action and cytoprotective characteristics, have a hypolipidemic effect, and are beneficial in dyslipidemia due to compounds such as gallic and chlorogenic acid, rutin, and catechin, quercetin, luteolin, and isorhamnetin. In cardiovascular diseases and associated disorders, white mulberry and black mulberry fruits are studied more than the leaves because anthocyanins present in mulberry fruits are already validated for their beneficial action in cardiovascular disease prevention. Nevertheless, anthocyanins often interact with other phytochemicals, showing synergistic biological effects while making contributions from individual components that are difficult to decipher.