**Bioavailability and Sustained Plasma Concentrations of CoQ10 in Healthy Volunteers by a Novel Oral Timed-Release Preparation**

#### **Alessio Martucci 1,\* , Delia Reurean-Pintilei <sup>2</sup> and Anamaria Manole <sup>2</sup>**


Received: 8 February 2019; Accepted: 25 February 2019; Published: 28 February 2019

**Abstract:** Coenzyme Q10 (CoQ10) is a natural compound with potent antioxidant properties. Its provision through diet does not always allow adequate levels in the human body, and supplementation is often necessary. This bioavailability study intended to explore the plasma concentration levels of a novel CoQ10 oral preparation (COQUN®, Coenzyme Q10 Miniactives Retard 100 mg capsules) mimicking assumption on a regular basis. Twenty-four healthy adults tested a single dose of CoQ10 100 mg in one day to assess bioavailability. After a one week wash-out period, they were randomly assigned (1:1) to continuous administration for four weeks: Group A (*n* = 12) 100 mg once a day (OD); and Group B (*n* = 12) 100 mg twice a day (BID). During the single dose phase, Cmax was observed at 4 h, and the mean values of AUCt and Tmax were 8754 μg/mL·h and 4.29 h, respectively. The multiple dose phase showed increasing plasma levels up to 7 days after the start of administration, and sustained high concentrations during the all administration period. No relevant adverse events were reported. These results show that Miniactives® technology can release CoQ10 to allow high constant blood concentrations without a sharp decrease. This may be the first step of evidence for a potential new antioxidative treatment in human chronic diseases deserving high CoQ10 levels.

**Keywords:** coenzyme Q10 (CoQ10); bioavailability; intestinal absorption; neuroprotection

#### **1. Introduction**

Coenzyme Q (CoQ), or ubiquinone, is a lipophilic, vitamin-like compound with exceptional biochemical properties, synthetized by prokaryotic and eukaryotic cells. CoQ10 is the lipid form produced by the human body, where Q10 indicates the number of isoprenoid subunits in the lipid tail attached to the quinone ring of the coenzyme [1]. CoQ10 can also be obtained from diet, mainly from meat, poultry, and fish, and in much less quantity from fruits, vegetables, cereals, and dairy products [2].

CoQ10 is a physiological component of the human mitochondrial electron transport chain, but its half-reduced and fully reduced forms allow CoQ10 to function as an antioxidant [3]. By virtue of its proven ability to change in a reduced form, CoQ10 has been shown to induce protective effects against lipid peroxidation in a ubiquitous manner in the human body, with special regard in organs and systems' tissues such as cardiovascular, nervous, and metabolic. Over the years, an ever-increasing number of diseases have been associated with mitochondrial dysfunction and oxidative stress.

Significant reduction of cardiovascular mortality, decrease of NT-proBNP blood levels, and improvement of cardiac function has been reported among elderly subjects after five years of combined supplementation of CoQ10 and selenium [4]. The long-term (two years) beneficial effects of CoQ10 supplementation on symptoms improvement and reduction of major adverse cardiovascular events (i.e., cardiovascular and all-cause mortality, and incidence of hospital stay) have also been assessed in patients with chronic heart failure (CHF) [5].

Most of clinical evidence sustains that the glycemic control among individuals with type-2 diabetes mellitus (T2DM) can be improved by CoQ10 supplementation. CoQ10 administration at different daily doses (ranging 60–200 mg/day) and for different periods (eight weeks–six months) can determine increased insulin synthesis and secretion by pancreatic β cells, significant decrease of glycated hemoglobin level, and kidney protection against diabetic nephropathy. However, despite other studies reported, marginal or not significant clinical benefits of CoQ10 in glycemic control, it is now clear that mitochondrial dysfunction is secondary to oxidative stress that, most of time, can be successfully treated by adequate supplementation of CoQ10 in T2DM patients [6].

Promising results have been reported in the treatment of neurodegenerative disorders such as Parkinson disease (PD) and Huntington's disease (HD) with CoQ10. As these are chronic, progressive and non-regressive disorders, the goal of any treatment is to cause a slowing of the disease progression, since improvement and cure are not currently possible. That is why only the highest dose of CoQ10 slowed the functional decline of PD among the three dosages tested of 300, 600 or 1200 mg/day in subjects with the early stage of disease, and not yet requiring treatment for their disability [7]. Conversely, a chronic supplementation with 600 mg/day of CoQ10 did not produce any significant slowing in functional decline in patients with early HD [8].

An interesting field of CoQ10 neuroprotective research against reactive oxygen species focused on promoting mitochondrial functions and retinal ganglion cell (RGC) survival in ischemic retina under conditions of intraocular pressure elevation (glaucoma). Glaucoma is a progressive neurodegenerative disease of RGCs associated with axon degeneration in the optic nerve. During recent years, researchers became aware that traditional strategies of lowering intraocular pressure were often unsatisfactory to prevent progressive vision loss. Thus, the current trend of using neuroprotective strategies for the treatment of glaucoma is sustained by the growing evidence that glaucomatous neurodegeneration is analogous to other neurodegenerative disorders in the central nervous system [9,10]. Consistently, CoQ10 showed to significantly block activation of astroglial and microglial cells and apoptosis in ischemic retina in addition to protecting RGCs in animals [11], and to improving inner retinal function and visual cortical responses in humans [12].

Combination of appropriate formulations and dosages is a key factor to allow optimal absorption and achieve adequate blood concentration of CoQ10 to exert the expected clinical benefits. The importance of achieving an optimal CoQ10 bioavailability is justified by the possible risk of exposing treated subjects to a lack of efficacy in the case of underdosing. On the other hand, too high concentrations can induce toxic effects or increase the rate of adverse events. The clinical evidence suggests that CoQ10 bioavailability can greatly vary not only after different daily doses or dose strategies, but especially belongs to formulations used [13–17]. For instance, it was shown that an emulsified CoQ10 preparation can increase the intestinal absorption, being more permeable across cellular membranes and allowing a relatively low-dose administration [13]. Despite many other factors can influence plasma CoQ10 concentrations, such as serum lipoproteins levels, i.e., cholesterol, High Density Lipoprotein (HDL), and Low Density Lipoprotein (LDL)/Very Low Density Lipoprotein (VLDL) are carriers of CoQ10 in the circulation–diet, daily motion, time of day, human race, age, and gender, and some authors indicated that dissolution is probably the more important factor rather than release and absorption rate [14,15]. Lu and coworkers [14] administered the same single daily dose of CoQ10 (50 mg/day) to a small group of healthy Asian volunteers using two different formulations. The baseline plasma values, and after day 15 of treatment of CoQ10, were similar to the respective values observed in European subjects, but CoQ10 bioavailability was higher in subjects treated with

the sustained release tablets compared to regular tablets. Another colloidal CoQ10 preparation achieved astonishingly higher plasma levels compared to the same doses (120 mg/day) of other more conventional formulations [15].

Good intestinal absorption and the achievement of high peak plasma concentrations should not be the only objectives of an oral formulation of CoQ10. It is important to ensure that plasma concentrations remain constant over time, avoiding excessive fluctuations in bioavailability, especially if once-daily dosing is clinically required. The authors of Reference [16] showed that the plasma concentrations of the five formulations used, following a high peak reached after 2–4 h, returned to the same initial levels after 12 h from the administration. Moreover, the dosing strategy is another major important cause to reach adequately high plasma CoQ10 concentrations. A divided dose administration (e.g., BID) improves absorption by almost double, as compared with the same amount of active substance taken in one single dose [16]. The dose fractionation strategy should be carefully considered when high doses have to be administered or when high bioavailability should be achieved with a relatively small daily dose.

The aim of this bioavailability study was to determine the single (100 mg) and multidose dose (100 mg/day vs. 2 × 100 mg/day) pharmacokinetics (i.e., dosage effect and dosage strategy) of a novel CoQ10 preparation based on neutral micro-particles dissolution technology (i.e., formulation effects), a prolonged-release capsule administered orally to healthy volunteers. Bioavailability intended to explore the plasma concentration levels which might assure antioxidant effect if the novel CoQ10 preparation were taken on a regular basis.

#### **2. Materials and Methods**

#### *2.1. Study Design*

This was a single-center (Consultmed Iasi, IASI, Romania), open-label, single and multi-dose bioavailability study of an innovative CoQ10 oral formulation in 24 healthy adults. All subjects tested a single dose of 100 mg of CoQ10 in 1 day to assess bioavailability. Then, the subjects followed a 1 week wash-out period after which they were randomly assigned (1:1) to a 4 week period of continuous administration of CoQ10: Group A of 12 subjects with intake of 100 mg OD (after dinner); Group B of 12 subjects with intake of 100 mg BID (after lunch and dinner). The primary objective was to evaluate the best dosage between 100 mg OD or 100 mg BID of the novel CoQ10 oral formulation in order to reach a level of plasma concentration which might assure its antioxidant effect if taken on a regular basis. The secondary objectives were to evaluate the safety and tolerability of both the single 100 mg oral dose and the multiple doses of 100 mg OD and BID during the 1 month daily dose phase.

In order to participate in the study, each subject had to meet all major inclusion and exclusion criteria at screening and at check-in visits. Inclusion criteria: informed consent form (ICF) signed, both gender aged between 25–75 years, body mass index (BMI) between 20–29 kg/m2, fasting the night before enrolment for at least 10 h, healthy status, abstention from consumption of any food supplements except vitamin D and calcium at least 2 weeks before and during the study, consumption of dairy and cereal products, and willing to follow all study procedures. Exclusion criteria: intake of any prescribed medication within 2 weeks of the beginning of the study, hypotension, any clinically significant history of serious digestive tract, liver, kidney, cardiovascular or hematological disease, diabetes, gastrointestinal disorders, or other serious acute or chronic diseases, known lactose/gluten intolerances/food allergies, inadequate veins, or known contraindication to placement of a dedicated peripheral line for venous blood withdrawal, known drug and/or alcohol abuse, use of any form of nicotine or tobacco, mental incapacity precluding adequate understanding or cooperation, participation in another investigational study or blood donation within 3 months prior to or during this study.

During the study the following procedures were performed: Physical examination, vital signs recording (blood pressure, heart rate, temperature, and respiratory rate), body measurements (height and weight), 12 lead electrocardiogram (ECG), safety laboratory analysis (Haematology: Red blood cells, white blood cells, platelet, haemoglobin, and haematocrit; Biochemistry: hepatic transaminases, alcalin phosphatase, total cholesterol, LDL, and HDL cholesterol), concomitant medication recording, and adverse events monitoring. With special regard to the latter, mild insomnia, elevated levels of liver enzymes, rash, nausea, upper abdominal pain, dizziness, sensitivity to light, irritability, headache, heartburn, and fatigue were closely monitored.

#### *2.2. Pharmacokinetic Timing and Assessments*

When they arrived at the study center, subjects were hospitalized for at least 12 h for the single dose phase, and they remained at study site for approximately 24 h. At the end of this visit (visit 1), if no serious adverse event (SAE) occurred, subjects were dismissed and requested to return after 1 week (wash-out period) for the second study visit (visit 2), in order to initiate the multidose phase. Besides, subjects received a diary and were requested to report any possible adverse event experienced between V1 and V2, as well as any treatment taken for treating adverse events (AEs), if applicable. In the wash-out period subjects had to respect the same lifestyle regimen and no medication had to be taken, if not necessary.

During the single-dose phase plasma CoQ10 levels were measured before dosing (0 h) and over the next 12 h after intake: at 1, 2, 4, 8, and 12 h. Pharmacokinetic properties were measured accordingly: Area under the curve until the last observation (AUCt) (μg/mL·h), maximum plasma concentration (Cmax) (μg/L), time at which the Cmax was observed (Tmax) (hours), and elimination half-life (T1 2 ) (hours). During the multidose-dose phase, plasma CoQ10 concentrations were measured once at the following timepoints: V2 (day 0), V3 (day 7), V4 (day 14), and V5 (day 28) (Figure 1). The pharmacokinetic properties measured were AUCt, Cmax, Tmax. Samples were analyzed for plasma CoQ10 using an immunosorbent assay (ELISA; enzyme-linked immunosorbent assay) validated at the analytical laboratory (Consultmed Iasi Laboratory, IASI, Iasi county, Romania).

**Figure 1.** Flow-chart of blood sampling times (z) for the bioavailability assessment after a single dose of 100 mg and after multiple doses of 100 mg once a day (OD) or twice a day (BID) of CoQ10. During visits, V2, V3, V4, and V5 (multiple dose phase) only had one sample (at one timepoint) was collected. AUC: area under the curve; PK: pharmacokinetic; Cmax: maximum concentration; Tmax: the time at which the Cmax is observed; T1/2: half-life.

For pharmacokinetic analyses 4 mL of blood were collected in blood collection tubes. Each blood sample was allowed to clot for 20–25 min at room temperature. Then, they were centrifuged for 15 min at 1300 g at 4 ◦C. Afterwards the plasma was separated into the secondary sample tubes as follows: 0.5 mL plasma into two cryotubes, one to be sent to the pharmacokinetic laboratory and one as back-up. The plasma cryotubes were appropriately labeled (study code, treatment period, subject number, sampling time) and stored at −20 ◦C to −80 ◦C at the study center until shipment to the specified laboratory. The backup cryotubes were kept at the study center at least until the confirmation from the pharmacokinetic laboratory that the samples arrived in good conditions.

#### *2.3. Formulation Administered*

The CoQ10 formulation administered (COQUN®, Coenzyme Q10 Miniactives Retard 100 mg capsules, Visufarma S.p.A.) is a novel oral preparation based on an innovative modified release technology of active principles at certain time intervals. The basis in Miniactives® form is neutral microparticles of round shape, with dimensions between 400 and 500 microns. Each single particle is covered with one or more concentric layers of the active ingredients, and subsequently coated with a polymeric membrane suitable for obtaining a pre-established timed release. This technology leaves the time of the active ingredients absorption unchanged. This formulation gradually releases the active ingredients by diffusion, in a pre-determined time, thanks to a polymeric permeable and insoluble membrane coating each single particle, thus assuring a constant release.

#### *2.4. Ethical Conduct of the Study*

All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki revised in 2013, and the protocol was approved by the local Ethics Committee Comisia Locala de Etica Consultmed, Sos Paracurari n. 70, bl 550, parter, Iasi, Romania (Project identification code: VF-BAQ10/2018) on May 31st, 2018, and also approved by Romanian Ministry of Health (Ministerul Sanatatii, Str Cristian Popisteanu, *n*. 1–3, Bucaresti, Romania) on June 19th, 2018.

The study was registered at ClinicalTrials.gov (Internet). Bethesda (MD): National Library of Medicine (US) (Identifier NCT03819491).

#### *2.5. Statistical Analysis*

Due to the explorative aim of the study no formal power calculation has been attempted, and no hypotheses were pre-specified. Twenty-four subjects (12 for each arm) have been considered sufficient to obtain reliable results for the exploratory purposes of the study. Descriptive statistics and confidence intervals (CI) at 95% level are provided. In particular, continuous variables are presented as arithmetic mean values ± standard deviation (SD), median values with interquartile range, minimum, maximum, and coefficient of variation (CV); for categorical variables, the absolute and percentage frequencies are provided. When normality assumption hold, results of Student *t*-tests are presented in order to compare pharmacokinetics parameters in subjects assigned to the two treatment groups. The statistical software package used was SAS version 9.4.

The following analysis sets were considered in the study: Safety Population (SP), all randomized subjects who signed the informed consent and took at least one dose of study product; Intention-To-Treat Population (ITT), all randomized subjects; and Protocol Population (PP), all subjects who met all inclusion/exclusion criteria and who did not have any major protocol deviation.

#### **3. Results**

Twenty-seven subjects were screened in the study but 3 were screening failures. A total of 24 subjects entered the study, half (*n* = 12) allocated to CoQ10 100 mg OD oral intake and half (*n* = 12) to CoQ10 100 mg BID. All randomized subjects completed the study. The SP, ITT- and PP-population were composed of 24 subjects. The two treatment groups were comparable for baseline characteristics (Table 1). A complete listing of all demographic variables of each participating subject is reported in Table S1.


**Table 1.** Demographic features of subjects randomized in each CoQ10 treatment group.

SD: standard deviation.

#### *3.1. Single Dose Phase*

The plasma concentration curve of CoQ10 over time is shown in Figure 2. The distribution of plasma concentrations was wide as well as standard deviations, indicating quite different levels of plasma concentration among subjects. The mean baseline plasma CoQ10 concentration (0 h) was 649.8 (191.8) μg/L. However, a slightly increasing absorption phase of CoQ10 mean plasma values was observed until 4 h (772.1 μg/L), followed by a slow terminal decline until 12 h (696.3 μg/L) (Table S2A. Plasma concentration of CoQ10 by time (0, 1, 2, 4, 8, 12 h) during the single dose phase (V1)—ITT population; Table S2B. Individual plasma concentrations of CoQ10 (μg/L) in the single dose oral administration phase—ITT population).

**Figure 2.** Single dose phase: mean plasma concentrations of CoQ10 by time after 100 mg single dose oral administration— Intention-To-Treat Population (ITT) population.

The descriptive statistics for pharmacokinetic parameters in the single dose phase are reported in Table 2. The mean AUCt was 8754 μg/mL·h. The maximum registered value was almost the double the mean (15,394.54 μg/mL·h) while the minimum was 5277.62 μg/mL·h. Among other parameters, Tmax values showed very high fluctuation with a minimum of 0 h (i.e., the maximum concentration was reached before CoQ10 oral intake) and a maximum of 12 h. The standard deviation was 3.58 h and the coefficient of variation was 83%, indicating that subjects registered very different time of maximum concentration (Table S3. Individual pharmacokinetic parameters of CoQ10 in the single dose oral administration phase—ITT population).

Males showed higher mean plasma concentrations compared to females at each time point (i.e., males: 759.79, 894.74, 844.33, 915.58, 840.01, and 772.41 μg/mL; females: 594.79, 628.38, 706.88, 700.34, 657.84, and 658.28 μg/mL, at 0, 1, 2, 4, 8, and 12 h, respectively). In the samples, significantly different plasma concentrations of CoQ10 between males and females were registered (*p* < 0.001); on average, females had a plasma concentration of 182.16 μg/L lower than men.


**Table 2.** Descriptive statistics for pharmacokinetic parameters of CoQ10 in the single and multiple phase—ITT population.

\* h: Tmax during single dose phase, \*\* day: Tmax during multiple dose phase.

#### *3.2. Multiple Dose Phase*

The mean plasma concentrations of CoQ10 increased over time in both treatment groups (Figure 3). At each study visit subjects who were assigned to 100 mg BID (Group B) had higher mean values than subjects in Group A, indicating that the assumption CoQ10 twice a day gave a higher concentration in the body.

**Figure 3.** Multiple dose phase: plasma concentrations of CoQ10 by visit and by treatment group (Group A: 100 mg OD; Group B: 100 mg BID)—ITT population.

The two dosages of the same CoQ10 oral formulation followed the same pattern over time. In both groups, the mean values of plasma concentrations increased considerably from Visit 2 (Day 0) to Visit 3 (Day 7). At Visit 2 (Day 0), the mean plasma CoQ10 concentrations in Group A and B (701.95 ± 295.17 μg/L and 756.96 ± 201.17 μg/L, respectively) represent the mean plasma values after the end of the washout period. After day 7 (Visit 3), constant trends of high plasma levels were observed, which remained high during the rest of the multidose phase (21 days), with higher values for the Group taking 100 mg BID.

Additionally, in the multidose phase males showed higher plasma concentration of CoQ10 compared to females at each study visit. As in the single dose phase, the pattern over time was the same for men and women. The distribution of plasma concentration was very wide: Standard deviations and coefficients of variation—showing the extent of variability in relation to the mean of the population—were quite large indicating that levels of plasma concentration were different among individuals. The high inter-individual variability observed in this study could be probably due to a series of physiological conditions such as age, gender, and multiple administration time, which makes a subject very different from another. More likely, by reviewing the pharmacokinetics profile of each subject randomized in this study, exceptionally high pharmacokinetic values were observed. They belong to a 36-year-old man randomized to Group B, who registered values of plasma concentration far above from the mean of the total set of subjects. By considering this individual as an outlier and by excluding him from the analysis (*n* = 23), mean plasma concentration values (and SD) were smaller for Group B, as follows (μg/L): 743.83 (205.53), 1153.86 (290.86), 1118.12 (421.44), and 1155.94 (338.48) at visit 2, visit 3, visit 4, and visit 5, respectively (Table S4. Individual plasma concentrations of CoQ10 (μg/mL) in the multiple dose oral administration phase—ITT population).

The descriptive statistics for pharmacokinetic parameters by treatment group are reported in Table 2. The mean CoQ10 bioavailability (AUCt) in Group B (3459.05 μg/mL·h) was statistically higher than in Group A (2657.45 μg/mL·h) (*p* = 0.0345). Despite values of the other pharmacokinetics parameters (Cmax, Tmax) remained higher for subjects treated by CoQ10 100 mg BID compared to ones with the OD dosing scheme, no statistically significant differences between groups were detected. As previously described, by excluding the outlier subject from the analysis, the difference between the AUCt values of the two groups was no more statistically significant (*p* = 0.0548) (Table S5. Individual pharmacokinetic parameters of CoQ10 in the multiple dose oral administration phase—ITT population). In the scenario without this subject, there is evidence of a difference in the CoQ10 bioavailability (AUCt) between the two dosages of the novel oral formulation of CoQ10, but the low power of the study

due to the small sample size did not allow highlighting, albeit slightly, a significant difference when excluding him.

#### *3.3. Adverse Events*

The oral formulation of CoQ10 was well tolerated in all 24 healthy subjects; only 3 non-serious, moderate intensity AEs were reported during all the study period. The 3 AEs occurred in 2 subjects, both enrolled in the Group B (100 mg BID): (1) Intermittent dizziness of 5 days duration, possibly related to the oral preparation, and spontaneously resolved; (2) 1 day respiratory virosis, adequately treated, but unrelated to the study product; and (3) pultaceous angina of 5 days duration, unrelated to the CoQ10 oral preparation.

During the multiple dose phase (at visit 2 and visit 5), 21 subjects (9 in Group A and 12 in Group B) showed some biochemical values out of normal ranges, but none was considered clinically significant. Only 5 subjects—2 in Group A and 3 in Group B—showed clinically not significant out-of-range liver biochemical values. The ranges of values considered outside the normal ranges are shown in Table 3.

**Table 3.** Range of values of liver biochemical parameters outside the normal ranges observed during the multiple dose phase in both treatment groups—ITT population.


AST = Aspartate Aminotransferase; ALT = Alanine Aminotransferase; ALP = Alkaline Phosphatase; TC = Total Cholesterol; HDL = High-Density Lipoprotein; LDL = Low-Density Lipoprotein; and TGs = Triglycerides. \* Some subjects experienced more than one biochemical value outside the normal range of the reference laboratory.

Subjects randomized to treatment with 100 mg BID of CoQ10 did not show higher out-of-range liver values compared to subjects treated with the halved dose (100 mg OD). Both doses were safely tolerated. However, renal function tests were not monitored during the study because they were not included in the protocol requirements, and no blood samples were taken for the evaluation of basic plasma biochemistry.

#### **4. Discussion**

These results demonstrate that in human plasma high levels of CoQ10 can be achieved by administrating relatively low oral doses by the use of a novel timed-release oral formulation determining optimal intestinal absorption and sustained plasma concentrations over time. The combination of bioavailability and safety results obtained with two oral dosages of CoQ10 (100 mg OD and BID) contribute to the construction of a rationale for a clinical use of this novel formulation of CoQ10. The information can help clinicians to protect patients from the negative effects of lipid peroxidation, on the one hand preventing possible therapeutic failures due to CoQ10 underdosing, and on the other, the possible development of toxicity following administration of too high doses.

In the experience of Joshi and coll. the pharmacokinetic properties of two new oral CoQ10 formulations (i.e., fast-melting tablet and effervescent tablet) were not statistically different compared with those of commercial formulations (i.e., soft gelatin capsule and powder-filled hard shell) when administered at 60 mg in single dose fashion [16]. The mean Cmax values of the four formulations (around 80 μg/mL) measured over 12 h were essentially similar to the one of CoQ10 Miniactives Retard capsule we studied (83 μg/mL), but the mean Tmax values were almost halved (1.3 and 2.0 h for fast-melting and effervescent tablets, respectively) compared to the one of Miniactives capsule (4.29 h). It is hard to believe that the more rapid delivery of the two fast melting formulations can play a significant role in the clinical cure of diseases in which the main feature of long-term treatment should be to ensure consistently high levels of CoQ10 over time. Furthermore, the bioavailabilities of the four formulations (ranging 4.9–5.5 μg/mL·h) were far below the bioavailability of the one we tested (8.754 μg/mL·h). This can be probably explained mainly by the dissolution properties of the Miniactives® technology that allows more sustained plasma concentrations over time (until 12 h after dosing), as well as by the higher dose administered (100 mg) in our study.

When supplemented at 100 mg/day by oral formulation consisting of soya oil in soft gelatin capsule (Myoqinon® 100 mg CoQ10), CoQ10 achieved median plasma concentration of 2.5 mg/L (2500 μg/L) after a 2 month administration period [18], far above the median plasma level achieved at day 28 with Miniactives capsule (773.35 μg/L) administered at 100 mg/day in Group A. It is difficult to draw conclusions when comparing our results with those of Zita and coll. [18] due to the profound difference between the durations of oral administration of CoQ10 in the two studies (2 months vs. 1 month). In addition to this, no other pharmacokinetic parameters were reported by the authors of Reference [18]. Astonishingly, in another study [17], the same 100 mg/day CoQ10 soya oil in soft gelatin capsule (Myoqinon® 100 mg CoQ10) did not generate comparable pharmacokinetic results to those reported by Zita. After 20 days CoQ10 plasma concentration was approximately 0.9 mg/L (900 μg/L), a result much below the 2.5 mg/L reported in the study [18]. The mean plasma concentrations of CoQ10 after administration of oil/soft gel formulation used by Singh [17] are closer to those observed in our study after 28 days of CoQ10 100 mg/day administration (944.8 μg/L). Moreover, the author also highlighted the importance of the dosing strategy in addition to the daily dose. Divided dosages (2 × 100 mg) of oil/soft gel CoQ10 formulation caused a larger increase in plasma levels of CoQ10 (approximately >1.9 mg/L) than a single dose of 200 mg (approximately >1.3 mg/L) [17]. Our results show that, after 28 days of supplementation of Miniactives®, formulation 2 × 100 mg the mean plasma concentration was a little higher than 1200 μg/L.

With the aim to overcome the poor intestinal absorption, the bioavailability of a CoQ10 colloidal oral preparation was determined versus one oil-based formulation and two solubilizates in a single dose (120 mg) study. The mean Cmax colloidal formulation was the highest among the four formulations studied (6890 μg/L), as well as its AUC(0-10) (30,620 μg/mL·h). Nevertheless, the oil-based formulation and the two solubilizates showed rather high bioavailability (i.e., 4900, 6100, 10,700 μg/mL·h, respectively) [15]. When comparing the pharmacokinetic profiles of the four formulations with Miniactives® capsule, all achieved the peak of plasma concentration 4 h after the administration and maintained sustained levels afterwards. Once again, it is difficult to compare the results of different studies, since several factors may have contributed to the achievement of a particular bioavailability profile (e.g., study design, selection criteria, diet, analytical procedures, etc.). In the case of colloidal preparation, it is undoubted that it has favored the intestinal absorption of the conveyed CoQ10.

Very recently, a pharmacokinetic study highlighted the importance of inter-subjects variability in the plasma level of CoQ10 caused by significant variation of intestinal absorption of CoQ10 between subjects and irrespective of the oral formulation or molecular form administered [19]. The three commercial preparations tested (i.e., ubiquinol 150 mg capsule, ubiquinone 150 mg capsule and liposome ubiquinone 40 mg/2 sprays) showed plasma levels of CoQ10 ranging 5000–6000 μg/L at the 2 h interval, with ubiquinol preparation having the highest response, but a high inter-individual variation was observed for each preparation at every time interval. In our experience, this phenomenon has also been observed in both the single and multiple dose phase. After the single dose phase, the mean AUCt (8754.34 ± 2382.03 μg/mL·h) showed a very wide range (5277.62–15,394.54 μg/mL·h) and also mean Tmax had a high fluctuation, indicating different times of maximum concentration between subjects (coefficient of variation: 83%). During the multidose phase a very wide distribution of plasma concentration was observed, indicating quite different levels among individuals. Particularly, the exceptionally high pharmacokinetic values of a single subject (36-year-old man) randomized in the

group of 2 × 100 mg CoQ10 dose contributed to increase in the overall variability of pharmacokinetic results of the entire subject population. Despite the bioavailability values of CoQ10 of the two groups (Group A and Group B), they were not statistically different after exclusion of the outlier subject (*p* = 0.0548), however, there is evidence of a difference at limits of the significance threshold (*p* < 0.05) between the two groups. Probably, a clear difference did not emerge due to the small sample size studied.

During both single and multiple dose phases, males showed higher plasma CoQ10 concentration than females at each time point. CoQ10 baseline is naturally higher in men than in women [14,20], ranging 0.40–1.72 μmol/L (345.34–1484.94 μg/L) for males and 0.43–1.47 μmol/L (371.24–1269.11 μg/L) for females in European (Finnish) population [20]. Our results on baseline CoQ10 plasma levels (0 h) in both sexes are included in these normal ranges (males 759.79 ± 198.09; females 594.79 ± 168.63). During the multidose phase, the same pattern of plasma CoQ10 concentration was observed between genders (e.g., males 909.79 ± 241.54 μg/L, females 639.29 ± 203.81 μg/L at Visit 2; males 1173.32 ± 524.40 μg/L, and females 1061.03 ± 464.98 μg/L at Visit 5). In this study, the proportion between genders was unbalanced (i.e., 8 males/16 females). However, this disproportion did not appear to have influenced the plasma concentrations of CoQ10 according to the expected levels in males and females. In addition, another pharmacokinetic study reported the same numerical disproportion between the two genders, without any reported influence on the observed results [15]. Regarding the difference between genders in CoQ10 plasma concentrations, our results are in line with previous studies. The present experience and others in the literature support the conclusions that men can have better absorption and/or lower clearance than women [13].

The results presented in our bioavailability study suggest that Miniactives® timed release formulation of CoQ10 gradually released the active ingredient by diffusion, in a pre-determined time, thanks to a polymeric permeable and insoluble membrane coating each single particle, thus assuring a constant release. After having achieved the peak at 4 h, CoQ10 plasma concentrations did not undergo a sharp decrease and remained constantly high. The development of this technology was supported with the aim of creating an oral formulation able to ensure consistently high CoQ10 blood concentrations, useful for supporting a treatment strategy in the neuroprotection of RGC. In glaucoma, retinal neuroprotection can be significantly improved through maintenance of mitochondrial functions and survival of RGC by CoQ10, one of the most powerful antioxidant compounds. The potential clinical significance of this finding should be further evaluated.

In conclusion, based on the obtained results and on data available in literature regarding the expected average plasma levels of CoQ10, this exploratory study highlighted that both 100 mg OD or BID are safe and assure a plasma concentration of CoQ10 that remains high for the duration of the intake and that 100 mg COQUN® Miniactives® BID would be preferred than OD in reaching a higher plasma concentration of CoQ10. These positive results suggest that further studies are needed in order to investigate the antioxidative effects of COQUN® OS oral formulation in patients with specific diseases like glaucoma where the antioxidative effect of the CoQ10 is expected to be seen at the target organ.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2072-6643/11/3/527/s1, Table S1: Individual demographic variables—ITT population; Table S2A. Plasma concentration of CoQ10 by time (0, 1, 2, 4, 8, 12 h) during the single dose phase (V1)—ITT population, Table S2B. Individual plasma concentrations of CoQ10 (μg/L) in the single dose oral administration phase—ITT population; Table S3. Individual pharmacokinetic parameters of CoQ10 in the single dose oral administration phase—ITT population; Table S4. Individual plasma concentrations of CoQ10 (μg/L) in the multiple dose oral administration phase—ITT population; Table S5. Individual pharmacokinetic parameters of CoQ10 in the multiple dose oral administration phase—ITT population.

**Author Contributions:** Conceptualization, clinical study design and writing—original draft preparation, A.M. (Alessio Martucci) and D.R.-P.; plasma separation, performance of ELISA testing, collection of laboratory data, reading, amendment, and approval of the final version of the manuscript A.M. (Anamaria Manole) and D.R.-P.

**Funding:** This research and the APC were funded by VISUFARMA S.p.A., Via Canino 21, 00191 Rome–Italy.

**Acknowledgments:** CRO 1MED Via Campagna, 13 6982 Agno–CH managed the study.

**Conflicts of Interest:** A.M. (Alessio Martucci) is medical consultant for Visufarma S.p.A.; D.R.-P. and M.A. (Anamaria Manole) received a grant for the conduction of the study. The authors have no further conflicts of interest relevant to the content of this manuscript. The funders had no role in the collection, analyses, interpretation of data, in the writing of the manuscript, or in the decision to publish the results.

#### **References**


© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

### *Review* **Pharmacological Properties of** *Morus nigra* **L. (Black Mulberry) as A Promising Nutraceutical Resource**

#### **Sung Ho Lim and Chang-Ik Choi \***

College of Pharmacy, Dongguk University-Seoul, Goyang 10326, Korea; teruai0608@naver.com **\*** Correspondence: cichoi@dongguk.edu; Tel.: +82-31-961-5230

Received: 30 January 2019; Accepted: 18 February 2019; Published: 20 February 2019

**Abstract:** Mulberry plants belonging to the Moraceae family have been grown for the purpose of being the nutrient source for silk worm and raw materials for the preparation of jams, marmalades, vinegars, juices, wines, and cosmetics. *Morus nigra* L. (black mulberry) is native to Southwestern Asia, and it has been used as a traditional herbal medicine for animals and humans. In this article, recent research progress on various biological and pharmacological properties of extracts, fractions, and isolated active constituents from different parts of *M. nigra* are reviewed. *M. nigra* exhibited a wide-spectrum of biological and pharmacological therapeutic effects including antinociceptive, anti-inflammatory, antimicrobial, anti-melanogenic, antidiabetic, anti-obesity, anti-hyperlipidemic, and anticancer activities. *M. nigra* also showed protective effects against various human organs and systems, mainly based on its antioxidant capacity. These findings strongly suggest that *M. nigra* can be used as a promising nutraceutical resource to control and prevent various chronic diseases.

**Keywords:** *Morus nigra* L.; black mulberry; nutraceutical; pharmacological properties

#### **1. Introduction**

*Morus*, commonly known as mulberry, is the genus of a flowering plant belonging to the Moraceae family. They are widely distributed into subtropic regions of Asia (including Korea, Japan, China, and India), North America, and Africa [1]. In Asian countries, mulberry plants have been grown for the production of silk worms (*Bombyx mori* L.), because their leaves are a major and important nutrient source for silk worms [2]. Meanwhile, most European countries have usually used mulberry fruits to prepare jams, marmalades, vinegars, juices, wine, and cosmetic products [3]. Various parts of mulberry plants have also been used as traditional herbal medicines [4]. Diels-Alder-type adducts, flavonoids, benzofurans, stilbenes, and polyhydroxylated alkaloids are the most representative bioactive compounds identified from Sang-Bai-Pi (Chinese name for root barks of *Morus* species) [5]. Some previous review articles on *Morus alba* L. (*M. alba*), one of the most valuable plants rich in natural ingredients, have demonstrated that extracts, fractions and major constituents from *M. alba* exhibit numerous pharmacological activities such as antioxidant, anti-inflammatory, anticancer, antimicrobial, antifungal, skin-whitening, antidiabetic, anti-hyperlipidemic, anti-atherosclerotic, anti-obesity, cardioprotective, cognitive enhancing, hepatoprotective, anti-platelet, anxiolytic, anti-asthmatic, anthelmintic, antidepressant, and immunomodulatory activities [6–8].

*Morus nigra* L. (*M. nigra*), also called black mulberry, is native to Southwestern Asia. It has been grown throughout Europe and around the Mediterranean for centuries. Although biological and/or pharmacological activities of *M. nigra* have been relatively less studied compared to those of *M. alba*, several bioactive compounds isolated from *M. nigra* have also been used as herbal medicines for animals and humans due to their analgesic and anti-inflammatory effects [1]. Budiman et al. [9] briefly summarized chemical compounds isolated from various parts of *M. nigra* and their pharmacological activities. In this review article, we extensively covered recent research progress on biological and

pharmacological properties of *M. nigra* extracts, fractions, and active constituents, suggesting its potential and usefulness as a nutraceutical resource. Major biological and pharmacological therapeutic activities of *M. nigra* were summarized in Table 1.

#### **2. Antinociceptive Activity**

In 2000, de Souza et al. [10] firstly reported on the antinociceptive effect of morusin, the main prenylflavonoid of *M. nigra* isolated from acetonic extract of its root barks. Morusin showed a significant inhibitory effect on acetic acid-induced abdominal constriction responses and formalin-induced pain, and it also resulted in prolongation of the latency period in a hot plate test in mice. Because morusin is also purified from other mulberry plants, such as *M. alba* [11], *M. australis* [12] and *M. lhou* [13], this study result alone is insufficient to fully reflect the analgesic activity of *M. nigra*. Nine years later, Padilha et al. [14] investigated the antinociceptive effect of methylene chloride extract of *M. nigra* leaves in mice. Similar to the results of de Souza et al. [10], *M. nigra* leaves extract showed significantly and dose-dependently reduced acetic acid-induced writhing and formalin-induced pain and increased response latency period in a tail-immersion test and hot plate test without any acute toxicity when the dose of the extract was up to 300 mg/kg.

Two studies by Chen et al. [15,16] recently evaluated the antinociceptive properties of total flavonoid extracts and main active ingredients from fresh fruits of *M. nigra*. In the first study [15], total flavonoids from *M. nigra* showed dose-dependent decreases in the duration of formalin-induced pain-response behaviors. In the second study, three different mulberry fruits (*M. alba*, *M. nigra* and *M. mongolia*) were compared [16]. *M. nigra* fruits had more anthocyanin and flavonol contents than other species. The duration of the formalin-induced secondary pain phase (inflammatory phase) in the group treated with total flavonoid extract from *M. nigra* was significantly shorter than that in the control group. Reduced development of inflammatory cytokine interleukin-6 (IL-6) and an increased level of an anti-inflammatory cytokine IL-10 associated with the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and nitric oxide (NO) pathways were observed after treatment with *M. nigra* extract, suggesting the possible mechanism of its antinociceptive effects. Interestingly, the three main flavonoid ingredients (cyanidin-3-*O*-glucoside, rutin and isoquercetin) from *M. nigra* did not reduce the duration of formalin-induced pain individually, although they significantly decreased such duration when they were used as a mixture.

#### **3. Anti-Inflammatory Activity**

Inflammation is defined as a set of physiological defense mechanisms taking place in the body. However, inflammation is also considered an initial event of major chronic diseases such as cardiovascular, autoimmune, eye, age-related, neurodegenerative diseases, and cancers [17]. In this respect, inhibiting and controlling inflammatory responses in the human body can be one of fundamental approaches for treating chronic diseases.

As a follow-up research of a previous study on antinociceptive activity, Padilha et al. [18] evaluated the anti-inflammatory effects of methylene chloride extract of *M. nigra* leaves in male rats. *M. nigra* leaves extract significantly inhibited the volume of paw edema induced by intraplantar injection of carrageenan at a half-maximal inhibitory concentration (IC50) value of 15.2 mg/kg. *M. nigra* leaves also significantly inhibited the formation of granulomatous tissues in the chronic inflammation status using a cotton pellet-induced granuloma rat model (IC50 of 71.1 mg/kg). In the same year, Wang et al. [19] isolated three new compounds (mornigrol D, G and H) with six other known compounds (norartocarpetin, dihydrokaempferol, albanin A, albanin E, moracin M, and albafuran C) from the stem bark of *M. nigra* and assessed their anti-inflammatory activities by calculating the inhibition of releasing β-glucuronidase from rat polymorphonuclear leukocytes induced by platelet-activating factor. At a concentration of 10−<sup>5</sup> M, mornigrol D and norartocarpetin showed potent anti-inflammatory properties, showing inhibition rates of 65.9% and 67.7%, respectively. In 2014, Zelová et al. [20] investigated into the anti-inflammatory activities of two Diels-Alder adducts (soroceal

and sanggenon E) isolated from the root bark of *M. nigra*, by determining the attenuation of secretion of pro-inflammatory cytokines, tumor necrosis factor-alpha (TNF-α) and IL-1β, in lipopolysaccharide (LPS)-stimulated macrophages. Although sanggenon E significantly reduced the production of TNF-α compared to the vehicle control, both compounds failed to significantly affect the level of IL-1β.

Chen et al. [15] reported that the total flavonoid extract of *M. nigra* fruits can dose-dependently inhibit xylene-induced ear edema (edema rate 60.1% at a concentration of 200 mg/20 mL/kg) and carrageenan-induced paw edema (edema rate 9.5% at a concentration of 100 mg/20 mL/kg; 8.6% at a concentration of 200 mg/20 mL/kg) in mice. Levels of pro-inflammatory cytokines including IL-1β, TNF-α, NO, and interferon-gamma (IFN-γ) were also significantly decreased after the treatment of *M. nigra* fruit extract in mice with xylene-induced inflammation. In addition, *M. nigra* fruits extract significantly reduced levels of NO in LPS-stimulated RAW 264.7 cells without showing the cytotoxicity effect at the concentration of 50 to 100 μg/mL.

A very recent study [21] has shown that extracts of *M. nigra* pulps and leaves can improve survival rate and decrease the number of total leukocytes in bronchoalveloar lavage fluid in LPS-induced septic mice, indicating the reduction of inflammatory infiltrate in the lung. Although most hepatic and serum cytokine levels were not changed by the administration of *M. nigra* extracts, serum levels of TNF, an important mediator of sepsis, were significantly lower in the *M. nigra* extract-treated group than those in the septic animal group.

#### **4. Antimicrobial Activity**

Antibacterial activities of *M. nigra* leaves have been investigated in various organic fractions. Tahir et al. [22] reported that the ethyl acetate fraction of *M. nigra* leaves is active against four dental caries-causing bacterial strains: *Streptococcus mutans*, *Escherichia coli* (*E. coli*), *Staphylococcus aureus* (*S. aureus*), and *Bacillus subtilis* (*B. subtilis*). Also, the chloroform fraction showed antibacterial properties against *Pseudomonas aeruginosa* (*P. aeruginosa*) and *B. subtilis*, while the methanol fraction was only active against *B. subtilis*. No activity was observed for n-hexane or aqueous fraction. The inhibition rate of streptococcal biofilm formation (anti-adherence effect) by *M. nigra* ethyl acetate fraction was 87%. In another study conducted by Souza et al. [23], crude ethanol extract of *M. nigra* leaves exhibited bactericidal activities against *Bacillus cereus* (*B. cereus*), *Enterococcus faecalis* (*E. faecalis*), and *E. coli*, with minimal inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) less than 0.195 mg/mL for all. Potent antibacterial activities against *B. cereus* and *E. faecalis* were also observed for hexane, chloroform and ethyl acetate extracts (MIC values < 0.195 mg/mL for all). However, their measured MBCs were over 6 mg/mL. It was noted that chloroform extract exclusively showed a bactericidal effect against *Salmonella choleraesuis* (MIC and MBC value < 0.195 mg/mL, respectively). The antibacterial activities of the total flavonoid extract of *M. nigra* fruits were evaluated against three inflammatory pain-causing bacteria, *E. coli*, *P. aeruginosa* and *S. aureus*. Its fruit extract strongly inhibited all three strains, with MBC values of 2 mg/mL or less [16].

The antimicrobial activities of fresh juice of *M. nigra* fruits against five Gram-positive and three Gram-negative bacterial strains have been compared with conventional antibiotics [24]. Although 100 μL of *M. nigra* fruits juice produced generally smaller zones of inhibition (ranging from 9.98 to 19.87 mm) than other antibiotics treated at their standard doses, it showed a broad-spectrum antimicrobial effect against both Gram-positive and Gram-negative bacteria, having the highest inhibition against *P. aeruginosa*. Minhas et al. [25] investigated into the antimicrobial effect of five *M. nigra* fruits extracts classified by different solvents against 16 bacterial and 2 fungal strains in comparison with conventional antibiotics and antifungal agent nystatin. Ethanolic and acetone extracts of *M. nigra* fruits showed highly-sensitive inhibition (defined as 20 mm or more longer diameter of zone of inhibition) against *E. coli*, *S. aureus*, and *Neisseria* spp.; methanolic extract against *Klebsiella pneumoniae* and *Neisseria* spp.; and chloroform extract against *Serratia marcesscens*, *Staphylococcus epidermidis* (*S. epidermidis*), *P. aeruginosa*, and *S. aureus*. Similar to the results of Khalid et al. [24], *M. nigra* extracts had smaller zones of inhibition than those observed with conventional drugs.

In a recent study assessing antibacterial activities against two strains causing acne, *S. epidermidis* and *Propionibacterium acnes* (*P. acnes*), the ethanolic extract of *M. nigra* fruits had MIC values of 2.5% for both strains and MBC values of 2.5% and 5%, respectively [26]. As a follow-up approach, a comparative study was performed for extracts from three parts (stem barks, fruits and leaves) of *M. nigra* on their antibacterial effects against *S. epidermidis* and *P. acnes* [27]. *M. nigra* stem barks possessed the most potent antibacterial activities against both strains, with an MIC value of 4 mg/mL for *S. epidermidis* and 2 mg/mL for *P. acnes*. In addition, *M. nigra* stem barks extract induced nucleic acid, protein, and ion leakages and cellular membrane damages against *P. acnes*. These results suggest that the antibacterial effect of *M. nigra* stem bark is related to reduced cell membrane fluidity and bacterial cell wall destruction.

Mazzimba et al. [28] reported that six isolated constituents (oxyresveratrol, moracin M, cyclomorusin, morusin, kuwanon C, and a derivative of kuwanon C) from aerial parts of *M. nigra* show antibacterial activities against *S. aureus*, *B. subtilis*, *Micrococcus flavus*, *S. faecalis*, *Salmonella abony*, and *P. aeruginosa*, with morusin having the most potent activity against *B. subtilis* (MIC value 3.91 μg/mL).

Tuberculosis (TB), an infectious disease caused by *Mycobacterium tuberculosis* (*M. tuberculosis*), is one of the top 10 causes of death in the world. TB is a curable and preventive disease, but resistance against conventional antibiotic medications for *M. tuberculosis* has increased the number of cases of multidrug-resistant or extensively drug-resistant TB [29]. In this respect, demand for new medications with novel therapeutic targets such as protein tyrosinase phosphatases (PTPs) is growing [30,31]. Mascarello et al. [32] evaluated the anti-tuberculosis activity of Diel–Alder-type adducts from *M. nigra* root bark to determine their potential as candidates for *M. tuberculosis* PTP inhibitor. A total of eight compounds (Kuwanon L, G, and H; cudraflavanone A; morusin, oxyresveratrol; chalcomoracin; and norartocarpetin) were isolated from *M. nigra*. They all significantly inhibited *M. tuberculosis* PTP-B (Mtb PtpB) with IC50 values ranging from 0.36 to 8.42 μM. Further enzyme kinetic analyses for Kuwanon G and H, two of the most potent compounds, showed that both compounds competitively inhibited Mtb PtpB, with inhibitory constant (Ki) values of 0.39 ± 0.27 μM and 0.20 ± 0.01 μM, respectively. In addition, Kuwanon G inhibited the growth of *M. tuberculosis* inside macrophages by 61.3% at a non-cytotoxic concentration (10 μg/mL, corresponding to 14.4 μM of Kuwanon G), indicating that it is the most promising anti-tuberculosis constituent isolated from *M. nigra*.

Antimicrobial activity of *M. nigra* against *Candida* spp., the most common cause of fungal infections around the world [33], was assessed with aqueous and methanol extracts of its fruits, by using a disc-diffusion assay [34]. Of nine selected *Candida* spp., both extracts exhibited anticandidal effect against *Candida* (*C.*) *albicans*, *C. parapsilosis*, *C. tropicalis*, and *Geotricum candidum*, with lower MIC values observed for the methanol extract (0.625–2.5 mg/mL) than those for the aqueous extract (1.25–5 mg/mL).

#### **5. Anti-Melanogenic (Skin-Whitening) Activity**

Although melanin pigmentation in the skin is an important defense mechanism against ultraviolet radiation, abnormal melanin hyperpigmentation catalyzed by tyrosinase can cause several serious aesthetic problems [35–37]. As an anti-melanogenic strategy, tyrosinase inhibitors have become increasingly important for treating skin disorders associated with pigmentation and to improve skin-whitening.

Zhang et al. [38] investigated the inhibitory effect of 2,4,2',4'-tetrahydroxy-3-(3-methyl-2-butenyl) chalcone (TMBC) isolated from the stem of *M. nigra* on tyrosinase activity and melanin biosynthesis. TMBC dose-dependently and competitively inhibited mushroom tyrosinase-mediated L-dopa oxidation (IC50 value 0.95 ± 0.04 μM), which was more potent than kojic acid (IC50 value 24.88 ± 1.13 μM), a well-known skin depigmenting agent. Furthermore, TMBC significantly reduced the melanin content and cellular tyrosinase activity in B16 melanoma cells, although it increased mRNA levels of cellular tyrosinase. Zheng et al. [39] screened tyrosinase inhibitory properties of a total of 29 constituents isolated from roots of *M. nigra*. Among them, nine compounds (5'-geranyl-5,7,2',4'-tetrahydroxyflavone, steppogenin-7-O-β-D-glucoside, 2,4,2',4'-tetrahydroxychalcone, moracin N, kuwanon H, mulberrofuran G, morachalcone A, oxyresveratrol-3'-O-β-D-glucopyranoside and oxyresveratrol-2-O-β-D-glucopyranoside)

showed better tyrosinase inhibitory activities than kojic acid (IC50 value 46.95 ± 1.72 μM, with 2,4,2',4'-tetrahydroxychalcone having the highest activity (IC50 value 0.062 ± 0.002 μM, 757-fold lower IC50 than kojic acid). More recently, de Freitas et al. [40] reported that five different batches of standardized ethanolic extracts of *M. nigra* leaves all exhibited tyrosinase inhibitory activities, with IC50 ranging from 5.00 to 8.49 μg/mL.

Koyu et al. [41] tested the microwave-assisted extraction of fresh fruits of *M. nigra* in variable conditions for optimizing and maximizing tyrosinase inhibitory activity. Consequently, the highest tyrosinase inhibitory activity (IC50 value 1.44 mg/mL) was observed in the optimum microwave extraction system yielding the highest amount of anthocyanin content (13.28 mg/g cyanidin-3-glucoside equivalent), suggesting the important potential of anthocyanins on tyrosinase inhibition.

#### **6. Antidiabetic and Anti-Obesity Activity**

Diabetes mellitus is a chronic endocrine disorder characterized by hyperglycemia related to metabolic impairment of insulin production, secretion, and/or utilization. It is closely associated with the development of several important complications in cardiovascular, neurological and renal systems that can lead to increased morbidity and mortality in diabetic patients [42]. Various classes of antihyperglycemic agents are now available. However, some undesirable adverse effects such as hypoglycemia, gastrointestinal symptoms, weight gain and hepato-renal toxicity caused by the administration of these medications have been arousing interests on the discovery of new effective and safer naturally-occurring antidiabetic agents with different therapeutic pathophysiological mechanisms and targets [43–45].

*M. nigra* has also shown good antidiabetic effects on extracts and active constituents from some parts of this plant. Abd El-Mawla et al. [46] investigated the hypoglycemic efficacy of *M. nigra* leaf extracts and its cell suspension cultures treated with methyl jasmonate to induce accumulation of flavonoid contents in cell cultures. Extracts from *M. nigra* leaves dose-dependently decreased plasma glucose concentrations and increased insulin levels up to 500 mg/kg/day in streptozotocin (STZ)-treated diabetic rats. In addition, a slightly higher hypoglycemic effect was observed when rats were treated with extracts from cultured cells, indicating the additive action of flavonoids induced by methyl jasmonate. Hydroethanolic extracts of *M. nigra* leaves also significantly decreased serum fasting and 2-h glucose concentrations (at dose of 50 mg/kg) and increased serum insulin level (at dose of 10 mg/kg) in nicotinamide-STZ-induced type 2 diabetic rats [47]. Diabetes-induced changes in blood vessels may enhance the pathophysiological activity of metalloproteinases (MMPs). It is known that the inhibition of MMPs can improve insulin resistance and oxidative stress [48,49]. Araujo et al. [49] demonstrated the hypoglycemic potential of *M. nigra* leaves via reduction of expression and activity of MMP-2 in livers of diabetic rats. In addition, several phenolic compounds and isoprenylated flavonoids isolated from extracts of *M. nigra* twigs showed good antidiabetic activities, involving mechanisms of peroxisome proliferators-activated receptor gamma (PPARγ) activation [50] and α-glucosidase inhibition [51]. On the other hand, 3-week treatment of aqueous extract of *M. nigra* leaves failed to affect serum glucose levels in non-diabetic or diabetic pregnant rats [52].

Although there is no published report on the antidiabetic activity of black mulberry fruit yet, its effects on obesity, associated with increased risk of many chronic adverse health effects including cardiovascular diseases, dyslipidemia, non-alcoholic hepatic disease, cancer, and type 2 diabetes [53,54] have been evaluated by Fabroni et al. [55]. They demonstrated that 80% hydroethanolic freeze-dried extract of fruits of *M. nigra* had moderate total anthocyanin and total phenolic contents, with an IC50 value for pancreatic lipase inhibition at 6.32 ± 0.01 mg/mL.

#### **7. Anti-Hyperlipidemic and Anti-Atherosclerotic Activity**

Cholesterol is a lipid molecule that acts as a structural component of cell membrane modulating fluidity and permeability, and as a precursor for steroid hormone and bile acid synthesis [56]. At the same time, hypercholesterolemia, a typical type of hyperlipidemia characterized by excessive

accumulation of cholesterol in serum, is one of the crucial risk factors for coronary heart disease and atherosclerotic progression [57]. It has also been reported that reduction of low-density lipoprotein cholesterol (LDL-C) and improvement in levels of high-density lipoprotein cholesterol (HDL-C) can contribute to the anti-atherogenic condition [58,59].

Results from biochemical profile studies conducted by Volpato et al. [52] and Mahmoud [60] demonstrated that *M. nigra* extracts can decrease total cholesterol, triglyceride, LDL-C, and very low-density lipoprotein cholesterol (VLDL-C) levels and increase HDL-C in diabetic pregnant rats [52] and rats fed a high-fat diet [60]. Zeni et al. [61] evaluated the lipid-lowering effect of *M. nigra* leaf extract using Triton WR-1339-induced hyperlipidemic rats. The LDL-C level had significantly decreased after treatment with 100 mg/kg *M. nigra* infusion extract and HDL-C levels were restored in all groups treated with *M. nigra* extract at three different concentrations (100, 200 and 400 mg/kg), compared to those in the group only treated with Triton WR-1339. Atherogenic index and cardiac risk factor, indicators of likelihood of cardiovascular diseases associated with hyperlipidemia, were also decreased by *M. nigra* leaf extract. In another study by Jiang et al. [62], a high dose (210 mg/kg) of ethanolic extract of *M. nigra* fruit (EEBM) resulted in lowering mean body weight in rats fed a 6-week high-fat diet, which is comparable to the effect observed in the group treated with 5 mg/kg simvastatin. EEBM also dose-dependently improved serum lipid profiles, atherosclerosis indexes and lipid peroxidation compared to the control (high-fat diet-induced hyperlipidemic model) group. Histopathological changes in rat liver and thoracic aorta with reduction in the intima-media thickness of rat aortic arch after treatment with EEBM suggest that *M. nigra* fruit can effectively suppress the development and deterioration of atherosclerosis.

#### **8. Organ-Protective Activity**

#### *8.1. Neuroprotective Effect*

Turgut et al. [63] investigated the effect of *M. nigra* leaves extract on D-galactose-induced cognitive impairment and oxidative stress in mice. The results from the Morris water maze test showed significant and dose-dependent decreases in mean escape latency and time required to reach the target quadrant. Time spent in the target quadrant and number of times crossed the platform location were increased after the administration of lyophilized *M. nigra* extract, suggesting its potential neuroprotective role by preventing D-galactose-induced learning dysfunction and memory loss. *M. nigra* extract also showed DNA damage protection, reduced malondialdehyde (MDA) levels and augmented activities of three anti-oxidant enzymes, superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) in the serum, brain and liver of D-galactose-treated mice. These antioxidant and anti-aging properties are considered as one of key mechanisms of *M. nigra* in delaying neurodegenerative processes.

Dalmagro et al. [64] performed a forced swimming test (FST) and tail suspension test (TST) to evaluate antidepressant-like activities of *M. nigra* and its major phenolic compounds syringic acid in mice. Acute and subchronic oral administration of aqueous extract of *M. nigra* leaves significantly decreased the immobility time in FST and TST except for acute administration at a dose of 100 mg/kg extract in TST. Acute treatment with 1 mg/kg and 10 mg/kg and subchronic treatment with 1 mg/kg of syringic acid also significantly decreased immobility time in TST. Nitro-oxidative stress in the serum and brain was assessed by measuring thiobarbituric acid reactive substances (TBARS), nitrite, protein carbonyl content (PC) and non-protein thiol groups (NPSH) levels, with some inconsistent and controversial study results. A significant decrease of TBARS level was observed at acute doses of 3 mg/kg *M. nigra* extract. However, TBARS levels were oppositely increased at subchronic doses of 3, 10, and 100 mg/kg extract in the serum and at a subchronic dose of 3 mg/kg extract in the brain. Levels of nitrites in the serum were significantly decreased after subchronic administration of 10, 30 and 100 mg/kg extracts of *M. nigra* leaves, and nitrites in the brain were also decreased after subchronic treatment with the extract at doses of 30 and 100 mg/kg. In addition, subchronic treatment

with 1 mg/kg syringic acid resulted in significant changes in TBARS and nitrite levels in the serum and brain (all decreased, except TBARS level was increased in the brain). PC level was decreased after treatment with 30 mg/kg *M. nigra* extract and syringic acid. There was no significant change in NPSH level at all treatment conditions. Nevertheless, *M. nigra* leaf extract and syringic acid both exhibited good cell viabilities in hippocampal and cerebral cortex slices incubated with 100 mM glutamate, suggesting their proper neuroprotective effect against glutamate-induced toxicity.

#### *8.2. Hepatoprotective Effect*

Tag et al. [65] evaluated the hepatoprotective effect of the ethanolic extract of *M. nigra* leaves. With an IC50 value at 14.5 μg/mL in in vitro cytotoxicity to HepG2 (a well-differentiated human hepatocellular carcinoma) cell line, *M. nigra* leaf extract also significantly decreased levels of liver enzymes alanine aminotransaminase (ALT), aspartate aminotransaminase (AST), alkaline phosphatase (ALP), and lactate dehydrogenase (LDH) in male albino rats with methotrexate-induced hepatotoxicity. Hematosomatic index, defined as the ratio between liver- and body-weight and considered as an indicator for hepatic damage and liver inflammation, in the group co-treated with *M. nigra* extract and methotrexate, was also apparently decreased compared to that in methotrexate-only treated group. In histopathological studies, *M. nigra* treatment resulted in moderate enhancement in the hepatoprotection from methotrexate-related injury. Microscopic damage scores (hepatocyte degeneration, congestion, leukocyte infiltration, fibrosis, and total histopathology score) were significantly decreased when *M. nigra* extract was simultaneously administered compared to those in the group treated with methotrexate alone. In addition, methotrexate-induced progressive increases in collagen deposition of liver tissue were normalized by treatment with *M. nigra* leaf extract. Another study performed by Hassanalilou et al. [66] also showed that *M. nigra* leaf extract can lead to less fatty degeneration in liver tissue and smaller distension of hepatic cytoplasm due to fatty droplets in STZ-induced diabetic rats along with reduced fasting blood glucose, compared to glibenclamide, a well-known sulfonylurea antihyperglycemic agent.

Hepatoprotective activity of *M. nigra* fruits in carbon tetrachloride (CCl4, a well-known potent hepatotoxin)-treated HepG2 cells [67] and adult male Sprague-Dawley rats [68] have been reported. Extracts of *M. nigra* fruits dose-dependently and significantly reduced levels of hepatic enzymes AST, ALT and gamma-glutamyl transferase (GGT) compared to control (CCl4-treated group). At the same time, they significantly increased SOD and gluatathione peroxidase (GPx) enzymatic capacities and decreased expression levels and activities hepatic capsase-3 (a biomarker for cell apoptosis) and 8-oxo-2'-deoxyguanosine (a biomarker for oxidative stress) in rat liver tissues, indicating that the hepatoprotective effect of *M. nigra* fruits might be closely associated with its antioxidant activity [67,68].

#### *8.3. Renal-Protective Effect*

The effects of hydroalcoholic extract of *M. nigra* fruits on biochemical and histopathological changes in serum and kidney tissues have been evaluated in alloxan-induced diabetic rats [69]. Milder glomerular damage and no mesenchymal tissue expansion into renal glomerular vessels were observed in the group after 8 weeks of treatment with 800 mg/kg *M. nigra* fruit extract compared to those in diabetic and positive control (150 mg/kg metformin) groups. Although an increase in serum creatinine level was observed in the group treated with 800 mg/kg *M. nigra* extract, this group had lower serum glucose and urea levels compared to diabetic and positive control groups. These results suggest that *M. nigra* fruits have a protective effect on diabetic nephropathy and related kidney tissue injury. The extract of *M. nigra* leaves also significantly improved biochemical parameters reflecting kidney functions (serum creatinine, urea, and uric acid) and exhibited milder histopathological glycogen accumulation, fatty degeneration, and lymphocyte infiltration of renal convoluted tubules in STZ-induced diabetic rats compared to non-treated and glibenclamide-treated groups [66].

#### *8.4. Gastroprotective Effect*

Nesello et al. [70] reported that oral administration of methanolic extract from *M. nigra* fruits at a high dose (300 mg/kg) can protect gastric mucosa against acidified ethanol-induced acute gastric ulcer in female mice. This study result was confirmed by macroscopic and microscopic representative images, showing that the degree of epithelial damage in gastric tissue was decreased. To further investigate the underlying mechanisms for the gastroprotective effect, levels of lipid hydroperoxide (LOOH) and glutathione (GSH) in ulcerated gastric mucosa were quantified. *M. nigra* fruits extract prevented GSH depletion and promoted partial reduction of LOOH, suggesting its ability to ameliorate oxidative stress involved in the development of gastric injury by acidified ethanol. Because *M. nigra* fruits did not affect the activity of H+/K+-ATPase in their study, they have pharmacological advantages of being free from the risk of several side effects such as rebound acid hypersecretion, hypergastrinemia, gastric polyps, or atrophic gastritis [71] known to be associated with suppressed gastric acid secretion.

#### **9. Activity on Female Reproductive System**

De Queiroz et al. [72] investigated the estrogenic effect of *M. nigra* on the female reproductive system and embryonic development. Five different concentrations (25, 50, 75, 350, and 700 mg/kg) of hydroalcoholic extract of dried *M. nigra* leaves were administered in female Wistar rats for 15 days and their biological and clinical features were compared with the control group, in which distilled water instead of *M. nigra* extract was used as treatment. There were no significant differences in the number of deaths, clinical signs of toxicity, changes in food consumption, or body weight between groups, suggesting that *M. nigra* leaves did not cause maternal reproductive toxicity. Histological changes in ovarian structures, signs of edema, cystic follicles, retained oocytes, or thickened uterine epithelium were not observed. The number of corpora lutea, live fetuses, implants, resorptions, implantation, and pre- or post-implantation loss were not affected by the administration of *M. nigra* leaf extract either. Consequently, *M. nigra* exhibited no estrogenic effect or toxicity on the female reproductive system.

Another study conducted by Cavalcante et al. [73] showed that ethanolic extract of *M. nigra* fresh leaves at 0.1 mg/mL can improve percentages of follicular morphology, antrum formation, and fully grown oocytes, as well as the diameter of follicles compared to control group at 12 days after treatment. Furthermore, additive effects on follicular growth (described as follicular diameter increase and higher daily growth rate) were observed when *M. nigra* extract with supplemented medium and follicle-stimulating hormone (FSH) were used as co-treatment, indicating its capacity on ovine secondary follicle development.

#### **10. Anticancer Activity**

Cancer is a life-threatening disease state characterized by unregulated and permanent cell growth and proliferation [74]. Because of its ability to avoid programmed cell death (apoptosis) as one of the main driving forces for maintaining cancer cell proliferation, induction of apoptosis in cancer has been considered a reasonable strategy to treat cancer [75,76].

Morniga M, a mannose-specific jacalin-related lectin from the bark of *M. nigra*, can preferentially trigger the proliferation and activation of human T- and natural killer- (NK-)lymphocytes and dose-dependently induce cell death of α-CD3 activated T lymphocytes when compared with concanavalin A (Con A), a well-known mannose-specific legume leptin from *Canavalia ensiformis* [77]. Results from flow cytometry analysis have demonstrated that morniga M-induced cell death is probably associated with the apoptotic mechanism, suggesting the anticancer potential of morniga M via cell-death induction and immunomodulation as reported in previous studies with Con A [78,79]. Anticancer activities of morniga M were further investigated by Çakıro ˘glu et al. [80], in which they demonstrated that both *M. nigra* fruit extract and morniga M significantly and dose-dependently decreased cell viability against HT-29 cell line (human colorectal cancer). Another brief research by Qadir et al. [81] the demonstrated dose-dependent anticancer activity of n-hexane and aqueous

methanol extract of *M. nigra* leaves against HeLa cell line (human cervical cancer), with IC50 values of 185.9 ± 8.3 μg/mL and 56.0 ± 1.7 μg/mL, respectively.

Anti-proliferative and apoptotic effects of *M. nigra* fruits against several human adenocarcinoma cell lines have been reported [80,82,83]. Ahmed et al. [82] compared the anticancer effects between fresh and dried fruit extracts of *M. nigra* on MCF-7 cell line (human breast cancer). Study results have shown that both ethanolic extracts dose- and time-dependently inhibit cellular growth of MCF-7 cells; exhibit apoptotic morphological changes in their cytoplasmic membranes, cell bodies, and nuclei; induce DNA fragmentations and single strand breaks; and decrease mitotic indexes, with better pharmacological properties in fresh fruit of *M. nigra*. Turan et al. [83] evaluated the anticancer activities of *M. nigra* fruit extract on PC-3 cells (human prostate cancer). Dimethyl sulfoxide (DMSO) extract of *M. nigra* exhibited moderate cytotoxicity against PC-3 cells with an IC50 value of 370.1 ± 5.8 μg/mL. It significantly increased the cell number at G0/G1 phase and decreased the cell number at S phase, indicating that *M. nigra* fruits inhibited the progression of the cell cycle at the G0/G1 phase. *M. nigra* fruit extract at a high dose (666 μg/mL) significantly increased the number of necrotic, early apoptotic and late apoptotic cells compared to the untreated control group. It also dose-dependently decreased mitochondrial membrane potential and increased activities of caspase 3 and 7 (key mediators of apoptosis) in PC-3 cells [83].

#### **11. Antioxidant Activity**

Oxidative stress is characterized by an excessive increase in intracellular oxidizing species such as reactive oxygen species (ROS) involved in the loss of antioxidant defense capacity. It plays a critical role in various clinical conditions including aging, cancer, diabetes, atherosclerosis, chronic inflammation, neurodegenerative diseases, rheumatoid arthritis, human immunodeficiency virus (HIV) infection, ischemia and reperfusion injury, and obstructive sleep apnea [84,85]. Many researchers are interested in the antioxidant activity of naturally-occurring ingredients because phenolic compounds and flavonoids, the largest phytochemical molecules from natural resources, possess a variety of biological properties including antioxidant activity [86–89]. It has also been widely reported that mulberries are rich in anthocyanin constituents having remarkable antioxidant activities and other health benefits such as anti-inflammatory, antimicrobial, anti-obesity, antidiabetic, anti-hyperlipidemic, antihypertensive, cardioprotective (reduced risk of coronary heart disease and stroke), and anticancer effects [90–92].

Numerous researches have proven antioxidant properties of *M. nigra* with different in vitro methods, including DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging assay [15,23–26,28,61,70,93–113], ABTS (2,2'-azino-bis(3-ethylbenzothiazoline)-6-sulfonic acid) radical scavenging assay [15,91,94,99–103, 107,110,112–117], reducing power assay [15,99,113,118,119], superoxide anion radical (O2−) scavenging assay [15,118,120], hydroxyl radical (OH-) scavenging assay [15,113,120], lipid peroxidation assay [19, 52,60,62–64,70,121,122], antioxidant enzyme activity assay [21,49,52,62,63,67], β-carotene bleaching assay [23,119,123], ferric-reducing antioxidant power (FRAP) assay [24,85,91,95,100,102,104,107,110,111], protein carbonyl assay [49,64,96], GSH measurement [67,70,112], hydrogen peroxide (H2O2)-induced injury assay [113,121], NO radical scavenging assay [111,118], SOD-like activity [96], cupric-ion reducing antioxidant capacity (CUPRAC) assay [102,107,108,110], H2O2 scavenging assay [108,119], phosphomolybdenum assay [108,119], and ROS measurement [112].

#### **12. Other Pharmacological Activities**

Malik et al. [124] investigated the cardiovascular activity of aqueous methanolic extract of *M. nigra* fruit in frogs. Treatment of *M. nigra* fruit extract showed significant and dose-dependent decreases in heart rate without direct effects on the contractility of frog's heart. Results of phytochemical analysis revealed the presence of cardiac glycosides in *M. nigra* fruit, along with other active constituents including saponins, alkaloids, phenolic compounds, and flavonoids.

Crude extract and fractions of *M. nigra* fruits exhibit both in vitro and in vivo prokinetic, laxative, and antidiarrheal effects [125]. *M. nigra* extract significantly promoted the transit of charcoal meal through the small intestine, increased gastric emptying rate and the mean number of wet feces, and decreased

castor oil-induced diarrhea in mice. In in vitro studies, chloroform and petroleum ether fractions of *M. nigra* fruits dose-dependently inhibited carbachol- and potassium ion-induced contractions of rabbit jejunum while aqueous and ethyl acetate fractions showed stimulatory effects on guinea-pig ileum. Suppression of maximum responses of acetylcholine and calcium ion (Ca2+) by *M. nigra* fruits was also observed, and most gastrointestinal effects were conversely affected by concomitant administration of atropine, suggesting that the underlying mechanisms of these prokinetic, laxative, and antidiarrheal activities might be associated with cholinergic control and Ca2+ channel antagonism [125].

Fahimi and Jahromy [126] described the effects of *M. nigra* fruit juice on levodopa-induced dyskinesia in mice with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinson's disease. After 14 days of levodopa treatment, administration of 10 or 15 mL/kg of *M. nigra* fruit juice significantly decreased abnormal involuntary movement scale (AIMS) scores compared to levodopa treatment only.

#### **13. Drug-Food Interaction and Toxicity**

Food ingredients can cause drug-food interactions, most of which are pharmacokinetic interactions associated with the alteration in activities of drug-metabolizing enzymes or drug transporters [127]. A brief experimental report by Kim et al. [128] demonstrated that the fruit juice of *M. nigra* has a potent inhibitory effect of human liver microsomal cytochrome P450 3A (CYP3A) activity, with IC50 values for midazolam (a probe drug for CYP3A) 1'-hydroxylation of 2.96 ± 0.33% (*v*/*v*, with 20-min preincubation) and 6.22 ± 0.47% (no preincubation). Because approximately 30% of clinically used drugs including macrolide antibiotics, antiarrhythmics, benzodiazepines, immune modulators, human immunodeficiency virus (HIV) antivirals, antihistamines, calcium channel blockers, and statins are metabolized by CYP3A [129,130], concomitant intake of CYP3A substrates with *M. nigra* fruit can lead to an increase in plasma drug exposure.

Figueredo et al. [131] assessed the acute and subacute toxicities of *M. nigra* leaves extract in Wistar rats. A single or 28-day oral dose of ethanolic extract of *M. nigra* leaves did not cause any adverse effects. It did not induce abnormal behaviors or mortality. *M. nigra* extract resulted in some significant but non-toxic changes in biochemical profiles (decreased urea and AST in males; decreased total cholesterol and AST in females) and leukocyte parameters (increased neutrophils in males; decreased white blood cell in females). *M. nigra* leaves did not affect lipid peroxidation and changes in renal and hepatic CAT enzymatic activities.


**Table 1.** Summary of major biological and pharmacological therapeutic activities of *M. nigra*.


**Table 1.** *Cont.*

a, E, extract; F, fraction; C, isolated compound; J, juice. b, Cell suspension cultures of *M. nigra* extract were used.

#### **14. Conclusions**

*M. nigra*, especially its leaf and fruit parts, exhibited various pharmacological properties including antinociceptive, anti-inflammatory, antimicrobial, anti-melanogenic, antidiabetic, anti-obesity, anti-hyperlipidemic, and anticancer activities. *M. nigra* also showed protective and therapeutic effects on the central nervous system, liver, kidney, gastrointestinal tract, and female reproductive system. Most of these features were attributable to its antioxidant capacity due to abundant phytochemical constituents such as polyphenols, flavonoids and anthocyanins. These findings suggest that *M. nigra* can be used as a promising nutraceutical resource to control and prevent various chronic diseases. Given that most researches are performed in vitro and in animal models, further studies at the clinical level are required to establish the efficacy and safety of *M. nigra* in the human body.

**Author Contributions:** S.H.L. was responsible for collecting and summarizing literature data. C.-I.C. wrote and edited the manuscript.

**Funding:** This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (No. 2016R1D1A1B03933963).

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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