The magnitude of systolic blood pressure (SBP) response to acute change in dietary NaCl intake, the salt-sensitivity of blood pressure, increases with advancing age [4]. Specific determinants of the greater blood pressure responsiveness to dietary NaCl observed in older subjects remain to be identified. It has been proposed that salt ingestion results in an increase in plasma volume and natriuresis. It has been postulated for some time that endogenous substances are stimulated by increased Na intake and increase natriuresis by inhibiting renal tubular Na exchangers to lower the renal reabsorption of filtered sodium. The age-associated differences in circulating endogenous Na/K-ATPase inhibitors may be implicated in the age-associated increase in SBP and increased salt sensitivity of SBP in the elderly. Anderson et al. were the first to demonstrate in normotensive *humans* that following a change from a low to a high salt diet, a sustained increase in MBG synthesis occurs, and renal fractional sodium elimination increases and correlates directly with increased urinary MBG excretion. In contrast to the sustained increase in MBG synthesis on high salt diet, ouabain levels in these subjects increased only transiently [52].

#### **7. Endogenous Ouabain and Other CTS**

Ouabain is another cardiotonic steroid demonstrated in *human* and animal plasma. In *humans* it does not increase sodium excretion, but it does have a role in the adaptation to both sodium depletion and loading. Although a few studies have shown that high salt loading in normotensive *rats* stimulates the release of ouabain, other experiments performed in *dogs*, *rats* and *humans* did not confirm these findings. In 180 patients with untreated hypertension, plasma levels of endogenous ouabain did not change during 2 weeks of salt loading, but increased following 2 weeks of sodium depletion [53]. Recent studies indicate that endogenous ouabain might act as a central mediator of salt - sensitive hypertension. In Dahl salt-sensitive *rats*, an important interaction seems to occur between brain and peripheral cardiotonic steroids. After acute or chronic salt-loading, a transient increase in circulating endogenous ouabain precedes a sustained increase in circulating marinobufagenin concentration [54]. This observation led to the postulate that endogenous ouabain acts as a neurohormone, triggering release of MBG, which in turn increases in cardiac contractility, peripheral vasoconstriction and natriuresis by inhibiting the Na/K-ATPase. More recently it was demonstrated that, similar to observations in Dahl salt-sensitive *rats*, normotensive *humans* on increased salt intake exhibit a transient

increase in urinary endogenous ouabain excretion, which precedes a more sustained increase in renal MBG excretion [52].

Experiments in Milan hypertensive *rats*, which carry a mutation in the cytoskeletal protein adducin gene and exhibit increased circulating levels of endogenous ouabain, administration of the digoxin derivative rostofuroxin antagonized the effects of ouabain and lowered blood pressure [55]. The experimental data are promising and led to a clinical trial aimed to show the hypotensive effects of rostofuroxin in *humans*. The results in *humans*, however, could not demonstrate the blood pressure lowering effects after rostafuroxin administration [56].

There is substantial uncertainty as to whether the "endogenous ouabain" is indeed identical with the plant derived ouabain [57]. Although adrenals are supposed to be the source of the endogenous ouabain, the details of the adrenal biosynthetic pathway remain to be defined. A large portion of the data supporting the presence of "endogenous ouabain" is based on immunodetection. Cross-reactivity with similar compounds is an important issue in these methods. Some authors; however, failed to detect any measurable amount of true ouabain using state-of-the-art mass spectrometry [58]. This suggests that the "endogenous ouabain" may differ slightly from the plant ouabain. Further research is definitely needed in order to determine the exact structure of the compound referred to as "endogenous ouabain". Oubain-like immunoreactivity has been localized mainly to neuronal cells, especially hypothalamus [59]. In contrast, marinobufagenin immunoreactivity has been detected primarily in adrenals. It has been hypothesized that endogenous ouabain in the central nervous system responds to increased sodium load and increases sympathetic nervous activity resulting in hypertension [59].

Other CTS have been identified in mammalian tissues: marinobufotoxin [60], telocinobufagin [61], digoxin [62]. It is not known whether different CTS have different roles or are they different metabolites of a single active compound.

#### **8. Summary**

High dietary salt intake is a cause of elevated blood pressure and cardiovascular risk. However, it was demonstrated that even if the blood pressure did not increase on high salt diet, organ damage may still occur. Both effects are mediated (among other mechanisms) by endogenous digitalis-like cardiotonic steroids (Figure 2). They are released in order to maintain body sodium and act on the NaK-ATPase not only blocking the pumping mechanism but also triggering cellular responses leading to fibrosis.

#### **9. Future Perspectives**

Interfering with this pathway may present a new therapeutic target for treating hypertension and cardiovascular disease. Much work is needed before drug development is possible. Antibodies that bind cardiotonic steroids are not useful for long-term treatment of hypertension and cardiovascular events, although they could be useful in short-term situations like preeclampsia. Exact molecular mechanisms in CTS biosynthesis and their regulation will be studied further. Finding a way to influence differently the Na/K-ATPase blocking and signaling functions would be a major step forward in developing new medications in this pathway.

**Figure 2.** A possible mechanism of salt-induced hypertension and organ damage in humans. NaCl loading stimulates brain endogenous ouabain. Endogenous ouabain in the brain activates the local renin-angiotensin system (RAS) as well as sympathetic nervous system (SNS). These actions stimulate renin-angiotensin system in adrenal cortex and release of adrenocortical marinobufagenin (MBG). MBG is secreted in order to facilitate natriuresis, but at the same time MBG induces vasoconstriction which increases blood pressure and promotes fibrosis leading to permanent heart, kidney, and arterial damage and dysfunction.

**Funding:** The study was financed by a grant #UMO-2013/09/B/NZ5/02489 from the National Science Centre Poland.

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

#### **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* **Relationship of Circulating Irisin with Body Composition, Physical Activity, and Cardiovascular and Metabolic Disorders in the Pediatric Population**

**Leticia Elizondo-Montemayor 1,2,3,\*, Gerardo Mendoza-Lara 1,2, Gustavo Gutierrez-DelBosque 1,2, Mariana Peschard-Franco 1,2, Bianca Nieblas 1,2 and Gerardo Garcia-Rivas 1,3,\***

Received: 24 October 2018; Accepted: 10 November 2018; Published: 23 November 2018

**Abstract:** Exercise-induced irisin, a recently discovered myokine, has been linked to insulin resistance, obesity, and other diseases in adults; however, information in children is scarce and contradictory. We analyzed the limited evidence of irisin's effects in children and adolescents, and its association with body composition, exercise training, cardiovascular risk factors, and metabolic diseases, as well as the results of dietetic interventions. Both positive and negative correlations between irisin concentrations and body mass index, fat mass, fat-free mass, and other anthropometric parameters were found. Likewise, contradictory evidence was shown associating irisin plasma levels with cardiovascular and metabolic parameters such as glucose, insulin resistance, and cholesterol and other lipid and fatty acid plasma levels in healthy children, as well as in those with obesity and the metabolic syndrome. Gender, puberty, and hormonal differences were also examined. Furthermore, important contradictory findings according to the type and duration of exercise and of dietetic interventions in healthy and unhealthy subjects were demonstrated. In addition, correlations between mother–infant relations and circulating irisin were also identified. This review discusses the potential role of irisin in health and disease in the pediatric population.

**Keywords:** irisin; pediatric; children; cardiovascular disease; nutrition; diet; body composition; metabolic syndrome; obesity, neonates

#### **1. Introduction**

Böstrom et al., 2012 [1] discovered irisin as a myokine derived from fibronectin type III domain-containing protein 5 (FNDC5) that is regulated by PGC1-α in mice. PGC1-α has several effects related to energy metabolism, including the activation of PPAR-γ that regulates the expression of the uncoupling protein 1 (UCP1) and thermogenesis in brown adipose tissue. It also stimulates the expression of messenger RNA (mRNA) of FNDC5, a muscle gene product that encodes a type 1 membrane protein. Irisin is a 112-amino acid polypeptide that is proteolytically cleaved from FNDC5, which undergoes glycosylation and is then secreted into the bloodstream. The original findings of Böstrom et al., 2012 [1] showed that exercise in humans induced circulating irisin, which activated browning and thermogenic genes in white adipose cells through UCP1, while it downregulated genes

involved in white fat development. They hypothesized that irisin could play a role in increasing total body energy expenditure, reducing body weight, and improving obesity-related insulin resistance [1].

More recent information indicates that irisin is also an adipokine with endocrine and autocrine functions secreted by white adipose tissue, and to a lesser extent, visceral adipose tissue (VAT) in the subcutaneous adipose tissue (SAT) [2]. Furthermore, irisin has also been found to be secreted by muscle. The expression of FNDC5 in muscle is related to SAT and VAT irisin levels and to the expression of FNDC5 and UCP1 genes in SAT. Muscle expression of the FNDC5 gene was 200-fold higher than that of adipocytes, indicating a relationship between muscle and adipose tissue functions in metabolic diseases. The correlation between irisin levels and FNDC5 expression in adipose tissue demonstrates a positive feedback mechanism for autocrine or paracrine production of irisin by adipose tissue, which further increases its capacity to metabolize glucose and fatty acids [3] (Figure 1).

**Figure 1.** Irisin expression and function. Irisin is a myokine cleaved from fibronectin type III domain-containing protein 5 (FNDC5). Its expression is regulated by PGC1-α and it has been found to be secreted by adipose tissue and muscle. Irisin activates browning and thermogenic genes in white adipose cells through uncoupling protein 1 (UCP1), and downregulates genes involved in white fat development. PGC1-α: Peroxisome proliferator-activated receptor gamma coactivator 1-alpha. +: Positive influence.

Research has focused on finding mechanisms to explain the influence of irisin in the regulation of obesity, cardiovascular risk factors, metabolic syndrome (MS), and other related diseases. Irisin, named after the Greek messenger goddess Iris, has been linked to insulin resistance, obesity, exercise training, and cardiovascular and metabolic diseases in adults; however, the information on its role in children is scarce and contradictory. In this review, we discuss current knowledge in the pediatric population concerning irisin. The analysis also includes findings according to the type and duration of exercise in health and disease, those of dietetic and nutrition education interventions, as well as the influence of gender, puberty, and hormonal status on irisin plasma levels. In addition, the correlations between irisin and mother–newborn relationships are identified, along with the findings of irisin levels in plasma and tissues in other diseases in the pediatric population.

#### **2. Association of Circulating Irisin with Body Mass Index and Body Composition**

The associations of circulating irisin with body mass index and body composition parameters are shown in Table 1.

**Table**

*Int. J. Mol. Sci.* **2018**, *19*, 3727

(obese), OW (overweight),

correlation), −

(negative correlation), 0 (no correlation).

 SBP (Systolic blood pressure), UW

(underweight),

 vBMD (volumetric bone mineral density), WC (waist

circumference),

 WHR

(waist–hip ratio), + (positive

#### *2.1. Body Mass Index And Anthropometric Parameters*

The relationships between irisin, body mass index (BMI), and anthropometric parameters are still not completely understood. Literature findings have shown different correlations. Most clinical studies in adults have observed a positive correlation between irisin levels, weight, and BMI at both ends of the body weight spectrum [15–17]. Additionally, positive associations between irisin and body fat, waist circumference (WC), waist-to-hip ratio, and muscle mass have also been found [11,18,19].

However, the findings in children still differ substantially. In a study in Mexican children aged 2–6 years, plasma irisin levels were lower in the underweight group compared with the normal weight and obese groups. Irisin levels correlated positively with WC and BMI percentile, but after multiple linear regression analyses, the correlation remained only for the latter.

Irisin levels have also been found to be higher in obese children compared with healthy children [5,20]. Other studies have shown a positive relationship of circulating irisin with BMI and waist-to-hip ratio in Turkish children [7] and with BMI and WC in Korean adolescents [8]. Elevated irisin was independently associated with the risk of obesity even after adjusting for age, sex, physical activity, puberty status, tryglicerides, low density lipoprotein-c (LDL-c), and HOMA-IR [8]. In addition, both before and after a physical activity intervention program, irisin levels have been shown to have a positive association with BMI [6].

In contrast, in prepubescent Korean children, those with obesity tended to exhibit a lower irisin concentration compared with normal-weight children [9]. There was also a significant inverse correlation between irisin and both BMI and WC; although after adjusting for age and gender, this relationship remained significant only for BMI in the normal-weight group, but not in the overweight/obese group. However, in a study that included obese and normal-weight children, irisin was significantly correlated with BMI at baseline, but after a lifestyle intervention program, changes in BMI were not related to changes in irisin levels [10], concluding that during childhood irisin levels are not related to weight status.

#### *2.2.  One study showed no correlation between irisin levels and FFM [7], while in others, a negative association was found between irisin and FFM in German children and adolescents [11], as well as in amenorrheic athletes (AAs), though not in eumenorrheic athletes (EAs) [12]. In contrast, other researchers have demonstrated a positive correlation between irisin and FFM [8]. Furthermore, in another study, the positive correlation between circulating irisin and FFM was lost after multiple regression analyses, while there was no relationship with muscle mass [4].

#### *2.3.  Jang et al., 2017 [8] considered body fat mass to be the most important independent factor in this relationship. However, no correlation was found between irisin plasma levels and total fat mass in Mexican children [4], or in athletic and non-athletic lean female adolescents [12].

These contradictory findings in children and adolescents have been attributed to the different body composition of children compared with adults and to the variations that occur during growth development and puberty resulting from the interplay between total fat mass, muscle mass, and the fat/muscle mass ratio. The lower irisin levels shown in underweight children might reflect an adaptive response to conserve energy, while irisin could play an ambiguous role in people who are obese. The higher levels in obese children suggest a compensatory role to increase subcutaneous brown adipose tissue and energy expenditure, and to improve obesity-related insulin resistance. Additionally, the higher irisin levels observed in obese people might be attributed to irisin resistance.

#### *2.4. Bone Mineral Density*

Irisin also appears to play a role in metabolic bone health. Several biomarkers secreted by adipose tissue, skeletal muscle, or bone may affect bone metabolism and bone mineral density (BMD). In adults, negative associations between irisin levels and BMD have been found in patients with previous osteoporotic fractures [21] and with an increased risk of hip fractures [22], probably due to the positive influence of irisin in bone quality, but not in bone mass [23].

In contrast, in a study of prepubescent Finnish children, irisin was positively and independently associated with BMD after adjusting for age and sex, and after controlling for lean and fat mass [13]. Similarly, a positive association between irisin levels and spine, femoral neck, and whole body bone density *Z*-scores, total and trabecular volumetric-BMD, and strength estimates has also been demonstrated in adolescent female athletes [12]. Thus, it is possible for irisin to induce the metabolic effects of brown adipose tissue on bone strength [24] and cortical thickness [25], as previously reported.

#### **3. Association of Circulating Irisin with Physical Activity, Exercise Training and Dietetic Interventions**

Table 2 shows the associations of circulating irisin with physical activity, exercise training, and dietetic interventions.

Studies in children concerning the changes in circulating irisin levels induced by physical activity have demonstrated inconsistent results and certain limitations. Regular, moderate physical activity appeared to maintain higher irisin levels in normal-weight adolescents compared with their sedentary counterparts; although this difference was not observed in overweight/obese adolescents [8]. However, another study showed a negative correlation between aerobic exercise and irisin levels in Mexican children alongside the entire BMI spectrum [4].

Varying results have also been demonstrated in dietetic, nutrition education, and physical activity intervention studies. Evidence suggests that acute intervals of aerobic physical activity seem to increase irisin concentrations in subjects. This was confirmed after observing a 2.23-fold increase in irisin levels after as little as 15 min of intense ergometer activity in normal-weight and obese German children [11]. A similar result was observed in the EXIT intervention trial in obese Canadian adolescents [26]. Circulating irisin was observed to increase by 60% after one 45-min session of aerobic exercise, but no change was observed after one 45-min session of weight-training exercise.

Long-term exercise and diet counseling seem to induce contradictory results. Irisin concentrations were found to be higher after one year of diet counseling and a combined endurance and resistance physical activity intervention program in overweight and obese German children, which resulted in decreased BMI [27]. No correlation between changes in BMI and irisin concentration was found, concluding that irisin levels were the direct result of exercise, and they were not influenced by changes in BMI. Similarly, in another one-year nutrition education and physical activity intervention consisting of a weekly low-intensity program, irisin levels were higher in obese children without significant weight reduction, but no changes in irisin levels were observed in obese patients with significant weight loss [10]. The authors suggested that unidentified confounders could explain these findings.

In contrast, after a 4 to 6-week nutritional education, psychological counseling, and daily exercise intervention that resulted in significantly lower BMIs, although inconsistent changes in irisin concentration were observed, most of the patients displayed decreased irisin levels [11]. The same authors evaluated a 3-year longitudinal comparison between children who performed regular, increased, or competitive levels of physical activity and found no difference in irisin levels among the groups. Similarly, a non-significant tendency towards decreased irisin levels was observed in healthy children after an 8-month physical activity program consisting of five weekly sessions of 25 min of moderate intensity exercise compared with overweight/obese children [6].

**2.**Associationofcirculatingirisinwithphysicalactivityandexercisetraininganddieteticinterventions.

WC (waist

circumference),

 + (positive correlation), −

(negative correlation), 0 (no correlation).

Finally, AAs aged 14–21 years showed lower irisin levels than EAs and non-athletes (NAs), even after controlling for age, body fat, and lean mass. Irisin concentration correlated with higher resting energy expenditure in all subjects. No difference was found between EAs and NAs, suggesting that irisin response is an adaptive reaction to preserve energy by decreasing resting energy expenditure and brown adipogenesis in AAs [12].

Interpretation of the results of studies concerning the association between irisin and physical activity is difficult because of the different physical activity regimens in regard to intensity, duration, regularity, number of sessions, the tools used to measure such activity, the combined dietetic interventions, and the studied pediatric populations. Irisin concentrations appear to increase after acute bouts of aerobic exercise, but not after long-term programs, especially in obese populations. Thus, the original hypothesis by Böstrom et al, 2012 [1] that irisin increases energy expenditure and decreases weight status in adults could apply to short bouts of exercise in normal-weight children and adolescents. The possibility that during long-term exercise programs, the body adapts to irisin thermogenesis has to be considered. As irisin seems to not increase in obese children even after weight loss, the loss of muscle mass that occurs during weight reduction might be responsible for the lack of change in irisin concentrations.

#### **4. Association of Circulating Irisin with Cardiovascular and Metabolic Alterations**

The association of irisin plasma levels with metabolic changes in insulin resistance, glucose, triglycerides, cholesterol, fatty acid composition, and other variables is shown in Table 3.

#### *4.1. Insulin Resistance and Glucose Regulation*

The relationship between circulating irisin and insulin resistance and impaired glucose metabolism in children is not completely understood. In a cross-sectional study in healthy children from Saudi Arabia, a negative correlation between plasma glucose and irisin was observed, but after adjusting for gender, this correlation remained significant in girls only [28]. In contrast, in obese pediatric populations, several studies have demonstrated a positive correlation of irisin levels with insulin resistance and glucose levels. In two Turkish cross-sectional studies, circulating irisin exhibited a positive correlation with glucose, insulin levels, and HOMA-IR in obese children compared with normal-weight ones [5,7].

However, in a cohort of overweight/obese and normal-weight Brazilian children, irisin levels showed a positive correlation with glucose and insulin levels and with HOMA-IR in both groups, though this correlation remained significant only for insulin after multiple logistic regression analyses [29]. Likewise, no significant differences in serum irisin levels between obese children with and without insulin resistance were demonstrated in Italian children [20]. Both studies attributed the lack of observed differences to the small number of subjects analyzed. Contradictory results were shown between two different longitudinal weight loss interventional studies in obese German children. In a study by Reinehr et al, 2015 [10], irisin levels were found to be higher in obese children with impaired glucose tolerance compared with those with normal glucose tolerance at baseline. Additionally, positive correlations were found between irisin concentration and insulin levels, HOMA-IR, and glucose tolerance tests. Although the positive correlations persisted after the intervention, irisin levels were not associated with changes in BMI; rather, a correlation was observed in children entering into puberty, probably due to the effects of insulin resistance related to hormonal changes. In contrast, a study by Bluher et al., 2014 [27] found a 12% increase in irisin levels after weight loss in overweight/obese subjects, but no correlation was found between irisin and insulin levels, HOMA-IR, BMI, or fasting glucose levels either at baseline or after the intervention.

Regarding the role of irisin in type 1 diabetes mellitus (T1DM) in children and adolescents, an Italian cross-sectional study found irisin levels to be higher in T1DM patients than in controls. Circulating irisin was even higher in patients with subcutaneous insulin infusion compared with the ones on multiple daily injection treatments. Furthermore, irisin showed a negative relation with HbA1c%, serum glucose, and years since the diagnosis of T1DM [14].

**Table 3.** Association of circulating irisin with cardiovascular risk factors and metabolic alterations.

(oral glucose tolerance test), OW (overweight), PBF (percent body fat), SBP (systolic blood pressure), TC (total cholesterol, + (positive correlation), − (negative correlation),correlation).

#### *Int. J. Mol. Sci.* **2018**, *19*, 3727

Because irisin levels were negatively correlated with glucose levels only in healthy girls, glucose could be an independent predictor of circulating irisin, attributing the difference between genders to dissimilarities in circulating hormone levels, or to the difference in brown adipose tissue quantity. Additionally, entry into puberty rather than BMI might be responsible for increased levels of irisin. Despite the contradictory correlations of irisin concentration with insulin resistance and glucose levels in the obese population, irisin has been proposed as a marker to differentiate obese children from normal-weight children. The increased irisin levels observed in obese subjects may represent the body's compensation mechanism for the insulin resistance observed in this population by increasing insulin sensitivity. On the other hand, the increased irisin concentration may reflect a state of irisin resistance. The findings in T1DM children suggest that better metabolic control is related to higher irisin levels in this pediatric population.

#### *4.2. Cardiovascular Risk Factors and the Metabolic Syndrome*

Previous studies have reported a link between irisin and the MS in adults, but only a few studies have evaluated this relationship in the pediatric population. A significant positive correlation exists between irisin levels and BMI, WC, triglyceride levels, systolic blood pressure (SBP), and diastolic blood pressure (DBP); but an inverse correlation with HDL-c levels were observed in a study by De Meneck et al., 2018 [29]. However, after multiple regression analyses, the relationship remained significant only for WC. Similarly, a cross-sectional study by Jang et al., 2017 [8] found irisin levels to be positively correlated with SBP, WC, triglycerides, fasting plasma glucose, HOMA-IR, and LDL-c. Higher irisin levels increased the risk for obesity and MS by two-fold, even after adjusting for age, sex, physical activity, and puberty, but after adjusting for BMI, this odds ratio was lost. A positive correlation between circulating irisin levels and branched and aromatic amino acids was also found. The authors suggested that the metabolic actions of irisin start during childhood and that beta cell dysfunction and evolution towards metabolic diseases are driven by the interplay between circulating irisin and branched-chain amino acids, which highly influence adiposity, lipids, and glucose.

In contrast, a cross-sectional study in prepubescent children found circulating irisin to be positively correlated with SBP and DBP only, but an inverse correlation with other components of the MS was demonstrated. After adjustments, irisin concentrations were found to be significantly lower in overweight and obese children with the MS compared with those without the MS. The authors proposed an irisin concentration of 15.43 ng/mL as a cutoff value for MS distinction, suggesting that irisin might be used as a biomarker for the MS [9].

The explanations for the contradictory role of irisin in metabolic diseases in children have yet to be clarified. Although myocytes are responsible for exercise-induced irisin secretion, in the context of increased adiposity, fat cells may be the primary source of high circulating irisin observed in some obese individuals with the MS. Increased fat mass may stimulate irisin production as a means to counteract new set points in energy balance. Conversely, a decrease in adipose tissue browning in obese individuals with the MS may be related to lower circulating irisin. Notably, in studies that showed positive associations between the MS and irisin, only anthropometric parameters such as BMI and WC, but not metabolic parameters, remained significant after adjustments, supporting the role of adipose tissue and its association with irisin in children. Furthermore, although it has been suggested that circulating irisin might regulate energy expenditure in adults with altered glucose metabolism, this compensatory mechanism could be limited in children, particularly before puberty, as a consequence of a proportionally lower muscle mass compared with adult populations.

#### *4.3. Adipocytokines*

Contradictory results have also been demonstrated regarding the relationship between irisin levels and adipocytokines. Circulating irisin has been positively correlated with leptin, but negatively associated with adiponectin in both obese and normal-weight Korean children [8]. A negative correlation between irisin and adiponectin was also observed in obese and normal-weight Italian

children [20]. In an interventional study, a positive correlation between irisin and leptin was observed before and after an 8-month physical activity program in normal weight and obese children. The decrease in leptin after the exercise intervention showed a strong association with the decrease in irisin levels across all BMI subgroups [6]. In contrast, two studies found no correlation between circulating irisin and leptin, adiponectin, or resistin levels, either at baseline or after lifestyle interventions [27,30]. Concerning inflammatory cytokines, positive correlations between irisin and TNFα and IL-6 were observed in a cross-sectional analysis of the PANIC study [27,30].

The negative association between irisin and adiponectin levels may indicate that in states of low energy expenditure, decreased adiponectin might stimulate a compensatory increase in irisin in order to increase energy expenditure. However, the positive association between irisin and leptin levels, which both decrease after a physical exercise intervention, supports the hypothesis that irisin is produced by adipose tissue. Since there are some inconsistent results, the interplay between irisin and adipocytokines needs to be further investigated. The association of irisin levels with TNFα and IL-6 suggests that irisin levels could be related to a proinflammatory profile.

#### *4.4. Fatty Acids Composition*

Higher irisin levels have recently been associated with metabolically unfavorable fatty acid profiles in overweight and obese children, compared with normal weight ones, in a cross-sectional analysis from the ongoing PANIC study. In a subset of children, higher irisin levels were associated with polymorphism linked to an increased accumulation of hepatic triglycerides, suggesting that increased irisin levels may be intended to prevent lipid accumulation and progressive steatosis and fibrosis. Additionally, higher proportions of oleic acid, adrenic acid, and docosapentaenoic acid in plasma were associated with higher plasma irisin levels among overweight/obese children, which suggests that irisin is directly associated with increased activity of elongation and desaturation steps following the desaturation of linoleic acid. A possible association between plasma irisin and delta-6-desaturase activity in plasma cholesteryl esters was also found [31], which has previously been reported to be associated with insulin-resistant states [32]. Excess body fat could modulate these relationships through fatty acid-mediated cross-talk between metabolically active tissues.

#### *4.5. Energy Intake and Expenditure*

Recent studies have elucidated the interplay between new energy intake and expenditure regulators, such as oxytocin, which is involved in food intake regulation in the central nervous system. A positive correlation between circulating irisin and oxytocin levels was observed in AAs compared with EAs and NAs [33]. In contrast, in an obese population, irisin and oxytocin appeared to have opposite roles, as the first was found to be higher in obese and overweight pubescent children and adolescents compared with controls, while the opposite was found for oxytocin levels. Yet, a correlation between irisin and oxytocin was not studied by Binay et al., 2017 [7]. Thus, oxytocin signaling and the regulation of food intake may be more significant in high energy consumption situations, such as in AAs, while an inverse association is found in obesity.

#### **5. Association of Circulating Irisin with Gender, Puberty, and Hormonal Status**

Regarding the differences in irisin concentrations between genders, some studies in normal-weight subjects have concluded that circulating irisin levels were higher in lean girls than in lean boys. Noteworthy this difference between genders has not been observed in overweight or obese children [5,6,8,11,27,28]. As for puberty, a significant difference in irisin concentration between prepubescent and pubescent subjects was also observed in obese children. After one-year follow-up, an increase in irisin levels was found in five obese subjects that had begun puberty, compared with those who had not [10].

Exploring the relationship between irisin concentrations and hormonal levels, in a study of adolescent athletes, Singhal et al., 2014 [12] found no significant difference in sexual hormones between groups. However, irisin was found to correlate with a higher free androgen index in AAs, while a positive correlation between irisin and estradiol was observed in NAs. The meaning of these findings is not yet understood.

Most studies have concluded that gender differences in irisin concentrations in children and adolescents are not due to puberty status, but rather to total adipose tissue, brown adipose tissue, and metabolic activity, with girls having a higher body fat mass than boys. However, a contradictory conclusion was obtained in one of the previous studies stating that in the obese population, entry into puberty was the main factor involved in the increased irisin levels.

#### **6. Association of Circulating Irisin with Mother–Offspring Relationship and Gestational Age in Neonates**

Circulating levels of irisin in newborns are believed to be maternally inherited. In a cross-sectional study in Arab families, circulating irisin was found to be an inheritable trait between mother and offspring [34]. Others have found significantly lower irisin levels in newborns than in mothers. In a study of Mexican mother/newborn pairs with single, non-complicated pregnancies, only newborns from cesarean sections presented lower irisin concentrations than their mothers compared with labor-born neonates [35]. Arterial cord blood total antioxidant capacity, IL-1β, and IL1-RA levels positively predicted newborn irisin concentrations. Maternal IL-13 negatively predicted offspring irisin levels, while IL-1β positively predicted newborn irisin concentrations.

In another cross-sectional study in 70 pairs of newly-delivered Greek neonates and their mothers, irisin levels were also lower in the neonates [36]. A possible explanation for the higher irisin levels in labor-born neonates compared with cesarean-born ones could be related to physical stress during labor possibly mimicking physical activity. Additionally, differences in neonate and adult muscle mass could explain the lower irisin concentrations found in neonates compared with their mothers.

Associations between irisin concentrations and gestational age in newborns have been demonstrated. Comparing newborns from different gestational ages, many studies have found higher irisin levels in term infants compared with preterm newborns, as well as positive correlations with birth weight Z-scores. Measurement of irisin in umbilical cord blood has shown that irisin levels are lower in small for gestational age (SGA) newborns compared with appropriate for gestational age (AGA) and large for gestational age (LGA) neonates [36–38]. In contrast, in a study of Caucasian women, a higher irisin level in preterm infants compared with term infants was found [39]. Irisin levels were also significantly higher in maternal blood compared with umbilical cord blood. In intrauterine growth restriction (IUGR) subjects, fetal irisin concentrations were found to be significantly lower, compared with AGA controls and LGA neonates [40]. In the LGA group, fetal irisin concentrations were positively correlated with fetal insulin levels.

The lower irisin concentration in SGA and IUGR newborns might be attributable to their smaller muscle mass. It has been previously shown that IUGR neonates have impaired skeletal muscle growth [41], while SGA neonates have a smaller skeletal muscle mass [42] and total body fat percentage [43] compared with AGA newborns. Since irisin is needed for non-shivering thermogenesis, which is crucial for the adaptation of the newborn to the postnatal environment IUGR, and SGA neonates with low irisin levels might be predisposed to hypothermia at birth [40]. In addition, IUGR, together with LGA neonates, are at high risk for obesity and metabolic disorders, as well as for alterations in fetal adipose tissue development and hormonal dysfunctions [44]. These facts might explain the positive correlation between fetal irisin and insulin levels observed in the LGA group. Therefore, irisin might be an important metabolic factor during very early stages of life that may render these neonates susceptible to insulin resistance and the MS later in life.

#### **7. Association of Circulating Irisin and Other Diseases**

Although irisin has been studied in a few other organs and diseases in the pediatric population, its role remains unclear. High irisin immunoreactivity evaluated through immunohistochemistry was demonstrated in acute appendicitis biopsies from Turkish children. Positive correlations of irisin levels with urine, saliva, and serum, as well as with total white blood count, neutrophil percentage, and reactive C-protein have also been found. Irisin was higher in all samples from patients with appendicitis, compared with controls. In the post-operative period, irisin concentration reached baseline levels 72 h after surgery, suggesting that irisin secretion could be increased in response to the acute inflammation of appendix tissues [45].

Irisin was also studied in Egyptian children with epilepsy. Children with idiopathic epilepsy presented higher irisin levels compared with controls [46]. Plasma irisin showed a positive correlation with the severity of seizures and the duration of the disease, probably playing a role as a predictor of uncontrolled seizures. The authors hypothesized that the elevated irisin levels may play a protective role against the hypoxic effects of seizures, as has been demonstrated by Zhao et al., 2016 [47] and Mazur-Bialy et al., 2017 [48] in animal models.

**Figure 2.** Association of irisin concentrations with cardiovascular, metabolic, and anthropometric parameters, physical activity, and mother-infant relations in children. +: Positive correlation; −: Negative correlation; 0: No correlation; SFA: Saturated fatty acids; MUFA: Monounsaturated fatty acids; PUFA: Polyunsaturated fatty acids; SBP: Systolic blood pressure; WC: Waist circumference; LDL-c: Low density lipoprotein cholesterol; HDL-c: High density lipoprotein cholesterol; HOMA-IR: Homeostatic model assessment of insulin resistance; DM1: Diabetes Mellitus type 1; IL13: Interleukin 13; IL-1β: Interleukin 1 beta; IL1-RA: Interleukin 1 receptor antagonist.

Finally, in a study of prepubescent patients with Turner syndrome who underwent recombinant human growth hormone (rhGH) therapy, a significant increase in irisin levels was found after treatment, although no relationship between irisin and IGF-1 was observed before or after therapy [49]. Negative associations between irisin levels and metabolic parameters were found, while a positive association of irisin with HbA1c was identified. These results suggest that in treatment-naïve Turner syndrome patients, who are predisposed to the MS, the physiological role of irisin may be disrupted and that rhGH therapy may restore it.

Figure 2 summarizes the associations of plasma irisin levels with body composition, cardiovascular risk factors, the metabolic syndrome, diet and physical activity interventions, as well as neonates and infant–mother correlations in the pediatric population.

#### **8. Conclusions**

The role of irisin as a regulator of body composition, and cardiovascular and metabolic diseases, as well as its correlation with physical activity and dietetic interventions has been understudied in the pediatric population, and it is still poorly understood. Contradictory findings have been found regarding the association of irisin with BMI, WC, fat mass, muscle mass, cardiovascular risk factors, insulin resistance, fasting glucose, and lipid levels, as well as its role in obesity and the MS. Irisin levels have been found to differ by gender in lean children, but not in obese ones. Controversial hypotheses to explain these findings have also been explored.

Irisin might represent an adaptive response to preserve energy in children with decreased muscle and fat mass, such as those who are underweight, and in SGA and IUGR neonates. Meanwhile, in children and adolescents with obesity, cardiovascular risk factors and the MS, irisin might either increase energy expenditure through thermogenesis, or it may represent an insulin-resistant state, especially considering the negative association of irisin with adiponectin and its positive association with leptin. Furthermore, irisin has even been associated with a proinflammatory profile.

Increased irisin levels during short bouts of aerobic exercise may only represent increased energy expenditure, but the lack of response during long term regimens, which even include nutrition and diet counseling, may be attributed to an adaptive thermogenesis. Further research in the pediatric population is required to confirm the association of irisin concentration with body composition parameters, physical activity, and cardiometabolic diseases, as well as to elucidate the role of irisin and its underlying mechanisms as a regulator of the metabolic state.

**Funding:** This research was funded by the Center for Research in Obesity and Clinical Nutrition, Tecnologico de Monterrey (LEM), and by CONACYT grant number 256577, a1-s-48883 (GGR).

**Acknowledgments:** The authors wish to thank Luisa Perez-Villarreal for her support in the literature search as well as to the Center for Research in Clinical Nutrition and Obesity (Leticia Elizondo-Montemayor), to the Cardiovascular and Metabolomics Research Group (Gerardo Garcia-Rivas) and to CONACYT, grant number 256577, a1-s-48883.

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

#### **Abbreviations**

#### **References**

© 2018 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/).

#### *Article*

## **Ginseng Berry Extract Rich in Phenolic Compounds Attenuates Oxidative Stress but not Cardiac Remodeling post Myocardial Infarction**

**Mihir Parikh 1,2,3,†, Pema Raj 1,3,†, Liping Yu 1,4, Jo-Ann Stebbing 1,4, Suvira Prashar 1,4, Jay C. Petkau 1,4, Paramjit S. Tappia 5, Grant N. Pierce 1,2,3, Yaw L. Siow 1,3,4, Dan Brown 1,4, Heather Blewett 1,3,4,6 and Thomas Netticadan 1,3,4,\***

Received: 22 December 2018; Accepted: 20 February 2019; Published: 24 February 2019

**Abstract:** The cardioprotective effects of ginseng root extracts have been reported. However, nothing is known about the myocardial actions of the phenolic compounds enriched in ginseng berry. Therefore, this study was undertaken to investigate the effects of American ginseng berry extract (GBE) in an experimental model of myocardial infarction (MI). Coronary artery ligation was performed on Sprague–Dawley male rats to induce MI after which animals were randomized into groups receiving either distilled water or GBE intragastrically for 8 weeks. Echocardiography and assays for malondialdehyde (MDA) and TNF-α were conducted. Flow cytometry was used to test the effects of GBE on T cell phenotypes and cytokine production. Although GBE did not improve the cardiac functional parameters, it significantly attenuated oxidative stress in post-MI rat hearts. GBE treatment also resulted in lower than control levels of TNF-α in post-MI rat hearts indicating a strong neutralizing effect of GBE on this cytokine. However, there was no effect of GBE on the proportion of different T cell subsets or ex-vivo cytokine production. Taken together, the present study demonstrates GBE reduces oxidative stress, however no effect on cardiac structure and function in post-MI rats. Moreover, reduction of TNF-α levels below baseline raises concern regarding its use as prophylactic or preventive adjunct therapy in cardiovascular disease.

**Keywords:** *Panax quinquefolius*; ginseng berry; myocardial infarction; phenolic compounds

#### **1. Introduction**

Ginseng has been used for centuries in the traditional medicines in Asia. It is a herb derived from genus *Panax*, of family *Araliaceae* and has thirteen different species which are indigenous to Asia and North America [1]. Active components of ginseng include ginsenosides, saponins, polysaccharides, alkaloids, peptides, polyacetylenes, phenolics, and fatty acids [2,3].

Out of all the bioactive compounds of ginseng, ginsenosides have been more extensively studied compared to the phenolic compounds. However, phenolic compounds are now being investigated in several studies for their diverse biological actions [3]. Salicylic acid, p-coumaric acid, ferulic acid, cinnamic acid, and quercetin are some of the phenolic compounds identified in ginseng [3]. Unlike ginseng roots, the chemical composition of ginseng berry is less known. A comprehensive profile of the phenolic compounds found in Korean ginseng berry, root, and leaf has been reported [3]. In the Korean ginseng berry, chlorogenic acid was reported to be the predominant compound present, followed by gentisic acid and rutin [3]. In the North American ginseng berry, caffeic acid and chlorogenic acid were reported to be the active polyphenolic constituents in a study of the protective effect of ginseng berry extract against oxidant injury in cardiomyocytes [4]. In spite of reports of the presence of bioactives in ginseng berry, it is not commercially used and is often discarded as a 'useless by-products' [5,6].

A recent study showed that ginseng berry has higher total phenol content (including quercetin, rutin, and resveratrol) than the root [5]. Although both ginseng root and berry have pharmacological actions, in some instances berry has been found to be more effective. Ginseng berry has been reported to have a more potent antihyperglycemic action than the root at the same dose [7]. Ginseng berry has been shown to reduce coagulation of blood [8], improve insulin sensitivity [9], and regulate glucose metabolism [10]. Ginseng berry extract (GBE) was found to protect cardiomyocyte against oxidative stress by activating the antioxidant Nrf2 pathway [11]. An echocardiography study using ginseng root extract demonstrated a significant improvement in left ventricular function [12]. However, ginseng berry with its high phenolic content has not been evaluated for its effect on cardiac structure and function. Accordingly, the present study investigated the effect of a phenolic rich GBE on cardiac structure and function. Furthermore, the damage to the heart muscle resultant from a myocardial infarction (MI) triggers an immune response [13]. When this immune response is uncontrolled it can cause more damage to the heart. Phenolic compounds have been shown to modulate immune responses [14]; but there is a paucity of information on immunomodulatory effects of phenolic extracts from ginseng berry. Thus, our study also assessed immunomodulatory activity of GBE in the myocardial infarction (MI) model induced in rats by coronary artery ligation.

#### **2. Results**

#### *2.1. Phenolic Content and Antioxidant Capacity of GBE*

The total phenolic content of the GBE was 3586 ± 04 mg gallic acid equivalents/100 g dry weight using the Folin–Ciocalteu assay. Oxygen radical absorbance capacity (ORAC) assay was performed to assess the oxygen radical scavenging activity of the extract. As expected, GBE exhibited a strong antioxidant capacity with a value of 151,864 ± 883 μmol Trolox equivalents/100 g dry weight. The proximate analysis is presented in Table 1.

**Table 1.** Proximate analysis of ginseng berry extract.

#### *2.2. Body Weight and Heart Weight Characteristics after MI*

Biometrical characteristics were assessed to check whether the coronary artery ligation or treatment with GBE was able to produce any alterations. No significant changes in body weight and heart weight were observed between groups 8 weeks post-surgery. The percentage of scar weight was 21.31% less in GBE-treated MI animals, but not statistically significant when compared to the water-treated MI animals (Table 2). No differences were observed in the heart weight to tibia length ratio values between groups. Similarly, liver and lung weights also did not show significant differences between groups (values not shown).

**Table 2.** Biometrical characteristics of sham and MI animals with and without ginseng berry extract treatment for 8 weeks after coronary artery ligation.

Values presented are mean ± SEM. MI: Myocardial infarction; LV: Left Ventricle; Sham-W: Sham MI treated with distilled water (*n* = 8); Sham-G: Sham MI treated with GBE 150 mg/kg/body weight/day (*n* = 8); MI-W: MI treated with distilled water (*n* = 12–14); MI-G: MI treated with GBE 150 mg/kg/body weight/day (*n* = 12–14).

#### *2.3. Lack of Improvement in Cardiac Structure and Function with GBE Treatment*

M-mode echocardiography was carried out to assess the effect of GBE on the left ventricular remodeling at 4 and 8 weeks post-MI. At 4 weeks, left ventricle (LV) internal diameter (LVID) values at systole and diastole were comparable between water- and GBE-treated sham animals (Figure 1A,C). In contrast, the water-treated and GBE-treated MI groups had significantly higher LVID values when compared to the water-treated sham group indicating left ventricular dilatation (Figure 1A,C). A similar trend was observed at 8 weeks with significantly high LVID values at systole and diastole in the water-treated and GBE-treated MI groups in comparison to water-treated sham group (Figure 1B,D). However, no changes were observed in LVID after GBE treatment in MI group (vs. water treated MI group) at 4 and 8 weeks (Figure 1A–D). LV volumes were also observed to be altered. MI animals treated with water and GBE showed significantly higher end-diastolic volume (EDV) and end-systolic volume (ESV) when compared to water treated sham animals (Figure 1E–H). There was no difference observed between sham animals treated with water and GBE, for EDV and ESV. GBE treatment of MI animals did not produce significant changes in EDV and ESV when compared to the water-treated MI animals (Figure 1E–H).

**Figure 1.** *Cont*.

**Figure 1.** Effect of ginseng berry extract on ventricular chamber dilatation and remodeling. (**A**) LV internal diameter at diastole (LVIDd) at 4 weeks; (**B**) LVIDd at 8 weeks; (**C**) LV internal diameter at systole (LVIDs) at 4 weeks; (**D**) LVIDs at 8 weeks; (**E**) end diastolic volume (EDV) at 4 weeks; (**F**) EDV at 8 weeks; (**G**) end systolic volume (ESV) at 4 weeks; (**H**) ESV at 8 weeks. Values presented are mean ± SEM. Bars with differing letter are significantly different, *p* < 0.05. Sham-W: Sham MI treated with distilled water (*n* = 8); Sham-G: Sham MI treated with GBE 150 mg/kg/body weight/day (*n* = 8); MI-W: MI treated with distilled water (*n* = 12–14); MI-G: MI treated with GBE 150 mg/kg/body weight/day (*n* = 12–14).

To check for left ventricular hypertrophy along with the increased LV dilatation, the thickness of interventricular septal thickness (IVS) and LV posterior wall (LVPW) was measured. IVS thickness at systole was observed to be significantly lower at 4 weeks (Figure 2C), and at both systole and diastole at 8 weeks (Figure 2B,D), in the water- and GBE-treated MI animals when compared to water treated sham animals. LVPW thickness values were also found to be significantly low at systole in the water-treated MI group when compared to water treated sham group (Figure 2H). At both 4 and 8 weeks, values for IVS and LVPW thickness were found to be comparable between water and GBE treated MI groups (Figure 2A–H).

**Figure 2.** Effect of ginseng berry extract on cardiac hypertrophy. (**A**) Interventricular septal thickness at diastole (IVSd) at 4 weeks; (**B**) IVSd at 8 weeks; (**C**) IVS at systole (IVSs) at 4 weeks; (**D**) IVSs at 8 weeks; (**E**) LV posterior wall thickness at diastole (LVPWd) at 4 weeks; (**F**) LVPWd at 8 weeks; (**G**) LV posterior wall thickness at systole (LVPWs) at 4 weeks; (**H**) LVPWs at 8 weeks. Values presented are mean ± SEM. Bars with differing letter are significantly different, *p* < 0.05. Sham-W: Sham MI treated with distilled water (*n* = 8); Sham-G: Sham MI treated with GBE 150 mg/kg/body weight/day (*n* = 8); MI-W: MI treated with distilled water (*n* = 12–14); MI-G: MI treated with GBE 150 mg/kg/body weight/day (*n* = 12–14).

Cardiac function was assessed by using M-mode echocardiography. Ejection fraction (EF), a measure of cardiac contraction (systolic heart function), was identical between water- and GBE-treated sham animals. The water-treated and GBE-treated MI groups showed significantly lower EF at 4 and 8 weeks when compared to water treated sham group (Figure 3A,B). EF values were not significantly different between the water- and GBE-treated MI groups (Figure 3A,B). Another parameter of cardiac contraction, fractional shortening (FS), was also significantly reduced in the water- and GBE-treated MI groups when compared to water treated Sham controls (Figure 3C,D). No significant differences were noted for FS values between water and GBE treated MI groups (Figure 3C,D).

**Figure 3.** Effect of ginseng berry extract on systolic heart function. (**A**) Ejection fraction (EF) at 4 weeks; (**B**) EF at 8 weeks; (**C**) fractional shortening (FS) at 4 weeks; (**D**) FS at 8 weeks. Values presented are mean ± SEM. Bars with differing letter are significantly different, *p* < 0.05. Sham-W: Sham MI treated with distilled water (*n* = 8); Sham-G: Sham MI treated with GBE 150 mg/kg/body weight/day (*n* = 8); MI-W: MI treated with distilled water (*n* = 12–14); MI-G: MI treated with GBE 150 mg/kg/body weight/day (*n* = 12–14).

Doppler echocardiography was used to examine the changes in cardiac relaxation (diastolic heart function), post-MI. At 4 and 8 weeks, no changes were observed in the parameters assessing diastolic heart function, mitral valve (MV) E and A wave velocity in all groups (Figure 4A,B). At 4 weeks, the values of another diastolic heart function parameter, Isovolumic relaxation time (IVRT) was also found to be similar in all the groups (Figure 4C). However, IVRT was observed to be significantly increased in the water- and GBE-treated MI groups when compared to water treated sham controls at 8 weeks (Figure 4D). GBE-treated MI animals had values comparable to water-treated MI animals (Figure 4D).

#### *2.4. Reduction in Oxidative Stress and Inflammation with Ginseng Berry Extract Treatment*

The 8-week water treated MI animals demonstrated significantly increased MDA levels in the heart tissue when compared to water-treated sham animals (Figure 5A). The GBE treated MI group showed a significant decrease (29%) in MDA levels when compared to the water-treated MI group (Figure 5A). Although, values for TNF-α were comparable between 8-week water treated sham and MI groups, a significant reduction of ~94% was observed in 8-week GBE-treated MI and sham animals when compared to water-treated sham and MI animals, respectively (Figure 5B).

**Figure 4.** Effect of ginseng berry extract on diastolic heart function. (**A**) Mitral valve E- to A- wave ratio (MV E/A) at 4 weeks; (**B**) MV E/A at 8 weeks; (**C**) Isovolumic relaxation time (IVRT) at 4 weeks; (**D**) IVRT at 8 weeks. Values presented are mean ± SEM. Bars with differing letter are significantly different, *p* < 0.05. Sham-W: Sham MI treated with distilled water (*n* = 8); Sham-G: Sham MI treated with GBE 150 mg/kg/body weight/day (*n* = 8); MI-W: MI treated with distilled water (*n* = 12–14); MI-G: MI treated with GBE 150 mg/kg/body weight/day (*n* = 12–14).

**Figure 5.** Effect of ginseng berry extract on oxidative stress and inflammation. (**A**) Malondialdehyde (MDA); (**B**) tumor necrosis factor-α (TNF-α). Values presented are mean ± SEM. Bars with differing letter are significantly different, *p* < 0.05. Sham-W: Sham MI treated with distilled water (*n* = 8); Sham-G: Sham MI treated with GBE 150 mg/kg/body weight/day (*n* = 8); MI-W: MI treated with distilled water (*n* = 12–14); MI-G: MI treated with GBE 150 mg/kg/body weight/day (*n* = 12–14).

#### *2.5. Immune Cell Phenotypes*

Using forward vs. side scatter we can identify viable lymphoid cells. Cell shrinkage, one of the first indicators of apoptosis, can be identified by a decrease in forward light scatter. There was no difference among groups in the proportion of cells that appeared in the non-viable lymphoid region.

In the viable lymphoid region (Figure 6), MI had no effect on the proportion of total T-cells (identified as CD3+), helper T-cells (identified as CD3+CD4+CD8−), cytotoxic T-cells (identified as CD3+CD4−CD8+) or activated T-cells (identified as CD3+CD25+) and activated cytotoxic T-cells (identified as CD3+CD4−CD8+CD25+). MI did result in an 8.0% lower proportion of activated helper T-cells (identified as CD3+CD4+CD8−CD25+) and 9.8% lower proportion of T-regulatory cells (identified as CD3+CD4+CD8−CD25+foxp3+) compared to sham-operated rats. GBE treated rats had a 9.4% higher proportion of T-cells and 13.2% higher proportion of cytotoxic T-cells, but a 7.4% lower proportion of helper T-cells compared to water treated rats. There was no effect of GBE on the

proportion of activated T-cells, activated helper T-cells, activated cytotoxic T-cells and T-regulatory cells (Table 3).

**Figure 6.** Immune cell phenotyping. (**A**) Definition of viable lymphocytes (Lymph) and non-viable lymphocytes (NV); (**B**) Definition of CD3 binding after gating on Lymph; (**C**) Definition of CD4 and CD8 after gating on CD3<sup>+</sup> Lymph; (**D**) Definition of CD25 binding after gating on CD3+ Lymph; (**E**) Definition of Foxp3 and CD25 after gating on CD3+CD4+CD8<sup>−</sup> Lymph; (**F**) Definition of CD25 binding after gating on CD3+CD4−CD8+ Lymph.

In the non-viable lymphoid region compared to the viable lymphoid region, there were lower proportions of all T-cell phenotypes, except for helper T-cells (Figure 7). Rats that had an MI had a 30% lower proportion of activated cytotoxic T-cells compared to sham-operated rats in the non-viable region. There was a trend towards a lower proportion of T-regulatory cells after MI compared to sham (*p* = 0.06), but no significant difference in the proportion of total T-cells, activated T-cells, helper T-cells, activated helper T-cells, or cytotoxic T-cells in the non-viable region. Compared to water-treated controls, rats treated with GBE had a 29.4% higher proportion of T-cells and a 10% lower proportion of

activated Helper T-cells in this region. There was no difference in the proportion of activated T-cells, helper T-cells, T-regulatory cells, cytotoxic T-cells and activated cytotoxic T-cells (Table 3).

**Figure 7.** Representative flow cytometry plot for non-viable lymphocytes. (**A**) Definition of CD3 binding after gating on non-viable lymphocytes (NV); (**B**) Definition of CD4 and CD8 after gating on CD3+ NV; (**C**) Definition of CD25 binding after gating on CD3+ NV; (**D**) Definition of Foxp3 and CD25 after gating on CD3+CD4+CD8<sup>−</sup> NV; (**E**) Definition of CD25 binding after gating on CD3+CD4−CD8+ NV.

#### *2.6. Cytokines Production*

ConA stimulated splenocytes from rats that had an MI when compared to controls, did not show a significant difference in IL-2, IFNγ, IL-10 or TNFα concentrations produced or between GBE treatment and control (Table 4).

Unstimulated samples were below the lower limit of detection for IFNγ (6.8 pg/mL) and TNFα (27.7 pg/mL). Unstimulated concentrations of IL-2 and IL-10 were not affected by MI or GBE treatment (Table 4).

(value >3×

standard deviation of mean) were removed: 1 from MI water.

 water group;

 >3×

**Table4.**SupernatantcytokineconcentrationsfromconcanvalinA-stimulatedandunstimulatedsplenocytes. Valuesinterleukin-10 (IL-10); tumor necrosis factor α (TNFα); ND = not detectable. 1 IL-2 UNS 4 samples from sham/water, 2 from sham/GBE, 4 from MI/water, 6 from MI/GBE weredetection limit of 0.46 pg/Ml; 2 IFNγ ConA 1 outlier removed from sham/water group; 3 IL-10 ConA 2 samples from MI/water and 1 from sham/water were below detection limit19.4 pg/mL; 4 IL-10 UNS 5 samples from sham/water, 3 from sham/GBE, 5 from MI/water, 6 from MI/GBE were below detection limit of 19.4 pg/mL.

 below

 of

#### **3. Discussion**

The present study is the first to report that ginseng berry phenolic extract has a strong antioxidant effect despite no effect on cardiac structure and function in the condition of a MI.

Although there is no information in the literature on the effects of ginseng berry on heart structure and function in any settings of cardiovascular disease, some studies conducted with Korean ginseng (*Panax ginseng*) have shown that GBE exerts anti-atherosclerotic [15], anti-diabetic, and anti-obesity effects [16]. Unlike ginseng root extract, GBE has been found to inhibit the mRNA expression of interleukins 1β and 6 inflammatory markers and it is likely through this mechanism that it exerts its antihyperglycemic and anti-obesity effects [17]. GBE also increases prothrombin time, indicating potential atherothrombotic effects [8].

Ginseng consists of phenolic compounds which include phenolic acids and flavonoids with strong antioxidant activity, but these are less characterized as compared to the ginsenosides. Chung et al. reported 4–9-fold higher phenolic compounds in the 3–6 year-old Korean ginseng berry (*Panax ginseng*) compared to the same age roots [3]. A higher phenolic content has been observed in the older Korean ginseng berries when compared to younger ones; the total amount increasing by 20–48% with an increase in the cultivation year. DPPH free-radical-scavenging activity (DPPH activity) of the 3–6 years old Korean ginseng berry is 3–5 fold higher than the root, suggesting the strong antioxidant activity of the berry [3]. In this context, an extract derived from American ginseng berries protected cardiomyocytes from oxidative stress induced by H2O2 and antimycin A [8]. An approximately 60% reduction in cardiomyocyte death was observed with the American GBE, clearly demonstrating that it can salvage cardiomyocytes from the oxidative injury [18]. The proposed mechanisms for the American GBE antioxidant actions included direct free radical scavenging activity and stimulation of NO synthesis [18]. This study, therefore, examined the antioxidant potential of North American GBE (*Panax quinquefolius*) in addition to examining its cardioprotective potential in an experimental model of MI.

The present study demonstrated a significant reduction in ventricular function after a MI. The reasons behind this adverse outcome include an impaired contractile function as well as infarct expansion leading to ventricular dilatation. Two-dimensional echocardiography performed on patients with an acute transmural MI has demonstrated dilatation of the infarct zone. This infarct expansion phase, which occurs due to the slippage of necrotic myocardial fibers, starts as early as 3 days after infarction [19]. This early regional dilation occurring in the infarcted zone results in an overall left ventricular dilatation. The ventricular dilatation may continue until 30 months after infarction. Unlike the early expansion phase, both infarcted and non-infarcted segments are affected during the chronic phase dilation [20]. This disproportionate cardiac dilatation alters left ventricular topography and is associated with poor prognosis for long-term survival of MI patients [19]. Left ventricular dilatation has been associated with the progression of cardiac dysfunction as cardiac and stroke indices decrease. Of note, ventricular dilatation results in and is not the result of deteriorating cardiac function [21]. Left ventricular function is usually assessed by ejection fraction and end diastolic and systolic volumes. Ejection fraction has been observed to have a strong effect on mortality. With the increase in left ventricular internal diameter, there are substantial increases in end systolic and diastolic volumes with consequently increased stress on ventricular wall. Earlier studies with gated scintiphotography revealed an increase in left ventricular end-systolic volume and decrease in ejection fraction in MI patients compared to normal subjects [22]. Three months follow-up demonstrated increased values of LV ejection fraction in patients with clinical improvement and decreased values with clinically worsened cases [22]. Moller et al demonstrated that echocardiographically determined ejection fraction was a powerful predictor of mortality during a median follow up of 19 months [23]. A progressive increase was noted in the cardiac mortality in patients with ejection fraction below 40% [24]. Another important determinant of the ventricular function post-MI is the end systolic volume (ESV). Consideration of ESV along with EF adds more predictive power for mortality risk

stratification of MI patients [25]. Echocardiographic analysis revealed that GBE treatment does not prevent infarct-related impairment in cardiac structure and function.

Reactive oxygen species produced during oxidative stress damage membrane lipids, proteins, DNA thereby causing apoptosis of cardiomyocytes and eventually resulting in cardiac dysfunction [26]. Infiltration of inflammatory cells in the myocardium after an ischemic event initiates an exaggerated inflammatory response, this further accelerates and worsens ventricular remodeling by increasing myocardial injury [27]. Higher levels of the inflammatory marker TNF-α have been associated with ventricular dilation and cardiac fibrosis. Our results demonstrate a significant decrease in the LV levels of TNF-α and MDA due to GBE treatment, indicating that it can reduce the inflammatory and oxidative response in MI. The substantial attenuation of cardiac oxidative stress observed in MI rats could be associated with the robust antioxidant capacity of GBE, as determined by its oxygen radical scavenging activity. Despite exerting strong antioxidant effects, however, GBE was unable to ameliorate cardiac remodeling and rescue cardiac function in MI rats.

The large GBE mediated decline in cardiac inflammatory response observed in the present study is consistent with a previous report which showed that treatment with Korean ginseng berry (*Panax ginseng*) suppressed the expression of TNF-α and thereby reduced atherosclerotic lesions [15]. Chronic use of TNF-α blockers has nevertheless been associated with increased risk of cardiovascular complications. A deficiency in TNF signaling for an extended period of time can cause immune system defects. The cardiac effects of TNF-α are biphasic. While high levels of TNF-α is associated with apoptosis, the basal level of TNF-α is required to maintain cytoprotective Nrf2 signaling [28]. Kurrelmeyer et al reported the importance of TNF signaling in protecting cardiomyocytes against ischemic injury [29]. The protective mechanism against hypoxic damage could be via activation of protein kinase A which stimulates SERCA2a thereby reducing intracellular calcium concentration during calcium overload [30]. Thus, TNF-α signaling helps in pumping calcium ions in the sarcoplasmic reticulum and restoring cytosolic calcium back to baseline levels [30]. It is possible that the reduction of TNF-α levels due to GBE observed in the present study may be as a consequence of: (a) reduced expression of the TNF-α gene and a lessened production of TNF-α protein, or, (b) increased production of soluble TNF-α receptors, which could bind and inactivate TNF-α [31]. However, the results of our examination of T cell phenotypes and function and ex-vivo cytokine production showed no differences in the immune response to GBE.

#### *Limitations and Future Opportunities*

This study has certain limitations that should be taken into account while interpreting the observed findings. Despite the study being sufficiently powered for analysis, the sample size may have been too small as the observed effect size was small. A preliminary pharmacokinetic study would have assisted in appropriate dose selection and treatment regimen. In addition, more than once daily administration might have resulted in a sufficient plasma steady state concentration that could have produced a different outcome. Furthermore, pretreatment with GBE may have resulted in an adequate cardiac tissue distribution of polyphenols necessary for potentiating the endogenous antioxidant system during an ischemic insult. The results of our study open up opportunities for further investigation including a comparative study of a polyphenol rich extract from ginseng berries vs. polyphenol rich extract from roots, and other parts of the plant.

#### **4. Materials and Methods**

#### *4.1. Ginseng Berry Pulp Extract*

Ginseng berries from three-year-old *Panax quinquefolius* L. (North American ginseng) were provided by C & R Atkinson Farms Ltd, St. Williams, ON, Canada. The berries were stored at −20 ◦C until they could be freeze-dried in smaller batches. After freeze-drying berries, the seeds were removed and the pulp was ground to a fine powder. Pulp extracts were prepared in batches at a ratio of 10 g ground pulp per 200 mL of 80% methanol. The slurry was mixed at room temperature using a rotary shaker, at 200 rpm for 1.5 h. The mixture was centrifuged at room temperature for 15 min at 10,000× *g*, saving the pellet for further extraction. The supernatant was collected and stored at −20 ◦C and the remaining pulp went through two additional rounds of extraction with 80% methanol. All supernatants were pooled together and stored at −20 ◦C. The solvent was removed by rotary evaporation and the remaining aqueous extract was freeze-dried. Multiple extracts were performed to produce sufficient ginseng berry pulp extract for the study. Dried extracts were further ground to a fine powder and combined to form a homogeneous product for the study. The pH of the dried extract was 4.5 and the final product was stored at −80 ◦C

#### *4.2. Quantification of Phenolic Content and Antioxidant Activity*

To ensure complete mixing of dried samples three biological replicates were prepared. A quantity of 0.125 g dried powder of each of these replicates (a, b, and c) was dissolved in 3 mL of autoclaved <sup>18</sup> ͉ Milli-Q water. Tubes were vortexed, sonicated for 10 min and then centrifuged at 10,000<sup>×</sup> *<sup>g</sup>*. The supernatant was used for subsequent measurement. The assay was performed in a 96 well plate by combining reagent and sample in sodium carbonate buffer. By using a protocol modified from Ainsworth and Gillespie [32], the total phenolic content was determined by spectrometry using SpectraMax M5 (Molecular Devices, San Jose, CA, USA) microplate reader at 765 nm wavelength. A method modified from Gillespie et al [33] was used to determine the oxygen radical absorbance capacity (ORAC) using 2,2-azobis(2-amidinopropane) dihydrochloride (Wako Chemicals, Richmond, VA, USA) as a peroxyl generator. The decline in fluorescence of fluorescein was measured kinetically for 60 min using a SpectraMax M5 microplate reader and the area under the curve was calculated for each sample.

#### *4.3. Proximate Analysis*

The core proximate and dietary chemical composition of the sample was determined using standard AOAC methods for ash (AOAC 923.03), fat (AOAC Am5-04), crude fiber (AOAC Ba 6a-05), crude protein modification (AOAC 990.03) and moisture (AOAC 930.15). All tests were conducted by the Central Testing Laboratory (Winnipeg, MB, Canada).

#### *4.4. Animal Study*

This study protocol was approved by the University of Manitoba Office of Research Ethics and Compliance and Animal Care Committee and was done in accordance with the guidelines by the Canadian Council for Animal Care. Male Sprague Dawley rats (150–175 g; Charles River Laboratories, Quebec, Canada) were housed in a temperature and humidity controlled room with a 12 h light/dark cycle. Rats were anesthetized with 1–5% isoflurane with oxygen at a flow rate of 2 L min−<sup>1</sup> and kept in a surgical plane of anesthesia with 2% isoflurane during surgery and subjected to permanent ligation of the left anterior descending artery (LAD) to induce MI or to sham surgery. A left thoracotomy was done, and the heart was gently exposed from the pericardial sac through the incision. The left anterior descending coronary artery (LAD) was located and occluded with 6-0 polypropylene silk suture at about 2 mm from aortic root. The suture was tied and the ligation was estimated to be successful when the anterior wall of the left ventricle turned pale. The heart was repositioned, then chest compressed to remove any air from the cavity and the incision was closed using a purse string suture. Sham-operated animals that served as normal control were subjected to similar surgical procedures except that the LAD was not ligated. Buprenorphine 0.05 mg kg−<sup>1</sup> was administered pre- and post-surgery (2 times a day for 2 days) subcutaneously as an analgesic agent to all rats. All surviving sham and MI rats were assigned to the following 4 treatment groups: (1) Sham MI—distilled water as vehicle (Sham-W); (2) MI—distilled water as vehicle; (MI-W); (3) Sham MI—ginseng berry extract 150 mg/kg/body weight/day (Sham-G); (4) MI—ginseng berry extract 150 mg/kg/body weight/day (MI-G). The 4 groups consisted of sham (*n* = 8) and MI (*n* = 12–14) rats and received the

treatments by oral gavage for 8 weeks. The sample size was calculated using G\*Power statistical software with the power of study kept at 80%. Animals were regularly monitored for well-being.

#### 4.4.1. Transthoracic Echocardiography

All experimental rats were weighed and anesthetized with 3% isoflurane in a chamber, and then kept under 1.5–2% isoflurane throughout the procedure. An echocardiogram was obtained at 4 and 8 weeks post-surgery by 2D guided M-mode and Doppler modalities with a 13 MHz probe (Vivid E9; GE Medical Systems, Milwaukee, WI, USA) as described by us earlier [34]. 2D M-mode parasternal short-axis view images were obtained to determine systolic functional parameters such as the percentage of left ventricle (LV) ejection fraction (EF), fractional shortening (FS), and end-systolic volume (ESV) and end-diastolic volume (EDV). Doppler measurements included isovolumic relaxation time (IVRT), mitral valve (MV) E wave, A wave, and E wave deceleration time. The cardiac structural parameters such as interventricular septal thickness (IVS), LV posterior wall thickness (LVPW), and LV internal diameter (LVID) at diastole and systole were determined from parasternal short-axis view images. All echocardiographic images were analyzed to calculate the listed parameters using EchoPAC software (GE Medical Systems, Milwaukee, WI, USA). All measurements were performed and averaged from three cardiac cycles to account for interbeat variability.

#### 4.4.2. Biological Sample Collection and Analysis

All animals were anesthetized with ketamine/xylazine (9.0 mg per 100 g and 0.9 mg per 100 g IM). The depth of anesthesia was assessed by pedal withdrawal reflex. The blood sample was collected from the inferior vena cava by opening the thoracic cavity and the heart was immediately excised. The whole heart was rinsed in PBS, LVs, septum, and fibrotic scar tissues were separated, weighed and flash frozen in liquid nitrogen.

Percentage of infarct (scarred/fibrotic) LV tissue was calculated by dividing the weight of scarred LV tissue by whole weight of LV tissue as described previously [35]. Evidence of overt heart failure was assessed by determining the presence of ascites and by calculating the lung and liver wet-to-dry weight ratio in all rats.

To determine MI-associated oxidative stress and inflammation, the levels of the lipid peroxidation product, malondialdehyde (MDA), and tumor necrosis factor-α (TNF α) as a proinflammatory marker were assessed in the heart tissue using commercial kits.

#### *4.5. T-cell Phenotypes and Function*

To assess the effect of MI and GBE treatment on T-cell phenotypes and activation state and T-cell function, the following experiments were conducted.

#### 4.5.1. Isolation of T-cells

Single cell suspensions were obtained by pressing spleens through a 100μm cell strainer using the barrel of a sterile syringe into a sterile Petri dish containing Hank's buffered saline supplemented with 10 mM-HEPES, 4% fetal bovine serum, and 1% antibiotic/antimycotic at pH 7.4. Erythrocytes were lysed with ammonium chloride buffer. Cells were subsequently washed and re-suspended in label buffer (PBS containing 23 mM-sodium azide and 2% fetal bovine serum). Cell count and viability was completed using a Nexcelom AutoT4Plus. Cell concentration was adjusted to 1 × 107 cells/mL in label buffer [36].

#### 4.5.2. Cytokine Determination

Splenocytes were resuspended in RPMI-1640 supplemented with 10mM-HEPES, 10 mM-sodium bicarbonate, 1 mM-sodium pyruvate, 2 mM-gluatmine, 0.1 mM-non-essential amino acids, 50 μM-2-mercatpoethanol, 1% antibiotic/antimycotic and 5% fetal bovine serum at pH 7.2 at a

concentration of 1 × 106 splenocytes/mL and incubated at 37 ◦C and 5% CO2 with 2.5 <sup>μ</sup>g/mL Concanavalin A (ConA), or unstimulated for 48 hours. After incubation, samples were centrifuged at 400 g at 4 ◦C for 5 min to pellet cells. Supernatants were stored at −80 ◦C until analysis. The concentration of IL-2 (lower limit of detection (LLOD) 0.46 pg/mL), IFNγ (LLOD 6.8 pg/mL), IL-10 (LLOD 19.4 pg/mL), and TNFα (LLOD 27.7 pg/mL) were measured in cell culture supernatants using a cytometric bead array on a FACSCanto II flow cytometer. All samples were analyzed in duplicate with CV <10% according to the BD Cytometric Bead Array Mouse/Rat Soluble Protein Master Buffer Kit Instruction Manual [37].

#### 4.5.3. Phenotyping

T-cell phenotypes were determined by flow cytometry using isolated splenocytes. Monoclonal antibodies against rat CD3 (FITC label, clone 1F4, isotype mouse IgMκ), CD4 (PE-Cy7 label, clone OX-35, isotype mouse IgG2a,κ), CD8 (PerCP label, clone OX-8, isotype mouse IgG1,κ), CD25 (APC label, clone OX39, isotype Mouse IgG1,κ), foxp3 (PE label, clone FJK-16s, isotype rat IgG2a,κ) were obtained from BD BioSciences (Mississauga, ON, Canada). Antibodies were incubated with 1 × 106 cells/mL for 30 min at 4 ◦C in the dark. Cells were washed, and then incubated in Foxp3 fixation/permeabilization working solution at 4 ◦C for 18 h. Following incubation cells were washed with the permeabilization buffer and incubated with mouse CD16/CD32 antibody at 4 ◦C for 5 min to block non-specific binding. Foxp3 was added to cells and incubated at 4 ◦C in the dark for at least 30 min. Finally, treated cells were washed using the permeabilization buffer and re-suspended in PBS. Data were acquired on a FACSCanto II flow cytometer using the 488 nm and 633 nm lasers. Figure 6 shows representative flow cytometry plots. Forward versus side-scatter plots were used to gate on intact lymphoid cells and non-viable cells. The data were collected in list-mode format with the analyses based on 100,000 cells satisfying the light scatter gate for lymphocytes using Cell Diva software (v8.0.1). Unstained cells were used to assess auto-fluorescence, isotype controls to assess background staining, and single color samples were employed to adjust color compensation [36].

#### *4.6. Statistical Analysis*

All values are represented as means ± SEM. Two-way analysis of variance (ANOVA) test was used to determine the effect of surgery (factor 1) and treatment (factor 2) and their interaction (SAS, version 9.4, SAS Institute, Cary NC, USA). Unpaired t-test was utilized for comparison between 2 groups Significant differences among means were determined using LSmeans. Differences were considered significant at *p* ≤ 0.05.

#### **5. Conclusions**

The results of this study suggest that although GBE exerted potent antioxidant activity, it was unable to recover cardiac function in post-MI rats.

**Author Contributions:** The authors were responsible for the following aspects of this paper: conceptualization, T.N.; methodology, T.N., Y.S., and H.B.; formal analysis, M.P.; investigation, P.R., J.S., S.P., J.C.P., and L.Y.; resources, D.B., T.N.; data curation, L.Y.; writing—original draft preparation, M.P., P.R, H.B; writing—review and editing, M.P, P.R., G.N.P., Y.S., H.B., P.S.T., J.S., S.P., J.C.P., and T.N.; supervision, T.N.; project administration, L.Y., and T.N.; funding acquisition, D.B, Y.S., T.N., and H.B.

**Funding:** This research was funded by Agriculture and Agri-Food Canada.

**Acknowledgments:** We would like to thank the R.O. Burrell Laboratory for assistance with animal studies.

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

#### **References**

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2.1.1. Drug Loading Types

Electrospinning has different drug loading types, which determine the diverse structure and drug release kinetics. The drug loading procedure in electrospinning can be executed in different ways, including co-electrospinning, multi electrospinning, side-by-side electrospinning, co-axial, surface immobilization and emulsion electrospinning (Figure 2). In co-electrospinning, the drug molecules are mixed with the polymer solution before electrospinning. These electrospun fibers provide a uniform distribution of drugs/biomolecules and high drug/biomolecule loading. However, the biomolecule properties can be negatively affected when they are directly exposed to high voltage. On the other hand, blend electrospinning and side-by-side electrospinning help to solve the issue of drug and molecule solubility in common solvents. Moreover, multi-jets with more than two spinnerets represent a way of protecting the bioactivity of the drug. In addition, surface immobilization is another method, in which drug molecules are covalently bonded with the scaffolds via chemical or physical immobilization methods. In these chemical methods, the surfaces of the nanofibers are changed by introducing amines, carboxyl, hydroxyl or thiol; the physical methods involve the incorporation of Van der Waals, electrostatic and hydrophobic interactions. These methods of immobilization retain biomolecular activity, but the drug molecules in all electrospinning processes exhibit burst release kinetics. To overcome this, co-axial and emulsion electrospinning processes were introduced. Co-axial and emulsion electrospinning processes have been gaining increasing interest recently due to their promising ability to shield the biomolecules with the core and to minimize the drawback of the initial burst. They provide sustained release of the drug by minimizing the initial burst release by controlling the thickness and composition of the shells. The core/shell structure is generated utilizing a single-nozzle electrospinning unit employing emulsion input, commonly named emulsion electrospinning [42]. Another means of drug loading for sustained release of a drug is layer by layer, via the addition of drug in between the electrospun scaffolds; controlled release is promoted by the shield provided by the layer of polymer scaffolds. Therefore, co-axial electrospinning and multilayer electrospinning drug loading types provide a more sustained and controlled release of the drug.

**Figure 2.** Schematic representation of different types of electrospinning processes. Reproduced with permission from [42]. Royal Society of Chemistry, 2015.