**Dietary and Supplement-Based Complementary and Alternative Medicine Use in Pediatric Autism Spectrum Disorder**

**Melanie S. Trudeau 1,\* , Robyn F. Madden 1, Jill A. Parnell <sup>2</sup> , W. Ben Gibbard <sup>3</sup> and Jane Shearer 1,3**


Received: 3 July 2019; Accepted: 26 July 2019; Published: 1 August 2019

**Abstract:** Previous literature has shown that complementary and alternative medicine (CAM) is steadily increasing in autism spectrum disorder (ASD). However, little data is currently available regarding its use, safety, and efficacy in children with ASD. Thus, the purpose of this study is to describe the use of supplement-based CAM therapies in children between the ages of 4 to 17 years with ASD. This population-based, cross-sectional study evaluated children with ASD regarding supplement use. A total of 210 participants were recruited from a variety of sources including educational and physical activity programs, and social media to complete a questionnaire. Primary caregivers provided information on current supplement based CAM use. Data evaluated the proportion of children that used supplement therapies, the types of supplements used, reasons for use, perceived safety, and demographic factors associated with use (e.g., income, parental education, severity of disorder). Seventy-five percent of children with ASD consumed supplements with multivitamins (77.8%), vitamin D (44.9%), omega 3 (42.5%), probiotics (36.5%), and magnesium (28.1%) as the most prevalent. Several supplements, such as adrenal cortex extract, where product safety has not yet been demonstrated, were also reported. A gluten free diet was the most common specialty diet followed amongst those with restrictions (14.8%). Health care professionals were the most frequent information source regarding supplements; however, 33% of parents reported not disclosing all their child's supplements to their physician. In conclusion, the use of supplement therapies in children with ASD is endemic and highlights the need for further research concerning public health education surrounding safety and efficacy.

**Keywords:** Autism spectrum disorder; dietary supplements; pediatric; physician communication

#### **1. Introduction**

Autism spectrum disorder (ASD) is a group of heterogeneous chronic neurodevelopmental disorders characterized by qualitative impairments in social interaction, communication, and repetitive stereotyped patterns of behavior [1]. The etiology of these conditions is thought to be multifactorial, involving genetic, prenatal, and postnatal factors [2]. The Centre for Disease Control (CDC) reports that 1 in 59 children are diagnosed with ASD, with boys 4 times more likely to be diagnosed than girls. As such, ASD is the fastest growing developmental disorder in the United States [3].

Treatment for ASD focuses on educational and behavioral interventions such as applied behavioral analysis [4]. Psychotropic drugs are commonly prescribed to treat core behavioral symptoms, decrease maladaptive behavior, and support learning and development [5]. In addition to conventional treatment

options, some parents of children with ASD seek out complementary and alternative medicine (CAM) to treat symptoms. The National Centre for Complementary and Integrative Health defines CAM as "a diverse group of medical and health care systems, practices, and products that are not generally considered part of conventional Western medicine" [6]. Complementary approaches fall broadly into 3 categories: Natural products such as dietary supplements and special diets, mind and body practices, and other complementary health approaches [7].

Evidence regarding the use of CAM in the general pediatric population is limited. Studies in the United States have shown that the prevalence of pediatric CAM use in populations with illness or disease can range up to 76% [8]. However, these studies are limited in several ways. First, while many employ sound methodologies, they often provide differing definitions of what constitutes a CAM therapy. For example, in their review of 136 studies on alternative medicines, Surette et al. [9] found 39 studies that included vitamins, 13 studies that excluded vitamins, and 41 studies that made no mention of their inclusion or exclusion criteria. Further, many of the pediatric CAM studies are characterized by wide variation in study populations and size, prevalence measurements, and research methodologies, all of which hinder the formulation of evidence-based recommendations.

Though limited in number, some studies have examined CAM effects in ASD. Levy et al. [10] found that greater than 9% of children with ASD used potentially harmful CAM, such as chelation, antibiotics, or excessive amounts of vitamins. These findings are consistent with anecdotal evidence of dangerous products used to "cure" ASD. For example, in 2014, the supplementation market saw an explosion of *Miracle Mineral Solution,* a solution of sodium chlorite and hydrochloric acid (i.e., bleach) as a treatment option for ASD. The US Food and Drug Administration has issued several warnings about the product and the treatment has been linked to 1 death and several serious injuries; however, *Miracle Mineral Solution* is still widely available, with 1000 + followers on social media promoting its use [11].

From a public health perspective, supplement-based therapies and specialty diets, a subcategory of CAM, requires further evaluation. While many supplements such as melatonin, vitamins, gluten-casein-free diet, and omega 3 fatty acids may have few adverse effects, their safety and effectiveness in reducing ASD symptomology have not been reliably established [2,12,13]. Research estimates that up to 74% of children with ASD have been provided with CAM and that supplement-based therapies make up approximately 50% of CAM therapies used by this population [14].

Despite its popularity, disclosure of CAM use to physicians is often poor, with rates as low as 23% [15]. Concurrent use of CAM and prescription medications is widespread and poses a possible risk to patients who may be unaware of the potential for interactions [16]. Further, research has documented that knowledge of CAM use is important for health care professionals, as it provides insight into patient values and health beliefs. Importantly, considering patient values may assist in providing optimum care, especially in the context of supplements that pose a safety risk to patients [15]. Given the rates of concurrent use, in conjunction with lack of disclosure, there is a pressing need to assess pediatric CAM use and parental perceptions of these therapies.

As the prevalence of supplements and specialty diets are high and many are unsupported by research, a better understanding of the use of supplementation in pediatric ASD could help provide better integrative care by (1) informing the public and health care professionals about the prevalence and types of supplement therapies and specialty diets used in children with ASD; (2) assessing patient-physician communication and interactions surrounding supplement and specialty diet use; and (3) highlighting priorities for evidence-based clinical trials for supplements in ASD. Therefore, this study seeks to describe the use of supplement-based CAM therapies in children with ASD.

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

#### *2.1. Participants*

This study was approved by the Conjoint Health Research Ethics Board at the University of Calgary (REB17-0970). Inclusion criteria for this population-based cross sectional study included (1) a physician-confirmed diagnosis of autism spectrum disorder (including previous diagnostic labels of Asperger's syndrome, pervasive developmental disorder—not otherwise specified, childhood disintegrative disorder, or Rett syndrome) and (2) between the ages of 4 and 17.99 years inclusive. A sample size calculation (margin of error 8% and 95% confidence interval) revealed 150 participants were required [17]. Many of the children were cognitively and/or developmentally delayed; therefore, the parents/legal guardians served as a proxy for describing their child's use of supplements, in order to maintain consistency between responses. The parents/legal guardians of children with ASD provided written informed consent and completed the questionnaire on their behalf.

#### *2.2. Dietary Supplement Questionnaire*

Data about supplement use with regards to ASD was collected via self-report online and paper form questionnaires. A validity and reliability tested supplement use questionnaire [18,19] was modified for children with ASD. Supplements were defined as a product that contains a vitamin, mineral, herb or botanical, amino acid, concentrate, metabolite, or other dietary ingredients intended to add further nutritional value to the diet. Supplements may be found in many forms such as tablets, capsules, soft gels, liquids, or powders. Examples include multivitamins, supplementary minerals, protein powders, energy drinks, meal replacements, etc. This definition was based on the definition provided by the National Centre for Complementary and Integrative Health [6]. Specialty diets such as the ketogenic diet, low-carb diet, and gluten free diet were also evaluated. The response format for the survey contained several closed-ended questions, short answer, and 5 item Likert scale questions. Several questions provided participants with answers to select from as well as short answer boxes to provide their answers. A pilot-test was conducted on a small sample of parents (*n* = 34) to ensure clarity of content. A sample of the questionnaire can be found in Supplementary Data File S1, Table S1.

#### *2.3. Measures*

The key outcomes were the demographic variables of the child (i.e., age, sex, ethnicity, medical characteristics) and parent (i.e., income, education level), and the types of therapies (i.e., gluten free diet, omega 3 fatty acids, probiotics) used. Secondary outcomes included reasons why parents have or have not used supplement therapies for their children, the information sources consulted by parents regarding therapies, and the proportion of parents who perceive the therapies used as being safe. In addition, parent perceived satisfaction with their child's family physician or pediatrician, comfort level in discussing nutrition and supplements, number of supplements/dietary patterns disclosed to the physician (if applicable), and reasons why they might have chosen not to disclose supplements were quantified.

#### *2.4. Procedures*

Children were recruited from clinics at the Alberta Children's Hospital, Autism Calgary, and physical literacy programs around the city. Researchers also utilized schools that focus on inclusive and accessibility programs to recruit eligible participants. Recruitment posters were displayed throughout the facilities to describe the study, explain eligibility criteria, and provide contact information should parents of children with ASD want to participate. Researchers also contacted program organizers and asked permission to approach parents directly during recreation and instructional sessions and enroll them in the study. Many of these organizations promoted the study on their social media platforms, which provided an online consent form and a link to complete the survey.

#### *2.5. Statistical Analysis*

Data from the question 'has your child previously taken or currently taking any dietary supplements' was categorized as yes or no. Dietary supplement use data were categorized into groups based on sex, age (4 to 8 years, 9 to 13 years, and 14 to 17 years), and abilities. The age groups were based on dietary reference intake (DRI) values [20]. Ability groups were based on 4 ability categories (verbal, intellectual, social, and physical) in which parents ranked their child as "very weak/weak," "neutral," or "strong/very strong. Differences between sex, age, and ability groups were determined by a Fisher's exact test. All analyses were performed using SPSS statistics version 25 (IBM Corporation, Armonk, NW, USA).

#### **3. Results**

#### *3.1. Participant Characteristics*

A total of 210 parents agreed to participate in the study on behalf of their child(ren) and completed the questionnaire. Descriptive characteristics and demographic characteristics of the participants are outlined in Tables 1 and 2, respectively.



Participants and year of diagnosis are listed as a count (percentage of total); age is listed as a mean (standard deviation).


**Table 2.** Demographic characteristics.

The category "Multiracial" was created as a result of multiple parents indicating this option in the "other" category to reflect the demographic of our sample. Data is presented as a count (percentage of total).

#### *3.2. Dietary Supplement Use*

A total of 167 parents (79.5%) indicated that their child had previously taken or was currently taking at least 1 supplement (males 77.1%; females 86.8%, *p* = 0.168). Eighty-three percent (83%) of parents reported thinking supplements were safe, 3.8% reported that they did not consider them safe, and 13.3% were undecided or perceived supplements as safe under certain conditions (i.e., evidence-based, supervised by a health care professional, etc.). There were no statistical differences between sexes in perceived safety of dietary supplement use (*p* = 234).

The top 10 previous and current pediatric supplement use is summarized in Figure 1. A more extensive list of the supplements listed by the questionnaire can be found in Supplementary Data, Table S2. The 5 most common supplements were multivitamins (77.8%), vitamin D (44.9%), omega 3 (42.5%), probiotics (36.5%), and magnesium (28.1%). Other supplements such as alpha lipoic acid, sodium butyrate, N-Acetyl Cysteine, 5HTP, fluoride, methylfolate, adrenal cortex extract, selenium, milk thistle, liposomal curcumin, cannabidiol, and melatonin were also mentioned. Differences between sexes were found in calcium (male = 9.1%, female = 21.7%, *p* = 0.037) and vitamin K (male = 1.7%, female = 10.9%, *p* = 0.018). When analyzed by age group, significant differences were found in calcium (*p* = 0.049), vitamin B (*p* = 0.010), and energy drinks (*p* = 0.027). The percentage of children who have/are taking more than 1 supplement was 88.6%. In addition, a Pearson's correlation determined that there was no relationship between number of supplements used and years since diagnosis (*p* = 240). The average number of supplements per child was 4.49 and the mean year of diagnosis was 2013.

**Figure 1.** Dietary supplements commonly used in pediatric autism spectrum disorder (ASD). Male and female is percent within sex.

Of the 167 parents who indicated their child has or is currently taking supplements, 126 rated the perceived degree of change on their child's overall well-being between neutral and positive (69.1 ± 15.9) on a scale of 1 (negative impact) to 100 (positive impact). The most common supplements cited to have the largest impact on the child's health were melatonin, multivitamins, omega fatty acids, and magnesium.

#### *3.3. Dietary Supplement Reasons for Use*

Parental reasons for providing their children with supplements are outlined in Table 3. The top 3 reasons for consuming supplements were to enhance the child's diet, promote immune system function, and increase quality/duration of sleep. Improvement in gut health was listed 7 times under the "other" section. Parents also indicated several reasons for omitting supplements including: Inadequate

knowledge/information (*n* = 14), too expensive (*n* = 8), and may be considered harmful (*n* = 5). Six parents indicated that supplements were not necessary, as their child eats a balanced diet.


**Table 3.** Reasons for and against dietary supplement use.

Reasons for use are listed for the parents who indicated that their child had taken supplements (*n* = 167). Reasons against use are listed for the parents who indicated that their child had not taken supplements (*n* = 43). Data is presented as a count (percentage of total).

#### *3.4. Special Diets and Information Sources*

Current diet information is summarized in Table 4. The top 4 diets followed in our sample included no restrictions, gluten free, high carbohydrate, and lactose free, with a significant difference between sex in diets with no restrictions (male = 72.0%, female = 54.7%, *p* = 0.027), gluten free (male = 5.1%, female = 24.5%, *p* < 0.001), and lactose free (male = 4.5%, female = 17.0%, *p* = 0.006). Other diets such as the paleo diet, nut-free, dye-free, and low sugar diets were also mentioned. In addition, 6 parents also stated that their children were picky eaters and had "very limited diets."


**Table 4.** Special diet use in pediatric autism.

Diet data is presented as a count (percentage within sex) who follow each diet. Differences between sex were determined using a Fisher's exact test. *p* < 0.05 was considered significant. Significant differences are bolded. n/a, not applicable.

The primary information sources regarding dietary supplements used by parents are shown in Figure 2. Sixty-five percent (65%) of parents indicated that health care professionals (e.g., physician, nurse, nutritionist) were their primary source of information regarding dietary supplements. Published literature and media (e.g., news, magazines, journals) were listed as the second and third most popular sources of information. Social media was the least utilized source, with only 5.8% of parents indicating

use. Two participants did not indicate a primary information source and two other participants indicated "knowledge over the years," and "myself (pharmacist)" in the "other" section.

**Figure 2.** Primary information sources about dietary supplements. Data is presented as % of respondents. Participants could only choose one source or answer in the "other" section. One participant indicated in the "other" section that they use a combination of social media and health care professionals, so a "yes" was put into each category.

#### *3.5. Physician Communication*

Nearly all families (98.1%) indicated that they have a family doctor and/or a pediatrician. Eleven percent (11%) of families (*n* = 21) rated their overall level of satisfaction with the primary care physician as not satisfied or partially unsatisfied on a scale of 1 (not satisfied) to 5 (very satisfied), whereas 87% of parents (*n* = 169) rated their level of comfort in discussing supplements with physicians as comfortable or very comfortable. A large majority (72.4%; *n* = 152) had never met with a dietician. The frequency of disclosure of supplement use to a physician is summarized in Table 5. Results revealed that 33.5% of parents did not disclose all supplements to their physician. Several reasons were reported for omitting disclosure of supplementation use to primary care physicians including perceived "physician lack of knowledge", "no benefit", "too time consuming", and "scared of judgment".


**Table 5.** Disclosure of supplement use to physician.

Frequency data is presented as a count (percentage of total).

#### **4. Discussion**

The use of complementary and alternative medicine (CAM) is increasing among children and is common in those with chronic illness or disorders, such as ASD [13]. Critically, however, a complete profile of dietary supplement use in children with ASD is lacking; making it difficult to develop educational strategies. The present study is impactful, as it provides a fully powered assessment of supplement-based CAM therapies in children with ASD.

#### *4.1. Dietary Supplement Patterns and Special Diet Use*

In this study, 75.9% of the children sampled have consumed dietary supplements, supporting the upper end of the prevalence range of supplement use in pediatric ASD cited in previous literature [14]. As the most recent studies evaluating supplement use in children with ASD were conducted several years ago, it is possible that the high prevalence reported here reflects a continued increase in supplement use. Additionally, this study utilized a broad definition of supplements when compared to other studies, therefore, it is likely we captured a wider variety of supplements used by children with autism. Nevertheless, the reported high rates of dietary and supplement use in this study would indicate these products continue to be of interest as a complementary approach to standard treatment of care.

Multivitamins, vitamin D, omega 3 fatty acids, probiotics, and magnesium were the most common supplement therapies used. Research regarding the efficacy of these supplements in ASD populations requires further evaluation [21]. Multivitamins, for example, are considered a popular CAM therapy in ASD [22]. The rationale for this treatment is based on the frequently observed dietary deficiency of vitamins and micronutrients in children with ASD. Children with ASD are often deficient in calcium, vitamin D, vitamin K, vitamin A, vitamin E, zinc, vitamin B6, and tetrahydrobiopterin [21]. These deficiencies could be the result of food selectivity or altered gastrointestinal absorption. Adams et al. [23] conducted a double-blind randomized control trial to examine the effect of a common commercial vitamin supplement on observed improvements in parent-rated pre and post autism symptomatology. They found significant improvements in hyperactivity, tantrumming, overall, and receptive language, suggesting it as a reasonable adjunct therapy for children with ASD. However, no other study has evaluated or been able to replicate the effectiveness or safety of this biological therapy. Similarly, preliminary studies on probiotics have shown improvement in core symptomatology in ASD [21], but they have been minimally replicated and several other studies have denounced their effects. It is also possible that there are responders and non-responders to individual treatments, further complicating interpretation.

Recently, vitamin D has been proposed as a potential treatment for ASD [24]. In 2015, an open trial demonstrated significant improvements in autism rating scales following 3 month vitamin D3 supplementation [25]. However, this small sample study has been the only experimental study to demonstrate the potential efficacy of vitamin D in children with ASD, highlighting the need for more wide-scale studies to critically validate the efficacy of vitamin D before drawing any definite conclusions.

Collectively, these supplements require more systematic and rigorous research. As a result, there is little evidence to support the use of any nutritional supplement or dietary therapy for children with ASD [26]. Furthermore, some of the other supplements reported in this study confirm anecdotal reports regarding the consumption of dangerous biological therapies. The Food and Drug Administration has issued several health warnings about adrenal cortex extract, for example, and has deemed it "unsafe and ineffective for labeled indications for human use" [27–29]. Evidently, there are gaps in the transmission of scholarly literature to quality educational materials for families, as many children continue to consume dietary supplements that are unsupported by research. In addition, several parents indicated that they were undecided about the safety of supplements and listed inadequate knowledge about supplements as the number one factor barring use.

Research evaluating specialty diets (e.g., gluten free casein free, lactose free, etc.) shows similar ambiguity. More specifically, there are few studies that demonstrate conclusive results in the gluten free and casein (or lactose) free diets reported in this study [30]. Many are small in size and lack strict dietary controls, both common problems in conducting dietary research in children, which limits the ability of researchers to drawn firm conclusions. Consequently, many studies regarding specialty diets point to the need for further research and illustrate how clinicians often find themselves unable to offer the most up-to-date and scientifically credible information to their patients. Of note, the ketogenic diet has emerged as a leader in specialty diets for ASD in the past several years and has offered promising, though preliminary, results in both animal and human studies [31–33]. Seven participants reported utilizing this diet.

#### *4.2. Physician-Patient Communication*

Sixty-five percent (65%) of parents disclosed that their primary source of information regarding supplements were health care providers. In addition, 72% of families indicated that they had never met a dietician, signifying that many are relying on their physician for quality information regarding supplements and special diets. However, 33% admitted to not disclosing all supplements to their physician due to perceived physician lack of knowledge, no apparent benefit, the time commitment, and fear of judgment. Alarmingly, as 36.7% reported taking prescribed medication, a lack of disclosure may pose a risk to patients who may be unaware of the potential for interactions with concurrent CAM use. An open, patient-centered, non-judgmental approach is recommended for physicians when discussing supplement therapies [34]. This study highlights that patients would like to receive information about CAM from their conventional health care team, underscoring the importance of clinician knowledge about CAM and emerging research findings.

#### *4.3. Limitations*

There are a couple areas to consider when examining the limitations of this study. While the study provides novel information about the use of supplement therapies and special diet use in children with ASD, the study is limited as the majority of its participants are from Canada, therefore, may not be generalizable to other geographic regions. Further, as this is a descriptive study, it does not provide causal information regarding the effect of individual supplement therapies (e.g., omega 3 fatty acids causing relief of gastroenteritis symptoms). Finally, social desirability, a common bias where respondents answer in a way viewed favorably by others, may have influenced the data.

#### **5. Conclusions**

Supplement use continues to be a prevalent form of CAM used in ASD. While a variety of supplements and dietary interventions are utilized, the scientific consensus remains that there is currently little evidence to support the use of any nutritional supplement or dietary therapy for children with ASD. Future investigation into the effects of individual supplements on physiological and psychological functioning to determine optimal supplementation strategies in ASD is required.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2072-6643/11/8/1783/s1, File S1: Dietary Patterns and Supplement Use in Pediatric Autism, Table S1. All Supplement Use in Children with ASD, Table S2. Supplements listed in the "Other" Category by Parents.

**Author Contributions:** Conceptualization, M.S.T., R.F.M. and J.S.; Data curation, R.F.M.; Formal analysis, R.F.M. and J.A.P.; Funding acquisition, M.S.T.; Investigation, M.S.T.; Methodology, M.S.T.; Project administration, M.S.T.; Supervision, J.A.P., W.B.G. and J.S.; Validation, W.B.G.; Writing—original draft, M.S.T.; Writing—review & editing, M.S.T., R.F.M., J.A.P., W.B.G. and J.S.

**Funding:** This research was funded by Alberta Innovates Health Solutions, Alberta. (M.S.T. Alberta Innovates Research Studentship).

**Acknowledgments:** The authors would like to acknowledge Jodi Siever for her assistance with the statistical analysis, and Sarah Tabler, Yegor Korchemagin, and Madeleine Brulotte for their assistance with data collection and distribution. The authors would like to acknowledge Taking Strides for its continued support and promotion of participation.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

#### **References**

1. American Psychiatric Association. *Diagnostic and Statistical Manual of Mental Disorders*, 5th ed.; American Psychiatric Pub.: Philadelphia, PA, USA, 2013; Volume 21, ISBN 9780890425541.


© 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* **Exploring the Science behind** *Bifidobacterium breve* **M-16V in Infant Health**

#### **Chyn Boon Wong <sup>1</sup> , Noriyuki Iwabuchi <sup>2</sup> and Jin-zhong Xiao 1,\***


Received: 9 July 2019; Accepted: 24 July 2019; Published: 25 July 2019

**Abstract:** Probiotics intervention has been proposed as a feasible preventative approach against adverse health-related complications in infants. Nevertheless, the umbrella concept of probiotics has led to a massive application of probiotics in a range of products for promoting infant health, for which the strain-specificity, safety and efficacy findings associated with a specific probiotics strain are not clearly defined. *Bifidobacterium breve* M-16V is a commonly used probiotic strain in infants. M-16V has been demonstrated to offer potential in protecting infants from developing the devastating necrotising enterocolitis (NEC) and allergic diseases. This review comprehends the potential beneficial effects of M-16V on infant health particularly in the prevention and treatment of premature birth complications and immune-mediated disorders in infants. Mechanistic studies supporting the use of M-16V implicated that M-16V is capable of promoting early gut microbial colonisation and may be involved in the regulation of immune balance and inflammatory response to protect high-risk infants from NEC and allergies. Summarised information on M-16V has provided conceptual proof of the use of M-16V as a potential probiotics candidate aimed at promoting infant health, particularly in the vulnerable preterm population.

**Keywords:** *Bifidobacterium breve* M-16V; infant health; clinical efficacy; probiotics; gut microbiota

#### **1. Introduction**

Gut microbiota has become an important aspect of human health. Gut microbes regulate host intestinal, immunological and metabolic activities through their wide array of modulatory capabilities and enzymatic armoury [1]. Recent advances in microbial research have revealed the importance of early gut microbiome for neonatal health development and disease pathologies [2]. Aberrations of infant gut microbiota—a state of altered microbial composition and functionality—are associated with adverse health-related consequences including asthma [3], necrotising enterocolitis (NEC) [4], eczema [5] and inflammatory bowel disease [6] in neonatal stage or later in life.

Microbial ecosystem is established during the first three years of life for which a host–microbe symbiotic interaction that mutually benefits both is initiated [7]. It has been implicated that a number of extrinsic factors, such as gestational age, delivery mode and feeding types, are affecting the process of microbial colonisation in newborns [7,8]. Initial neonatal gut microbial colonisation represents a crucial window of opportunity for shaping a healthy gastrointestinal tract and immune system [9], and positive modulation of gut microbiota during this critical period could be an effective preventative approach against immune-mediated and microbiome-related disease pathologies. Consequently, probiotics intervention is receiving significant attention as a non-invasive attempt to optimize the infant microbiota as a means to improve health or prevent disease.

Probiotics are defined as "live microorganisms, which when administered in adequate amounts, confer a health benefit on the host" [10]. Studies over the last decade have demonstrated that

probiotics supplementation could promote gut microbial colonisation and prevent or treat diseases in infants [11,12]. These reports have led to a massive application of probiotics in a range of products including foods, infant formula, dietary supplements, and pharmaceutical products for promoting infant health. Nevertheless, many of the marketed probiotic products encompass limited well-consolidated regulatory oversight and a lack of human substantiation of efficacy [13]. Moreover, the safety and effects of probiotics in the vulnerable preterm population remain relatively limited and inconsistent [14,15]. Therefore, a detailed review of the scientific basis of a specific probiotic strain has emerged as an important aspect for an optimised selection of suitable probiotic candidates for use in infants.

*Bifidobacterium breve* M-16V (designated as M-16V) is a commonly used probiotic strain in infants for modulation of gut microbiota as a means to support healthy growth and promote health. Some evidence suggests that M-16V can stimulate bifidobacterial colonisation, alleviate allergic disorders and protect premature infants against NEC. Nevertheless, despite its nutritional and medicinal benefits, a comprehensive review of its specific clinical effects for infant health is still lacking. In this review, we discuss the effects of probiotic administration on infant health, with specific attention to the probiotic strain M-16V. We conducted a systematic survey for publications related to M-16V using the databases including MEDLINE [16], EMBase [17], medical journal web [18] and JDreamIII [19] from inception to 12 May 2019. Search terms were: M-16V OR M16V and the languages used were English and Japanese. A total of 60 articles including in vitro, preclinical and clinical studies were extracted (two review and 58 original articles). Among them, 31 were on the single strain of M-16V, five were on the probiotics mixture with other strains, and 24 were on synbiotics (Supplementary Tables S1 and S2). Herein, we summarise the significant effects of M-16V on premature birth complications and allergic disorders from the most relevant in vitro, animal and clinical studies. We believed that improved understanding of the role of M-16V in governing development of healthy gut microbiota during early life would inform rational therapeutic application of probiotics aimed at promoting infant health, especially in the vulnerable preterm population, and ultimately preventing chronic diseases later in life.

#### **2. Probiotics for Infant Health**

Probiotics intervention has gained overwhelming popularity over the last two decades as a potential nutritional supplementation approach to promote and maintain a healthy gut milieu and protect against dysbiosis in early life [20]. Accumulating evidence suggests that manipulation of the microbiota with the use of probiotics at an early stage may lead to an appropriate microbial colonisation and could have long-lasting impacts on child and adult health [21]. Probiotics that have been commonly given to neonates and infants include species of *Bifidobacterium* and *Lactobacillus*. Among them, *Bifidobacterium* is thought to be a keystone taxon in infant gut microbiota that plays a vital role in regulating immunological and physiological functions [22].

Bifidobacterial species have been isolated from the gastrointestinal tract of humans and animals as well as a few that have been isolated from human vagina, oral cavity, breast milk, sewage and foods, and could be categorised into two major groups; bifidobacterial species of human origins as human-residential bifidobacteria (HRB), whereas other species which are the natural inhabitants of animals or environment as non-HRB [23]. It has been demonstrated that bifidobacterial species of different residential origins display different levels of adaptability and functionality in the infant gut [23]. Of note, *B. longum* subsp. *infantis* (*B. infantis*), *B. longum* subsp. *longum* (*B. longum*), *B. bifidum*, and *B. breve*, which are frequently isolated from infant intestines and are referred to as infant-type HRB [23,24], have a large repertoire of genes for the utilisation of human milk oligosaccharides (HMOs) [25,26]. Studies have reported that infant-type HRB are capable of utilising HMOs with different metabolic pathways and degrees of degradation, highly compatible with human breast milk and tolerant to lysozyme [25,27], demonstrating how well adapted they are to the transmission routes and growth conditions in the infant gut. In fact, infant-type HRB have been shown to be the exclusive members of healthy breastfed infants [28,29], while formula-fed infants are also colonised

with species that are commonly isolated from adult intestines (adult-type HRB) such as *B. adolescentis* and *B. pseudocatenulatum* [30], implying the strains of infant-type HRB could be better probiotic candidates for infant use.

Several studies have demonstrated the use of infant-type HRB, including the strains of *B. breve* [31], *B. longum* [32], *B. infantis* [33] and *B. bifidum* [34], as probiotics for therapeutic purposes in neonates and infants. Administration of infant-type HRB probiotic strains in the first stage of life may result in the prevention of NEC and reduction in the risk as well as treatment of infectious and atopic disease [11,12]. Despite the promise, questions and concerns have been raised about the safety and clinical efficacy of probiotics administration, especially if the product is destined for use in infants. It is increasingly apparent that not all probiotics are equally safe, and the effects demonstrated with one strain cannot be extrapolated to another strain, even if they belong to the same species [35]. Of note, among many infant-type HRB probiotic strains that have been studied, M-16V possesses a proven track record of safety and a number of beneficial attributes that make it an attractive probiotic candidate for infant use. The following paragraphs will review the safety and specific health benefits of M-16V in infants within the field that seek to provide rigorous preclinical characterisation and substantial clinical evidence of M-16V for successful probiotics selection.

#### **3.** *Bifidobacterium breve* **M-16V as Infant Probiotic**

#### *3.1. Origin and Characteristics*

M-16V was originated from the gut of an infant in 1963 and was first commercially available in Japan in 1976 with the launch of Vinelac dietary supplement. In 1982, M-16V was added to a growing-up powdered formula called Yochien-Jidai in Japan and has since been incorporated in several other products including term and preterm infant formula.

M-16V is a non-motile, non-spore forming, rod-shaped anaerobic Gram-positive bacterium. It was identified as *B. breve* based on morphological, physiological and genetic characteristics. M-16V is highly accessible to human gastrointestinal tract with strong adherence activity [36]. In addition, lyophilised powder of M-16V manufactured by Morinaga Milk Industry Co., Ltd. possesses excellent stability during storage and high survivability in finished products such as powdered formula until consumption [37].

#### *3.2. Safety*

M-16V is well-evaluated for safety and has met the safety standards regulated by the Food and Agriculture Organization of the United Nations/World Health Organization (FAO/WHO) guidelines for the evaluation of microbes for probiotics use in foods [38]. In 2013, M-16V attained not only FDA-Notified Generally Recognized as Safe (GRAS) status for food uses (GRN No., 453) [39], but also GRAS status for infants (GRN No., 454) [40]. In addition, in 2016, M-16V has been included in the list of authorised probiotic strains for infant's food in China, in which M-16V is the only infant-type HRB strain among the nine strains in the list [41]. To date, there has been broad use of M-16V in low birth weight infants to reduce the risk of preterm birth complications in more than 120 neonatal intensive care units (NICU) in affiliated hospitals in Japan, Australia, New Zealand and Singapore [42–44].

Comprehensive safety evaluation of M-16V, which includes functional, genomic, and in vivo analyses, demonstrated that M-16V is a non-pathogenic, non-toxigenic, non-haemolytic and non-antibiotic resistant probiotic bacterium that does not contain any plasmids and does not display harmful metabolic activities [36,40,45,46]. M-16V produces L-lactic acid but no D-lactic acid. In addition, M-16V was reported to possess conjugated bile salt hydrolytic activity [36]. M-16V was able to hydrolyse conjugated bile acids taurocholic and glycocholic acid to the primary bile acid (cholic acid) and glycochenodeoxycholic and taurochenodeoxycholic acid to chenodeoxycholic acid, while the production of hepatotoxic and carcinogenic secondary bile acids (deoxycholic and lithocholic acid) was not detected upon complete biotransformation of bile salts [47]. These results resolve the concern about the safety of administering a secondary bile acids-producing bacterium.

Studies on acute and chronic toxicological features of M-16V revealed that both single and repeated oral administration of M-16V did not cause death and any toxic symptoms in a rat model [45]. For instance, groups of 10 male and 10 female three-week-old Crj:CD (SD) rats were orally administered with a single dose of M-16V at 6000 mg/kg (1.4 <sup>×</sup> 1012 CFU/kg) or 3000 mg/kg (6.9 <sup>×</sup> 1011 CFU/kg) and examined for acute toxic symptoms for 14 days. There were no gross abnormalities or histopathological findings attributable to the treatment in all organs throughout the test period, although slightly lower body weight was observed in male rats administered a high dose of M-16V as compared to the control on days 8 and 10. Furthermore, oral administration of M-16V with a 90-day repeated dose (2.3 <sup>×</sup> <sup>10</sup><sup>11</sup> CFU/kg/day) to five-week-old Crj:CD (SD) IGS rats revealed no adverse effects attributed to M-16V during the study period. M-16V induced no significant histopathological changes in all organs examined. These findings demonstrate the absence of acute and chronic toxicity by consumption of M-16V. Additional in vitro tests showed that M-16V did not possess mucin degradation ability [48]. Taken together, these studies support that M-16V is safe for use as a probiotic in humans.

#### **4. E**ff**ects of M-16V on Premature Birth Complications**

Prematurity, prolonged hospitalisation, immunodeficiency, antibiotics use and delayed enteral feeding are challenging ways to begin life for preterm infants [49]. Premature infants are at elevated risk to develop multiple health comorbidities; one of which is the devastating necrotising enterocolitis (NEC) [50]. It is a major cause of morbidity and mortality in extremely preterm infants that is associated with severe sepsis and intestinal perforation [51]. Although the exact aetiology and pathogenesis of NEC remain elusive, perturbation of the gut microbiota, leading to a hyperinflammatory response, appears to be a key factor that predisposes neonates to NEC [52]. Premature infants often present with an immature gut and exhibit delayed gut colonisation with beneficial commensal bacteria such as *Bifidobacterium* and *Bacteroides*, where instead they are more susceptible to colonisation by *Enterobacteriaceae* and *Enterococcus* [53,54]. Moreover, the use of antibiotics in premature and low birth weight infants disturbs the colonisation patterns of *Bifidobacterium* and shifts the gut microbial composition toward a high abundance of Proteobacteria, with a decreased in the overall diversity of the infant's gut microbiota [55–57]. To this end, the neonatal period has; therefore, emerged as an opportune time for preventive M-16V probiotics intervention to promote bifidobacterial colonisation, facilitate the development of gut mucosal immune system and improve infant health.

#### *4.1. Preclinical Studies*

Several animal studies have demonstrated the potential role of M-16V in improving the maturation of intestinal immune system and promoting bifidobacterial colonisation during early infancy. In a neonatal Lewis rat model, oral supplementation with M-16V (4.5 <sup>×</sup> <sup>10</sup><sup>8</sup> CFU/100 g of body weight/day; *n* = 8) during suckling period (days 6 to 18) showed potential in enhancing the homing process of naïve T cells to the mesenteric lymph nodes (MLN) and the retention of activated T cells in the intraepithelial (IEL) compartment [58]. The control group (*n* = 8) was administered with a matched volume of mineral water. Administration of M-16V increased the proportion of cells bearing toll-like receptor 4 (TLR4) in the MLN and IEL compartments, and enhanced the percentage of the integrin αEβ7+ and CD62L+ cells in the MLN and that of the integrin αEβ7+ cells in the IEL, as compared to the control. However, M-16V did not exert a systemic immune-enhancing effect in which the proportions of the main lymphocyte subset in spleen were not significantly affected by M-16V. In addition, M-16V induced no harmful effects on the rats wherein no significant differences were observed in the growth curve of the control and M-16V groups. Administration of M-16V significantly increased the levels of intestinal immunoglobulin A (IgA) as compared to the control, indicating M-16V could also strengthen the humoral intestinal immune response.

Furthermore, M-16V has also been reported to be able to regulate immune responses and appear to exert anti-inflammatory effects in rats at different developmental periods [59,60]. Oral administration of M-16V (5 <sup>×</sup> 108 CFU/day) to F344/Du rats significantly reduced the expression of inflammatory molecules during the newborn period (days 1 to 14) and regulated the expression of co-stimulatory molecules during the weaning period (days 21 to 34) [60]. In addition, the numbers of *Bifidobacterium* were also significantly increased in both the caecum and colon during the newborn period but not during weaning, as compared to the control groups [60].

Similarly, significant improvements in inflammatory conditions were also observed in DSS-induced colitis F344/N rats administered with M-16V (2.5 <sup>×</sup> 109 CFU/day) during weaning period (from postnatal days 21 to 34), as compared to the control rats. M-16V showed potential in altering systemic T-cell immune functions and suppressing inflammatory responses in colitis rats during the weaning period [59]. Taken together, these preclinical studies imply that supplementation with M-16V may aid in the development of intestinal immunity and prevention of intestinal inflammation during early infancy.

#### *4.2. Clinical Studies*

Multiple implementation cohort studies have demonstrated the potential effect of early administration of M-16V in improving bifidobacterial colonisation in preterm infants (gestation < 33 weeks) [42,61] and low birth weight infants (<2250 g) [32,42,62,63]. Earlier detection and longer maintenance of a bifidobacteria-dominant gut microbiome were observed in M-16V-supplemented infants. For instance, in a randomised, double-blind, placebo-controlled trial involving 159 preterm neonates (gestation < 33 weeks) ready to commence or on feeds for <12 h, supplementation of M-16V (3 <sup>×</sup> 109 CFU/day) for three weeks significantly increased the levels of faecal *B. breve* as compared to placebo control where the *B. breve* counts were below detection level [42]. M-16V supplement was well-tolerated by all enrolled preterm neonates with no adverse effects including probiotic sepsis and deaths. These findings suggest that M-16V is a suitable probiotic strain for routine use in preterm neonates to promote the acquisition of beneficial commensal bacteria.

Another randomised, placebo-controlled trial involving 30 preterm low birth weight infants, with mean gestation 32.8 weeks and birth weight 1486 g, also revealed a positive effect of M-16V on early gut colonisation with commensal *Bifidobacterium* spp. [63]. The subjects were randomly divided into three groups; (A) subjects received M-16V supplementation within several hours (mean: 7.2 h) of birth, (B) subjects received M-16V supplementation >24 h (mean: 36.5 h) after birth, and (C) subjects who were fed normally without M-16V supplementation as control group. Intragastrical administration of M-16V (1.6 <sup>×</sup> 108 CFU in 0.5 mL of 5% glucose sterile distilled water, twice daily) until the subjects were discharged from the hospital remedied the delayed bifidobacterial colonisation in both groups A and B, while no *Bifidobacterium* was detected in eight out of ten infants in group C during the observation period of seven weeks [63]. Notably, a significant earlier detection of bifidobacteria and a significant decrease in the cell numbers of *Enterobacteriaceae* were observed at two weeks after birth in infants administered with M-16V within several hours of birth (group A), indicating timing of administration of M-16V is highly important for which the earlier the administration of M-16V to preterm low birth weight infants, the better the effects of M-16V in promoting the colonisation of bifidobacteria and reducing the susceptibility to colonisation by potentially harmful bacteria.

A comparative, non-randomised controlled, prospective trial involving 44 low birth weight infants (body weight 1000–2000 g), who were ready for feeds within seven days of birth, administered with either single strain of M-16V (5 <sup>×</sup> <sup>10</sup><sup>8</sup> CFU/day) or probiotics mixture containing three bifidobacterial strains, M-16V, *B. infantis* M-63, and *B. longum* BB536 (5 <sup>×</sup> 108 CFU/day of each strain), for six weeks has also revealed a significant increase in the detection rates and cell numbers of bifidobacteria in the faeces [32]. Notably, administration of the three-species probiotics mixture resulted in an earlier formation of a bifidobacteria-dominant microbiota and a significantly lower level of *Enterobacteriaceae* than those administered with M-16V alone [32]. It was noted that not only the total cell numbers of bifidobacteria but also the cell numbers of M-16V was higher in infants administered with the three-strain probiotics mixture than those administered with M-16V alone. This study suggests that M-16V may act synergistically and cooperatively with other *Bifidobacterium* strains to confer a more remarkable beneficial effect in premature infants. Nevertheless, the comparison between probiotics and control groups from the different timeline in this trial may introduce bias that tends to compromise the efficacy of M-16V and is likely to result in unfair comparisons. Additionally, two comparable pilot studies involving ten very low birth weight premature infants (<1250 g) administered with either M-16V or *B. longum* at a dose of 5 <sup>×</sup> 108 CFU/day for eight weeks have also suggested a potential capability of M-16V to colonise in the premature gut [62,64]. Supplementation with M-16V had a longer colonisation rate than those with *B. longum*, for which, while M-16V was found to colonise the premature gut as early as week two after birth and remain dominant, the administered strain of *B. longum* was not detected from week six after birth [62,64]. Collectively, these data have exemplified that M-16V is potentially beneficial at promoting early colonisation of bifidobacteria and may; therefore, support healthy growth in premature infants.

Furthermore, M-16V has also been evaluated for the preventive effects on NEC, death and late-onset sepsis in premature infants; however, the clinical findings are not conclusive [43,44,65]. The first evidence of the potential preventive effects of M-16V on NEC came from a non-randomised clinical trial involving 338 infants (220 extremely low birth weight (ELBW) and 118 very low birth weight (VLBW) infants) receiving M-16V supplementation (1 <sup>×</sup> 10<sup>9</sup> CFU/day in raw breast milk or formula milk) started within several hours (mean 7.2 h) after birth and continued until discharged from NICU, and 226 infants (101 ELBW and 125 VLBW infants) as a historical control [44]. The study revealed that administration of M-16V was potentially effective at reducing the incidence of NEC in ELBW and VLBW infants as compared to that in the historical control group. A significant reduction in morbidity and mortality rate, as well as the mortality due to infection, was also observed in ELBW and VLBW infants receiving M-16V supplementation [44]. These encouraging results have suggested a potential role of M-16V in protecting premature infants from NEC and infection. However, the use of historical control from another timeline in this trial may introduce bias that tends to compromise the efficacy of M-16V and is likely to result in unfair comparisons.

More recently, M-16V was reported to be associated with decreased incidence of "NEC ≥ Stage II" and "NEC ≥ Stage II or all-cause mortality" in preterm neonates <34 weeks [43]. The study was a retrospective cohort study involving 835 preterm neonates as historical control and 920 preterm neonates receiving M-16V routine probiotics supplementation (3 <sup>×</sup> 109 CFU/day in 1.5 mL breast milk or sterile water) started when the infants were ready for enteral feeds and continued until the corrected age of 37 weeks. The initial daily dose for neonates <sup>&</sup>lt;28 weeks was 1.5 <sup>×</sup> <sup>10</sup><sup>9</sup> CFU/day until reaching feeds of 50 mL/kg/day. It was noted that M-16V significantly lowered the incidence of NEC in preterm VLBW neonates born <34 weeks, while the incidence of NEC was lower but not statistically significant in those born <28 weeks, although the small sample size used [43]. Despite the encouraging results, the trial may introduce potential bias in comparisons with the historical control drawn from another timeline.

In addition, a recent strain-specific systematic review revealed that the significant efficacy of M-16V to reduce the risk of NEC remains controversial [65]. It was concluded that current evidence is limited regarding the potential of M-16V as a probiotic for preterm neonates, albeit the meta-analysis of non-randomised controlled trials showed a significant effect of M-16V intervention in NEC [65]. No significant benefits on stage ≥2 NEC, late-onset sepsis, mortality and postnatal age at full feeds were reported in the meta-analysis of randomised controlled trials. Well-designed and adequately-powered randomised controlled trials are needed for definite confirmation. Nonetheless, all clinical studies included in the systematic review have concluded that M-16V supplementation was not associated with probiotic-associated sepsis in this vulnerable population [65], suggesting the risk of developing sepsis related to M-16V administration in the setting of severe illness to be relatively low. In fact, issues on *B. breve* sepsis in immunocompromised infants [66,67] and meningitis caused by other strain

of *B. breve* in preterm infants [68] have been reported. Another systematic review using a network meta-analysis approach showed that only few probiotic strains have statistically significant effects in reducing mortality, NEC, late-onset sepsis, and time until full enteral feeding [69]. M-16V was one of the many studied probiotic strains that did not show significant efficacy in preterm birth complications, reflecting a lack of adequately-powered randomised controlled trials to precisely define the clinical efficacy [69]. Further large and well-powered trials are needed to evaluate the effectiveness of M-16V in preventing NEC.

Taken together, these clinical studies underscore the potential roles of M-16V as a promising infant probiotic that could potentially impact the incidence, morbidity and mortality associated with NEC (Table 1).


**Table 1.** Summary from clinical studies of the effects of M-16V on premature birth complications.


**Table 1.** *Cont.*


**Table 1.** *Cont.*


**Table 1.** *Cont.*

BW, birth weight; CFU, colony-forming units; Non-RCT, non-randomised controlled trial; *B. breve*, *Bifidobacterium breve*; *B. infantis*, *Bifidobacterium infantis*; *B. longum*, *Bifidobacterium longum*; SGA, small for gestational age; IUGR, intrauterine growth retardation; LBW, low birth weight; VLBW, very low birth weight; ELBW, extremely low birth weight; NEC, necrotising enterocolitis; SCFAs, short-chain fatty acids; TGF-β, transforming growth factor-beta.

#### *4.3. Potential Mechanisms of Action*

Colonisation by commensal bifidobacteria during early life is indispensable for the normal development and growth of the gastrointestinal tract, particularly for epithelial barrier function and mucosal immunity [72,73]. A high abundance of bifidobacteria may contribute to improved health status and protect premature infants from diseases [74]. In fact, instability of the microbiome and a lack of bifidobacteria have been reported to be associated with NEC [74]. Towards this end, it seems likely that M-16V may potentially reduce the risk of developing NEC in premature infants by promoting the colonisation of bifidobacteria. Additional studies have been deployed to understand the mechanisms by which M-16V potentially reduces the risk of developing NEC [60,75]. In an experimental rat model of NEC, oral administration of M-16V was found to be effective at reducing the pathological scores of NEC and promoting survivability via modulation of TLR expressions and suppression of inflammatory responses [75]. Multiple reports have suggested that functional expression of TLRs is critical in the dynamic interaction between the host epithelium and the microbiota that enables normal intestinal epithelial development and immune homeostasis [76–78]. Differences in the expression of TLRs may; therefore, alter a host's response to a commensal or pathogenic microorganism [79]. Specifically, TLR4, which recognises the lipopolysaccharides of Gram-negative bacteria, was demonstrated as the key mediator in NEC development [76]. Increasing evidence suggests that NEC develops in response to an exaggerated pro-inflammatory signalling upon activation of TLR4 in the mucosa of the premature gut, leading to increased enterocyte apoptosis, mucosal injury, intestinal ischemia, and bacterial translocation [76,77,79,80]. It has indeed been demonstrated that TLR4 is expressed at higher levels in the premature infant gut than the full-term intestine [76,81]. Importantly, oral administration of M-16V to the experimental NEC rats significantly normalised the expression of TLR4, enhanced the expression of TLR2, and rectified the increased expression of pro-inflammatory cytokines, including interleukin-1β (IL-1β), IL-6 and tumour necrosis factor alpha (TNF-α) that resulted from NEC induction [75]. The superior anti-inflammatory effects of M-16V in colonic inflammation have also been demonstrated in an in vivo study using F344/Du rat pups models, wherein the expression of inflammation-related genes, including lipoprotein lipase (Lpl), glutathione peroxidase 2 (Gpx2) and lipopolysaccharide-binding protein (Lbp), was significantly reduced in the colon during the newborn period [60].

Furthermore, M-16V was also able to restore the tight junction barrier function by stimulating TLR2 expression and consequently protect the host against the development of NEC [75]. It has been reported that enhanced TLR2 expression by probiotics treatment could contribute to the down-regulation of TLR4 signalling that is activated by NEC [82,83]. Of note, aberrant TLR4 signalling was found to have a direct role in the breakdown of the gut barrier in NEC. Enhanced TLR4 signalling impairs mucosal repair and weakens the integrity of the gut, allowing for bacterial translocation and the downstream inflammatory response, which in aggregate lead to NEC [77]. Remarkably, M-16V showed potential in protecting the experimental NEC rats from intestinal barrier dysfunction via suppression of the NEC-induced elevated expressions of tight junction-related proteins, including ZO-1, claudin-1 and occludin [75].

Studies have also shown that daily M-16V supplementation may potentially facilitate the development of gut immune function and attenuate inflammation in preterm infants [58,59,70]. Administration of M-16V (1 <sup>×</sup> 10<sup>9</sup> CFU in 0.5 mL of 5% glucose solution), starting several hours after birth, twice daily, was shown to be capable of significantly elevating the levels of serum transforming growth factor beta-1 (TGF-β1) and enhancing the expression of TGF-β signalling molecule Smad3, while suppressing the levels of Smad antagonist, Smad7 in 19 preterm infants (mean birth weight of 1,378 ± 365 g and mean gestational age of 31.3 ± 3.16 weeks) as compared to the control on day 28 [70]. TGF-β1 is an important immune regulatory cytokine that prevents adverse immunologic reactions in infants. It exerts potent anti-proliferative and anti-inflammatory effects by activating Smad signalling pathway that mediates cell cycle arrest and induction of apoptosis [84]. Deficiency in TGF-β1 or its receptor has been implicated in fulminant inflammatory disease that proves lethal in the first week of life [85]. The encouraging result obtained in the clinical study has; therefore, implied that M-16V may assist the development of mucosal immunity and attenuate inflammatory reactions in preterm infants through upregulation of TGF-β signalling.

In addition to interacting with the immune system, M-16V may also potentially protect premature infants against gut mucosal injury and NEC through the production of short chain fatty acids (SCFAs) that can affect the health and integrity of the intestinal epithelial and immune cells [71]. In a randomised controlled trial involving 66 premature infants (birth weight ranged from 414 to 2124 g and gestation age ranged from 23 to 36 weeks), the effects of oral administration of M-16V on faecal SCFAs were evaluated. Based on birth weight, the infants were divided into three groups: 22 extremely low birth weight infants (ELBW; <1000 g), 22 very low birth weight infants (VLBW; <1500 g), and 22 low birth weight infants (LBW; <2500 g) and within each group, the subjects were further randomly divided into M-16V-supplemented or control groups. Administration of M-16V (1.6 <sup>×</sup> 108 CFU in 0.5 mL of 5% glucose sterile distilled water) at time of normal feeding, twice daily for four weeks led to an intestinal environment where the levels of butyrate was significantly decreased in ELBW and VLBW infants, while the ratio of acetate to total SCFAs was significantly increased in ELBW, VLBW, and LBW infants as compared with those of the control groups [71]. The exact significant contribution of such changes in the levels of SCFAs to premature infant health upon M-16V administration remains unclear. Although evidence is limited, higher acetate level in infants, which is often associated with a high abundance of bifidobacteria, has been reported to potentially improve intestinal immunity and promote epithelial cell barrier function [86,87]. Nevertheless, the healthy composition of an infant faecal metabolome remains understudied.

The premature gut is known to have structural and biochemical deficiencies which predispose infants to NEC. Although bacterial production of SCFAs plays an important role in the intestinal maturation and functions, it has been reported that overproduction of certain SCFA could be associated with an increased risk of NEC in premature infants [88]. Study has suggested that *Clostridium* spp., for which the abundance was higher in premature infants, may be implicated in NEC through excessive production of butyrate as a result of colonic lactose fermentation [89]. Overproduction of butyrate may cause gut mucosal injury and lead to intestinal inflammation in premature infants [90,91]. However, numerous studies have also demonstrated the importance of butyrate for colon health and its beneficial effects on intestinal inflammation and barrier integrity [92–94]. Further studies are warranted to resolve the contradictory roles of butyrate and to investigate the association between reduction of butyrate production by M-16V and protection against NEC.

Taken together, the findings from both animal and clinical studies have shed lights into the potential protective mechanisms of M-16V against NEC in premature infants. It is evident that M-16V may potentially reduce the risk of developing NEC in premature infants by promoting bifidobacterial colonisation, modulating the expressions of TLRs and inflammatory responses, and aiding in the development of mucosal immunity (Figure 1).

**Figure 1.** Administration of *Bifidobacterium breve* M-16V showed potential in reducing the risk of developing necrotising enterocolitis (NEC) in premature infants. M-16V stimulates the colonisation of bifidobacteria and could potentially improve the intestinal environment and gut barrier function. Additional mechanistic studies revealed that M-16V may assist the development of mucosal immunity through up-regulation of transforming growth factor-beta (TGF-β) signalling in premature infants and attenuate inflammatory reactions by modulating the expressions of toll-like receptor 2 (TLR2) and TLR4. IL-1β, interleukin-1β; IL-6, interleukin-6; TNF- α, tumour necrosis factor alpha; ↑, increased; ↓, decreased.

#### **5. E**ff**ects of M-16V on Allergic Disorders**

The prevalence of allergic diseases in infants has increased strikingly worldwide in the past few decades [95]. While the pathogenesis of allergic diseases is likely to be multifactorial, deviations in gut colonisation during early life are possible major factors promoting abnormal postnatal immune maturation [96]. The hygiene hypothesis suggests that insufficient or aberrant microbial stimulation during the critical neonatal period may lead to an exaggerated adaptive immune response and reduced tolerance [97]. Although compelling evidence for microbiota associations with allergic disease and related conditions is emerging, a causal relation between specific bacterial taxa and the development of allergy remains unclear. Several studies have reported differences in gut microbiota composition and lower abundance of bifidobacteria and lactobacilli in the infant's gut precede the onset of allergic manifestations [98,99]. In addition, multiple cohort studies suggested that high abundance of *Escherichia coli* or *Clostridium di*ffi*cile* was associated with the development of eczema or atopy [100,101], while a low gut microbial diversity and an elevated *Enterobacteriaceae* to *Bacteroidaceae* (E/B ratio) in early infancy may contribute to the development of food allergy [102]. In this instance, a notable higher abundance of Firmicutes particularly *Clostridium* spp., *Blautia* spp., and a lower abundance of Actinobacteria in the early gut microbiota has also been described to contribute to the development of allergic diseases such as food allergy in infants [103], and type 1 diabetes in children [104]. On this basis, modulation of gut microbiota during early life through M-16V intervention has emerged as a potential measure to prevent allergic disorders in infants.

#### *5.1. Preclinical Studies*

The anti-allergic capability of M-16V in allergic airways disease, food allergy, and chronic asthma has been consolidated in a number of in vitro and animal studies [105–110]. In a bacterial strains comparative study assessing the capability of a panel of six bacterial strains (M-16V, *B. infantis* NumRes251, *B. animalis* NumRes252 and NumREs253, *Lactobacillus plantarum* NumRes8 and *L. rhamnosus* NumRes6) to alleviate allergic symptoms in ovalbumin (OVA)-sensitized BALB/c mice, M-16V was identified as the most effective strain in reducing allergic response [105]. Remarkably, in contrast to the other tested bifidobacteria, only the oral treatment with M-16V significantly inhibited the airway reactivity to methacholine and reduced acute allergic skin reactions to OVA. These discrepancies emphasise that the immuno-modulatory activity of probiotic strains is highly strain-specific.

Numerous studies have also shown that a synbiotic intervention, comprising M-16V and a galacto–fructooligosaccharide (GOS/FOS) mixture, was protective against the development of symptoms of oral sensitization with whey in mice model [110]. The promising effect was confirmed in an in vivo study demonstrating the partial prevention of skin reaction due to cow's milk allergy, following the probiotic administration in combination with specific β-lactoglobulin-derived peptides and a specific blend of short and long-chain fructo-oligosaccharides in mice [106]. Particularly, besides increasing the caecal content of propionic and butyric acid, the treatment with M-16V synbiotic formulation increased the expression of IL-22, which plays an antimicrobial role in the innate immune response and on the anti-inflammatory cytokine IL-10 in the Peyer's patches [106].

Additional preclinical studies revealed that administration of M-16V alone (109 CFU) [108], or in combination with non-digestible oligosaccharides (scFOS, lcFOS and pectin-derived acidic-oligosaccharides (AOS)) [109], could suppress pulmonary airway inflammation in murine OVA-induced chronic asthma model. M-16V treatments (both single-strain and synbiotic interventions) reduced T cell activation and mast cell degranulation, modulated expression of pattern recognition receptors, cytokines and transcription factors, and reduced airway remodelling [108,109]. More specifically, the treatments induced regulatory T cell responses in the airways by increasing IL-10 and Foxp3 transcription in lung tissue and systemically. These studies suggest that M-16V intervention, either as a single organism or as synbiotic, could be beneficial in the treatment of chronic inflammation in allergic asthma. Altogether, these findings laid the ground for the preventive and therapeutic effects of M-16V on allergic disorders.

#### *5.2. Clinical Studies*

Several interventional studies suggest that M-16V could promote bifidobacterial colonisation and prevent or reduce the severity of allergic diseases, including atopic dermatitis (eczema), food allergy, allergic rhinitis and asthma [111–113]. In a randomised controlled trial, oral administration of M-16V significantly improved the symptoms of atopic dermatitis in infants as compared to the control group [112]. The study randomly allocated 15 infants (aged 8.6 ± 4.5 months) with atopic dermatitis who had a *Bifidobacterium*-deficit gut microbiota to receive either lyophilised powder of M-16V (*<sup>n</sup>* <sup>=</sup> 8; 5 <sup>×</sup> 109 CFU/day) for one month or no M-16V supplementation as a control. It was noted that administration of M-16V was not only effective at alleviating the severity of allergic symptoms but also significantly increased the proportion of *Bifidobacterium* and decreased the levels of total aerobes in the gut microbiota of infants with atopic dermatitis [112]. Nevertheless, a significant correlation between alleviation of allergic symptoms and changes of the gut microbiota was not detected; suggesting M-16V may possess a direct immuno-modulatory effect on intestinal epithelial cells and not necessarily through the interaction with the gut microbiota.

Another clinical study involving 17 infants with cow's milk hypersensitivity with atopic dermatitis (aged 3.1–18.5 months) has also revealed the capability of M-16V supplementation (5 <sup>×</sup> 10<sup>9</sup> CFU/day for three months) to ameliorate allergic symptoms and improve gut microbiota composition [113]. The preventive effects of M-16V on allergic disorders have further been exemplified in a remarkable placebo-controlled, double-blinded and randomised trial involving 40 Italian children (mean age

9 <sup>±</sup> 2.2 years) treated with a probiotics mixture containing M-16V (1 <sup>×</sup> 109 CFU), *B. longum* BB536 (3 <sup>×</sup> <sup>10</sup><sup>9</sup> CFU) and *B. infantis* M-63 (1 <sup>×</sup> 10<sup>9</sup> CFU), for four weeks [111]. Administration of probiotics mixture protected the children against pollen-induced IgE-mediated allergic rhinitis and intermittent asthma and improved their quality of life, for which these parameters were worsened in the placebo group. This study implies that, in addition to its effectiveness as a single organism, as aforementioned, M-16V could also dampen allergic disorders when combined with other *Bifidobacterium* strains.

More interestingly, in an open trial, administration of a probiotics mixture including M-16V during pregnancy as well as in postnatal period tied to lower the risk of developing allergic disorders in infants [114]. The study involved 130 mothers who were provided with a daily powder formulation (two sachets daily, 1 g/sachet) containing M-16V and *B. longum* BB536 (5 <sup>×</sup> 10<sup>9</sup> CFU/g of each strain) one month before the expected date of delivery and postnatally to their infants (one sachet daily) for six months. Another 36 mother–infant pairs who did not receive the bifidobacterial supplementation were served as the control. Prenatal and postnatal supplementation with the bifidobacteria mixture significantly reduced the risk of developing eczema and atopic dermatitis in infants during the first 18 months of life as compared to the control group [114]. Additionally, the probiotics intervention (M-16V and *B. longum* BB536) resulted in slight changes in the gut microbial composition, wherein a significantly higher proportion of *Bacteroidetes* was observed in the microbiota of infants receiving the bifidobacteria mixture than in that of the control group at four months of age. The relative abundance of Proteobacteria was also significantly lower in mothers receiving the bifidobacteria mixture at the time of delivery than those in the control group, and was positively correlated with that of infants at four months of age. These findings implicate that supplementation with bifidobacteria mixture of M-16V and *B. longum* BB536 during pregnancy may modulate both the maternal and neonatal gut microbiota for prevention of allergies upset in infants later in life. Further studies are needed to elucidate the association between the probiotics-modulated gut microbiota and allergy development in infants. Collectively, these findings are cautiously promising with respect to the use of probiotics for the primary prevention of eczema in pregnant mothers of infants at high risk for developing allergy and in high-risk infants, as recommended in recent guidelines from the World Allergy Organization [115].

Furthermore, synbiotic intervention of M-16V has also been reported to be effective in preventing asthma-like symptoms in infants with atopic dermatitis [116]. The study was a double-blind, placebo-controlled, multicentre trial involving 90 infants with atopic dermatitis (aged <7 months) who received either an extensively hydrolysed formula containing M-16V (1.3 <sup>×</sup> 10<sup>9</sup> CFU/100 mL and a GOS/FOS mixture (90%/10%; 0.8 g/100 mL) or the same formula without synbiotics for 12 weeks. The follow-up period for this trial was one year. It was noted that the synbiotic intervention significantly reduced the prevalence of frequent wheezing and/or noisy breathing apart from colds as well as the usage of asthma medication as compared to the placebo group [116]. As a result, it seems to be likely that combining M-16V with prebiotics—synbiotic intervention—could result in stronger immunomodulatory effects for prevention against allergic disorders. Collectively, these findings serve as a basis to incorporate M-16V in prebiotics-supplemented infant formula as a means to promote infant health.

Taken together, these clinical findings support the notion that administration of M-16V can be a potential prophylaxis approach to improve immune tolerance and consequently protect high-risk infants from allergic diseases (Table 2), although larger clinical trials are needed for definite confirmation.


**Table 2.** Summary from clinical studies of the effects of M-16V on allergic disorders.


**Table 2.** *Cont.*

CFU, colony-forming units; IgE, immunoglobulin E; Non-RCT, non-randomised controlled trial; *B. breve*, *Bifidobacterium breve*; *B. infantis*, *Bifidobacterium infantis*; *B. longum*, *Bifidobacterium longum*; scGOS, short-chain galactooligosaccharides; lcFOS, long-chain fructooligosacharides.

#### *5.3. Potential Mechanisms of Action*

The mechanisms through which M-16V acts to protect infants against allergic disorders are not fully understood but clearly involve the contributions from M-16V to promote bifidobacterial colonisation, modulate Th2-skewed immune response and attenuate inflammatory reactions (Figure 2). M-16V has been shown to exert immuno-regulatory effect and anti-inflammatory capability in vitro, albeit the effect on allergic reaction has not been specifically demonstrated. M-16V was reported to interact with TLR2, upregulate the expression of ubiquitin-editing enzyme A20 in porcine intestinal

epithelial cells challenged with heat-killed enterotoxigenic *Escherichia coli*, and beneficially modulate the subsequent TLR4 activation by reducing the activation of MAPK and NF-κB pathways and the production of pro-inflammatory cytokines (IL-8, monocyte chemotactic protein (MCP)-1, and IL-6) [83]. Furthermore, in an experimental OVA-immunised mice model, oral administration of M-16V (5 <sup>×</sup> 108 CFU/0.5 mL/day/animal) for 21 days significantly reduced the serum levels of total IgE, OVA-specific IgE and OVA-specific IgG1 and ex vivo production of IL-4 by the splenocytes, as compared to control [117]. In addition, M-16V could potentially modulate the systemic Th1/Th2 balance in vitro wherein the production of OVA-induced total IgE and IL-4 was suppressed and the secretion of IFN-γ and IL-10 was induced by M-16V in a dose-dependent manner. Nonetheless, M-16V did not induce IL-12 production. It is; therefore, suggested that M-16V may have the potential to restore Th2 skewed immune response, which was at least partially independent of the Th1 cytokine induction [117].

**Figure 2.** *Bifidobacterium breve* M-16V could potentially promote bifidobacterial colonisation and may prevent or reduce the severity of allergic diseases in infants. Specifically, M-16V may suppress the differentiation naïve T-helper cells (Th0) into T-helper (Th) 2 cells and the production of Th2 cytokines such as interleukin-4 (IL-4) and IL-5, and subsequently attenuate allergic inflammation by reducing the production of immunoglobulin E (IgE) and IgG1 in B cells and the release of pro-inflammatory mediators including IL-6 and IL-8. In addition, M-16V could also potentially assist immune tolerance and attenuate allergic reactions in infants through modulation of TGF-β signalling. ↓, decreased; ↑, increased.

It has been suggested that the pathology of allergic disease is driven by the allergen-specific Th2 cytokines such as IL-4 and IL-5, which play a triggering role in the activation/recruitment of IgE antibody-producing B cells, mast cells and eosinophils [118–120]. Notably, in an OVA-allergic asthma mouse model, oral administration of M-16V (109 CFU/0.4 mL/day/animal) for 17 days prevalently reduced the number of eosinophils in the bronchoalveolar lavage fluid and reduced the levels of OVA-specific IgE and IgG1 and Th2 cytokines (IL-4 and IL-5) [105]. In addition, M-16V has also been shown to potentially assist immune tolerance and attenuate allergic reactions in premature infants through modulation of TGF-β signalling [70]. Altogether, these findings provide proof of the potential of M-16V in modulating Th2 skewed allergic immune response. Further in-depth studies are required to elucidate the exact mechanisms by which M-16V prevents and ameliorates allergic disorders in infants.

#### **6. Conclusions**

*Bifidobacterium breve* M-16V has emerged as a probiotic strain that exerts positive effects on infant health. With the data from in vitro animal and clinical studies, M-16V holds promise to treat adverse health-related conditions in infants, particularly the vulnerable premature populations, and possesses a proven track record of safety. Mounting evidence favours the use of M-16V as a worthy and suitable infant probiotic in early life for promoting a healthy gut microbial colonisation and maturation in premature infants and preventing the development of NEC and allergic diseases. Although the mechanistic insights supporting the use of M-16V are not robust, it has become clear that M-16V may modulate the gut microbiota, interact with TLRs and regulate inflammatory responses to reduce the risk of developing life-threatening diseases and immune-mediated disorders. Despite the promising results, many studies summarised here have multiple limitations such as potential bias in non-randomised controlled trials and small sample size. Therefore, additional well-designed randomised controlled trials with larger sample size are needed to serve as the basis for developing conclusive evidence on M-16V intervention in vulnerable preterm populations. In addition, further investigations are required for an increased understanding of the protective mechanisms of M-16V and to releasing the full potential of M-16V as a human probiotic in paediatrics.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2072-6643/11/8/1724/s1, Table S1. Summary of publications related to *Bifidobacterium breve* M-16V; Table S2. Articles related to *Bifidobacterium breve* M-16V.

**Author Contributions:** Conceptualization, C.B.W. and J.-z.X.; resources, C.B.W.; data curation, C.B.W.; writing—original draft preparation, C.B.W.; writing—review and editing, C.B.W., N.I. and J.-z.X.; visualization, C.B.W.; supervision, J.-z.X.

**Funding:** This research received no external funding. Our work was funded by Morinaga Milk Industry Co., LTD. Employees of Morinaga Milk Industry Co., LTD., C.B.W., N.I., and J.-z.X. received a salary from the company. The specific roles of these authors are articulated in the "author contributions" section. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

**Acknowledgments:** The authors are grateful to all the researchers whom we cited in this review for their significant and valuable research.

**Conflicts of Interest:** The authors, C.B.W., N.I., and J.-z.X. are employees of Morinaga Milk Industry Co., Ltd., which has several probiotic products marketed worldwide. This does not alter our adherence to Nutrients policies on sharing data and materials.

#### **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* **Nutraceutical Potential of** *Carica papaya* **in Metabolic Syndrome**

**Lidiani F. Santana 1, Aline C. Inada <sup>1</sup> , Bruna Larissa Spontoni do Espirito Santo 1, Wander F. O. Filiú 2, Arnildo Pott 3, Flávio M. Alves <sup>3</sup> , Rita de Cássia A. Guimarães 1, Karine de Cássia Freitas 1,\* and Priscila A. Hiane <sup>1</sup>**


Received: 21 June 2019; Accepted: 10 July 2019; Published: 16 July 2019

**Abstract:** *Carica papaya* L. is a well-known fruit worldwide, and its highest production occurs in tropical and subtropical regions. The pulp contains vitamins A, C, and E, B complex vitamins, such as pantothenic acid and folate, and minerals, such as magnesium and potassium, as well as food fibers. Phenolic compounds, such as benzyl isothiocyanate, glucosinolates, tocopherols (α and δ), β-cryptoxanthin, β-carotene and carotenoids, are found in the seeds. The oil extracted from the seed principally presents oleic fatty acid followed by palmitic, linoleic and stearic acids, whereas the leaves have high contents of food fibers and polyphenolic compounds, flavonoids, saponins, pro-anthocyanins, tocopherol, and benzyl isothiocyanate. Studies demonstrated that the nutrients present in its composition have beneficial effects on the cardiovascular system, protecting it against cardiovascular illnesses and preventing harm caused by free radicals. It has also been reported that it aids in the treatment of diabetes mellitus and in the reduction of cholesterol levels. Thus, both the pulp and the other parts of the plant (leaves and seeds) present antioxidant, anti-hypertensive, hypoglycemic, and hypolipidemic actions, which, in turn, can contribute to the prevention and treatment of obesity and associated metabolic disorders.

**Keywords:** blood glucose; food composition; metabolic syndrome; natural products; *Carica papaya*

#### **1. Introduction**

Plants with healing properties are utilized in folk medicine and, since remote times, have been considered traditional therapeutic approaches that have effects on health. They are also advantageous from a cost–benefit point of view [1]. Synthetic drugs used to be the first option for the treatment of several diseases. However, because of the adverse effects shown by long- or even short-term consumption, studies aiming at the use of alternative therapies in the treatment and prevention of diseases have increased considerably [2].

One alternative therapy includes the use of nutraceuticals, which, in turn, according to the existing regulations, cannot be categorized or defined either as food or a drug, but can be understood in the category of food supplements, with beneficial properties for health maintenance, in particular for some pathologic conditions. Therefore, a therapeutic approach, based on nutraceuticals for maintenance of health, resulted in a worldwide "nutraceutical revolution" [3].

Among plants with beneficial properties on health is *Carica papaya*, the well-known papaya. This fruit contains considerable concentrations of vitamins, bioactive compounds and a lipidic composition that reduces inflammatory markers and anti-platelet aggregation, protects against thrombogenesis and oxidative stress, and prevents hypercholesterolemia—factors that can be triggered by obesity [4,5].

*Carica papaya* is a popular fruit, and its largest production occurs in tropical and subtropical regions. According to the Food and Agriculture Organization of the United Nations (FAO) [6], over 6.8 million tons of the fruit are produced in the world annually, ca. 440 thousand ha. Central and South America, especially Brazil, are responsible for 47% of the fruit yield, produced year round, being an important source of nutrients with a low cost and great availability in the market.

*Carica papaya* is consumed worldwide, either in natura or processed as jam, sweets and pulp, and to aggregate the nutritional value, other parts of the plant (leaves and seeds) are added to some products in the form of teas and flours [7]. The pulp composition presents three important sources of vitamins with potential antioxidant action, A, C and E [8], besides minerals, such as magnesium and potassium, and B complex vitamins, such as pantothenic acid and folate [9], as well as the presence of food fibers [10]. Besides these nutrients, papaya contains the enzyme papain, effective in increasing intestinal motility and transit time, and is also utilized in the treatment of traumas, allergies and sport lesions [5]. Some studies observed the presence of proteolytic enzymes, such as chymopapain, with anti-viral, antifungal and antibacterial properties [5,11].

The seed contains phenolic compounds, such as benzyl isothiocyanate, glucosinolates, tocopherols (α and δ), β-cryptoxanthin, β-carotene and carotenoids [12,13], while the seed oil principally presents oleic fatty acid, followed by palmitic, linoleic and stearic acids [14]. The leaves have a high content of food fibers and polyphenolic compounds, such as flavonoids, saponins, pro-anthocyanins, tocopherol and benzyl isothiocyanate [15].

Considering the nutrients present in its composition, beneficial effects have been observed, with a significant improvement in the cardiovascular system, protecting against cardiovascular illnesses, heart attack and strokes [16]. Other studies have pointed out that this fruit is an excellent source of beta-carotene (888 IU/100 g), preventing harms caused by free radicals [17], besides exerting a role in the prevention of cardiovascular illnesses, diabetes mellitus (types 1 and 2) and in the reduction of cholesterol levels through its high content of fibers, which diminish fat absorption [5,18].

*Carica papaya* is a plant that is easily accessed and widely available. Furthermore, scientific studies have demonstrated the biological activities and medicinal applications of different parts of the plant. However, few studies have demonstrated the therapeutic potential in metabolic dysfunctions in experimental models specific to obesity. Therefore, the present study will investigate the nutritional value and bioactive compounds of the plant, as well as the existing medicinal uses and possible application in the metabolic syndrome.

#### **2. Nutritional Properties:** *C. papaya* **L.**

#### *2.1. Chemical Composition*

The tree *C. papaya* is native to Central and South America and is one of the most cultivated fruit plants in the world, especially in tropical and subtropical areas [6]. It is a herbaceous perennial plant, with a milky latex that can reach 12 m in height. It has a year-round fruit production, and each fruit weighs between 1000 and 3000 g [18] (Figure 1).

**Figure 1.** Images of *Carica papaya* L. (papaya CV Formosa): (**a**) Tree with leaves and green fruits, (**b**) female flower, and (**c**) ripe fruit with seeds and pulp. Photos: L. F. Santana.

The fruit of *C. papaya* is considered one of the most common fruits in relation to human consumption and provides a favorable cost benefit in consideration of its nutritional value, with a low caloric content (Table 1) and rich concentration of vitamins and minerals (Table 2) [19].

**Table 1.** Nutritional value of the macronutrients and fibers of *Carica papaya* L. (papaya) per 100 g of pulp of ripe fruit, seeds and leaves [5,18].


**Table 2.** Value of the minerals and vitamins of *Carica papaya* L. (papaya) per 100 g of ripe fruit pulp, seeds and leaves [7,19].


ND: not determined.

Among the most commercialized fruits, such as apple, banana, water melon, and orange, papaya has the highest concentrations of vitamin C (61.8 mg·100 g<sup>−</sup>1), vitamin A (328 mg·100 g<sup>−</sup>1), riboflavin (0.05 mg·100 g<sup>−</sup>1), folate (38 mg·100 g<sup>−</sup>1), thiamine (0.04 mg·100 g<sup>−</sup>1), niacin (0.34 mg·100 g<sup>−</sup>1), calcium (24 mg·100 g−1), iron (0.1 g·100 g−1), potassium (257 mg·100 g−1), and fiber (0.8 g·100 g−1), as well as presenting a low caloric value (32 kcal·100 g−<sup>1</sup> ripe fruit) and being one of the preferred fruits for weight loss. In addition, it has a high carotene content when compared with other fruits [5,20].

The green fruit is used in preparations, such as salads, cakes, ice creams and juice, without carotene [20], but with all of the other nutrients listed in Tables 1 and 2. Besides the ripe papaya pulp, the consumption of other parts, such as the seeds and leaves, is appropriate, since they have a higher nutritional value and more fibers [7]. The leaves and seeds present a higher carbohydrate content, compared with the fruit pulp, presenting 78.2 g and 436 g·100 g<sup>−</sup>1, respectively (Table 1), and the same is observed for the values of proteins (5.8 g and 2.63 g·100 g−1), lipids (1.4 g and 3.1 g·100 g−1) and fibers (13.1 g and 2.13 g·100 g<sup>−</sup>1). Consequently, they have a higher caloric value (seeds with 212.7 kcal and leaves with 348.6 kcal) [18].

Compared with the seeds and pulp, the concentrations of vitamins and minerals are different in the leaves, because they play an important role in fruit development [21]. For example, in relation to minerals, the contents of magnesium, iron, potassium and calcium are higher in the leaves (the leaves have 366.1 mg and the seeds 54.4 mg·100 g<sup>−</sup>1). Regarding vitamins, except for C, the leaves present a higher content, with the highest concentration in the pulp, as shown in Table 2 [7,22].

#### *2.2. Phytochemical Composition*

Different parts of the *C. papaya* plant, such as the fruits, seeds, roots, leaves, stem and latex were found to have important bioactive compounds, which, in turn, may exert medicinal effects. The methanolic extract of unripe fruits exerted antioxidant activity in vivo, for the presence of compounds, such as quercetin and β−sitosterol [20]. Other studies detected considerable quantities of total phenols (203 mg·100 g−<sup>1</sup> extract) [22] in the methanolic extract of the papaya pulp, while terpenoids, alkaloids, flavonoids and saponins were identified in the water extract [9] (Table 3). Besides, in papaya seed extracts, the presence of benzyl isothiocyanate [13] and expressive quantities of glucosinolates were observed [12].


**Table 3.** Main phytochemical compounds present in *C. papaya* L. (papaya): ripe fruit pulp, seeds and leaves [14,15,19,23,24].

Evaluating the oil extracted from the seeds, the main quantified fatty acid was oleic acid (71.30%), followed by palmitic (16.16%), linoleic (6.06%), and stearic acids (4.73%) (Table 3) [25]. The predominant tocopherols were <sup>α</sup> and <sup>δ</sup>-tocopherol, with 51.85 and 18.9 mg·kg–1, respectively. The <sup>β</sup>-cryptoxanthin (4.29 mg·kg–1) and <sup>β</sup>-carotene (2.76 mg·kg–1) were the quantified carotenoids, and the content of total phenolic compounds was 957.60 mg·kg–1 [26].

Studies showed that *C. papaya* leaves present tocopherol [24], lycopene [14], flavonoids [25] and benzyl isothiocyanate [23]. Another important study demonstrated that the phytochemical composition of ethanolic, methanolic, acetate and water extracts of *C. papaya* leaves is independent of the type of extract, detecting polyphenols, flavonoids, saponins and pro-anthocyanins, besides the antioxidant activity, evaluated by the method 1,1-diphenyl-2-picrylhydrazyl (DPPH). However, the water extract had superior values of polyphenols (23.1 mgGAE/g) and antioxidant activity (166 μgTE/g), while the ethanolic extract had the highest concentrations of flavonoids (17.1 mgCE/g), saponins (82.8 mgAes/g) and pro-anthocyanins (7.91 mgCE/g) [15].

#### **3. Medicinal Properties of** *C. papaya*

*Carica papaya* contains important nutrients (Tables 1 and 2) and bioactive compounds, such as antioxidants, vitamins, and minerals (Table 3), with nutraceutical characteristics and potential beneficial effects on health [5]. Studies evaluated the actions of *C. papaya* in recovery from drug-induced hepatoxicity in rodents [27–30], e.g., by carbon tetrachloride (CCl4), considered a potent inducer of toxic effects in the liver for being highly metabolized in bodily tissues because of the high reactivity of halogenated metabolites (CCl3 and Cl), and such activation of metabolites liberate the active oxygen species (ROS). Another drug in question was acetaminophen (600 mg·100 g−1), an analgesic and anti-pyretic, which causes acute hepatocellular damage that can be lethal if not treated [27].

Among the main effects that extracts of different parts of *C. papaya* demonstrated, in recovery from toxic effects on the liver, are the decrease in hepatic damage with the increase in antioxidant enzymes such as superoxide dismutase (SOD), glutathione (GSH), and catalase in the liver and decreases in the enzymes alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) [27–31]. Similar data were observed in nephrotoxicity induced by CCl4 in rats treated with *C. papaya* seed water extract, depending on the dose and time of treatment. The results showed a drop in biochemical parameters, such as the serum levels of uric acid, urea, and creatinine, besides the renal protecting ion, constated by histological evaluation after recovery from renal lesions [32].

Besides the effects on hepatic and renal toxicity, *C. papaya* displayed antimicrobial [33], anti-amoebic [34], anti-parasitic [12,13], and anti-malaria actions [11]. The use of *C. papaya* leaf water extract at different concentrations (25, 50, 100, 200 mg·mL<sup>−</sup>1) had antimicrobial activity on the inhibition of some human pathogens, such as *Escherichia coli*, *Pseudomonas aeruginosa*, *Kleibseilla pneumoniae*, *Staphylococcus aureus,* and *Proteus mirabilis* [33]. Another study, utilizing the same type of extract at the dose of 100 mg·mL−<sup>1</sup> found anti-amebic activity against *Entamoeba histolytica* [34]. Furthermore, *C. papaya* seeds had an activity on human intestinal parasites (*Caernorhabditis elegans*), without considerable side effects, owing to the presence of B-benzylisothiocyanate, a potent anti-helminthic [12]. Studies have shown the inhibitory effects on *Plasmodium falciparum* (malaria) in vitro, while the extract from the green fruit pulp of *C. papaya* demonstrated the highest anti-malaria activity, in comparison with different extracts of other tested plants [11].

Other studies showed the action of the water extract of *C. papaya* leaves (20 mg·mL−1) on proliferation inhibition in strains of solid tumor cells in trials in vitro, e.g., cervical carcinoma (Hela), breast adenocarcinoma (MCF-7), hepatocellular carcinoma (HepG2), lung adenocarcinoma (PC14), pancreatic carcinoma (Panc-1) and mesothelioma (H2452) in a dose-dependent manner, suggesting the anti-tumoral action of the extract. To determine whether the proliferation inhibition was associated with decreased cell viability, the water extract of *C. papaya* leaves was shown to inhibit proliferation responses of hematopoietic cell strains, including T-cell lymphoma (Jurkat), plasma cell leukemia (ARH77), Burkitt's lymphoma (Raji), and large-cell anaplastic lymphoma (Karpas-299). In addition, the *C. papaya* leaf extract showed immunomodulatory activity on peripheral human blood mononuclear cells [17].

Antiulcerogenic actions were verified with the use of *C. papaya* seed water extract (50–100 mg/kg), the same action being observed using the methanolic extract, showing gastro-protective activity in animals, in both prevention and treatment models of gastric ulcer [35]. In addition, the *C. papaya* seed extract was able to reduce the contractility of rabbit jejunum smooth muscle—the responsible compound being benzyl isothiocyanate [36].

Effective anti-inflammatory actions were verified by applying *C. papaya* leaf ethanolic extract (25–250 mg·kg<sup>−</sup>1) on carrageenan-induced paw edema in rats. However, after the ulcerogenic activity tests, the extract with the highest concentration produced a mild irritation of the gastric mucosa [37]. Besides the effects on inflammation, *C. papaya* showed wound healing properties. It is known that diabetic patients often have persistent difficulty in healing and require the delicate handling of wounds, demanding appropriate care. The topical use of the water extract of green fruits of *C. papaya* on wounds in diabetic rats, induced by streptozotocin (STPZ, 50 mg·kg−1), exhibited a 77% reduction of the wound, induced by excision, with faster epithelization, compared with the control group, which received Vaseline [10]. Similar results in the healing of wounds induced by excision were observed on alloxan-induced diabetic rats (150 mg·kg−1), which received a water extract of green fruits of *C. papaya*, the healing actions being attributed to the active component, papain, which led to the enzymatic debridement of wounds, and the fruit vitamin C content, since it is essential for the conversion of proline to hydroxyproline, a specific marker and component of the granulation tissue of the extracellular matrix in wounds [38,39]. In this way, besides possessing edible and tasty fruits, different parts of *C. papaya* are characterized by the quality of nutrients and bioactive compounds with medicinal properties that may be used in traditional medicine as an alternative or adjuvant in the treatment of some pathological conditions.

#### **4. E**ff**ects of** *C. papaya* **L. on Metabolic Syndrome**

Obesity consists of an excessive accumulation of body fat, which can represent a serious health risk and involves several ethological factors, including social, behavioral, environmental, cultural, psychological, metabolic, and genetic factors [40]. It is known that excessive fat accumulation, mostly visceral, can be an important condition in the development of metabolic dysfunctions, such as arterial hypertension, dyslipidemia and insulin resistance, and alterations conducive to the development of diabetes mellitus type 2, cardiovascular illnesses [41] and cancer [42], such as prostate [43] and colon rectal cancer [44]. Thus, the metabolic syndrome can be defined as the set of these risk factors, i.e., a cluster of metabolic disorders associated with obesity, including insulin resistance, atherogenic dyslipidemia and hypertension, which can lead to cardiovascular illnesses [45].

Since adipose tissue is a source of a great number of adipokines, such as the tumor necrosis factor (TNF-α), interleukin 6 (IL-6), monocyte chemoattractant protein, also known as chemokine ligand 2 (MCP-1/CCL-2), leptin, adiponectin, and resistin, among others [46], the larger the accumulation of adipose tissue, the higher the production of these adipokines. This leads to an imbalance in their secretion, with increased pro-inflammatory and decreased anti-inflammatory adipokines, stimulating the systemic and local inflammatory process, contributing to the development of insulin resistance [46]. Furthermore, the metabolic syndrome, besides being associated with the inflammatory process, is also related with the high production of reactive oxygen species (ROS) and, consequently, can induce insulin resistance [47,48], which is increasingly recognized as a key factor linking metabolic syndrome and liver steatosis; the last is associated with excessive fat accumulation in ectopic tissues, such as the liver, and increased circulating free fatty acids, which can further promote inflammation and endoplasmic reticulum stress [49].

For the treatment of obesity and its metabolic disorders which characterize the metabolic syndrome, there are various therapeutic approaches, either pharmacological or non-pharmacological treatments, and other methods, used as healing adjuvants. As such, the use of plants or fruits, reported since remote times as alternatives for the treatment and prevention of diseases, stand out in view of their high concentrations of vitamins, bioactive compounds and also lipidic composition, which reduces

inflammatory markers, aggregates platelet, protects against thrombogenesis and oxidative stress, and prevents hypercholesterolemia, hypertriglyceridemia and hyperglycemia, which can be triggered by obesity [5].

Considering the presence of vitamins, bioactive compounds and lipids of biological and nutritional importance in *C. papaya*, several studies (summarized in Table 4) evinced relevant effects of this plant in the treatment of metabolic dysfunctions, associated or not associated with obesity, which can be considered an alternative therapeutic approach in the treatment of the metabolic syndrome.

A preliminary study [50] demonstrated that the water extract of *C. papaya* seeds showed hypoglycemic and hypolipidemic activity in adult healthy male Wistar rats, without signs of acute toxicity. The groups received the water extract of *C. papaya* seeds, at concentrations of 100 mg, 200 mg and 400 mg/kg, and glibenclamide at 0.1 mg·kg−<sup>1</sup> by gavage for 30 days. The treatments at all doses of the extract led to decreased serum levels of fasting glycemia, triglycerides, total cholesterol, LDL-c, and VLDL-c, with increased HDL-c levels, depending on the dose, and responses similar to the effects of the positive control group (glibenclamide). Such a relation with the extract concentration was observed in the lowered atherogenic index, compared with the group receiving distilled water (10 mL/kg/day) and glibenclamide (0.1 mg/kg/day). The phytochemical analyses of the extract revealed the presence of alkaloids, flavonoids, saponins, tannins, anthraquinones and anthocyanosides, and the monitored animals showed a decrease insugars, related to the metabolic effects.

Another study [51] evaluated the effects of the water extract of *C. papaya* leaves (200 mg/kg to body mass), given by gavage in adult healthy male New Zealand rabbits, treated for 24 weeks, resulting in reduced body weight, concomitant with lowered levels of fasting glycemia during the trial. Moreover, over the supplementation period, the extract had a hepatotoxic effect, manifesting an increase in serum values of aspartate transaminase (AST), aspartate aminotransferase (ALT), gamma-glutamine transferase (γ-GT) and total bilirubin. Therefore, further investigations will be necessary to evaluate toxicological effects of the extract, especially on the liver, in order to, standardize doses time of administration and side effects for a safety administration.

While such metabolic effects were observed in healthy animals, other studies elucidated the hypoglycemic action in an alloxan-induced diabetes model [52–54]. Adenowo et al. (2014) [52] investigated alloxan-induced (150 mg/kg/body mass) diabetic Wistar rats, treated with an ethanolic extract of *C. papaya* leaves (250 and 500 mg·kg<sup>−</sup>1) by gavage for 21 days, and verified reduced levels of glycemia, total cholesterol, triglycerides and LDL-c, together with increased HDL-c levels, resulting mainly from the dose of 250 mg/kg to body mass. Furthermore, they verified that the extract diminished the serum concentrations of urea, creatinine, ALT and AST, as well as the parameters of diabetic animals receiving metformin. The data corroborate the study of Maniyar (2011) [54], where the water extract of *C. papaya* leaf (400 mg/kg/body mass), given by gavage for 21 days, showed a reduction in the levels of glycemia, triglycerides and total cholesterol in alloxan-induced diabetic rats (120 mg/kg/body mass), confirmed by Johnson et al. (2015) [53], who tested the water extract of seeds and leaves of *C. papaya* (400 mg/kg/body mass), by gavage for 28 days in an experimental model of diabetes (alloxan 150 mg/kg to body mass), having observed diminished levels of glycemia total cholesterol, hepatic enzymes, ALT, AST, urea, and creatinine. Nevertheless, regarding glycemic metabolism and hypoglycemic action, the seed extract was superior to the leaf extract.

Ezekwe et al. (2014) [55] applied the experimental model of alloxan-induced diabetes (120 mg/kg to body mass) in albino rats receiving a ration added to grated green *C. papaya* pulp, splitting the animals into three groups: control non-diabetic, diabetic and diabetic fed with added grated green *C. papaya* pulp for 28 days. The third group presented relevant effects on their metabolism, such as a reduction of weight, in the levels of glycosylated hemoglobin and in the lipidic profile, including low-density lipoprotein cholesterol (LDL-c), very low-density lipoprotein (VLDL-c), triglycerides and total cholesterol, and increased serum values of High-density lipoprotein cholesterol (HDL-c).

Metabolic effects were also observed in alloxan-induced (90 mg/kg/body mass) albino rats receiving the water extract of *C. papaya* root (500 mg/kg/body mass) and glibenclamide (5 mg/kg/body mass) by gavage for 21 days. The treatment with the extract showed improved parameters of glycemia already after 7 days of the trial, an improvement in the dyslipidemic parameters and recovery of hepatic tissues and renal dysfunction. The compounds identified include hexadecanoicacid, methylester, 10-octadecanoic acid, methyl ester, ergosta-5,22-dien-3-olacetate (3β, 22E), dianhydromannitol, 1,1,3,3,5,5,7,7,9,9,11,11-dodecamethylhexasiloxane, methyl-11-hexadecanoate, and octadecanoic acid. The compounds 10-octadecenoic acid, methylester, hexadecanoic acid, and methyl ester, were the phytochemicals most present in the root extract. Thus, they may have contributed to the cited metabolic effects [56].

Hypoglycemic effects were observed not only with the isolated administration of the leaf extract of *C. papaya* in alloxan-induced diabetic rats (180 mg/kg/body mass), but also combined with co-administrated reference antidiabetic drugs, such as metformin and glimepiride. The extract, the drug, or the combination drug + extract was administered daily by gavage in periods of a short and long duration, corresponding to 3 and 7 days, respectively. The concentrations of each product given to the animals were divided based on low and high doses, established in previous studies, as follows: extract low dose: 5 mg/kg and high dose: 10 mg/kg; glimepiride low dose: 0.2 mg/kg and high dose: 0.4 mg/kg; and metformin low dose: 50 mg/kg and high dose: 100 mg/kg, for 3 and 7 days. The same period of treatment was utilized for the combinations, glimepiride + extract and metformin + extract, and the dose combinations corresponded to high-high, high-low, low-high and low-low. The lowest concentration extract (5 mg/kg) reduced the glycemic level, but the highest concentration (10 mg/kg) accelerated the starting of the glimepiride activity, while the combination of all extracts with metformin diminished the glycemic levels after 24 h. Thus, the data demonstrated that the hypoglycemic activity of the *C. papaya* leaf extract was as effective as the hypoglycemic agents, metformin and glimepiride. However, the latter had a faster action onset, as the effect of the duration of application was dependent on the nature, i.e., on the activity strength, and on the dose. Besides, the interaction between the combination drug-extract was different for each group, since the action mechanisms of glimepiride differ from those of metformin [57].

In studies [58] on *C. papaya* on streptozotocin (STPZ)-induced diabetes, the crude ethanolic extract of *C. papaya* leaf (100 mg/kg/day), in comparison with the ethanolic extract of the leaves of *Pandanusam aryllifolius* (100 mg/kg/day) and the drug glyburide (10 mg/kg) by gavage for 6 days in albino mice with induced diabetes by STPZ (60 mg/kg/body mass), did not alter body weight. However, there was a reduction of glycemia, and the histology showed spleen cell regeneration, reduced the number of liver pyknotic nuclei and vacuoles, and recovered kidney cuboidal tissue. The phytochemical analyses indicated the presence of alkaloids, tannins, flavonoids and saponins, suggesting that these bioactive compounds are responsible for such effects.

A possible hypothesis for the metabolic actions of *C. papaya* extracts can be seen in the study by Juárez-Rojop et al. (2012) [59], utilizing the water extract of *C. papaya* leaves at three doses (0.75, 1.5 and 3 g·100 mL<sup>−</sup>1) in the drinking water of animals with induced diabetes by STPZ (60 mg/kg to body mass) and non-diabetic animals for a 4-week period. The results demonstrated that the extracts at 0.75 and 1.5 g·100 mL−<sup>1</sup> diminished the levels of glycemia, as well as the serum levels of total cholesterol and triglycerides. The regeneration of pancreatic islets, with a preserved cell size, was demonstrated, and yet, a rupture of hepatocytes and accumulation of glycogen and lipids was prevented. Besides, it was verified that the metabolites of nitric oxide (NO) were reduced in diabetic rats. However, with the application of the extracts, the NO levels rose. It is known that hyperglycemia and hyperlipidemia are characterized by the inhibition of endothelial NO Synthase (eNOS) and, consequently, can result in the formation of reactive oxygen species (ROS) in relaxation, depending on the damaged endothelium, with a high formation of free radicals, concomitant with a low effectiveness of antioxidant enzymes, leading to an imbalance between the formation and the protection against free radicals in the organism. Thus, the metabolic actions in that study could be related to the increased antioxidant activity of the extract, exerted in diabetic animals.

Previous studies [60] evaluated the phytochemical composition of *C. papaya* leaf extracts on the basis of chloroform, n-hexane and ethanol and verified that the chloroform extract presented steroids and quinones among its main components, which led to the choosing of this extract for the screening of biological activity in STPZ-induced diabetic rats (60 mg/kg/body mass).

Different doses of a chloroform extract (0, 31, 62, 125 mg/kg/body mass) of *C. papaya* leaf were given by gavage to diabetic and non-diabetic rats, and as the positive control group, diabetic rats were treated with insulin (5 U/kg, intraperitoneal) for 20 days. The data proved that the extract reduced the glycemic levels, the serum concentrations of triglycerides and total cholesterol and maintained the HDL-c at levels similar to those observed in non-diabetic rats. Furthermore, the concentrations of 31 and 62 mg/kg/body mass of the extract reduced the body weight and the levels of AST and ALT, without differences for the extract with the highest concentration (125/mg/kg/body mass).

Considering not just the systemic and biochemical actions, the extract was able to act on specific tissues, such as the pancreatic islets, either in diabetic rats induced by STPZ (60 mg/kg/body mass) and in vitro in cell cultures of pancreatic islets, which were found to be the actions of the chloroform extract of *C. papaya* leaves (31, 62, 125 mg/kg/body mass), applied by gavage for 20 days. The animals receiving the extract at concentrations of 31 and 62 mg/kg/body mass showed a reduction in fastening glycemia. On the other hand, the serum levels of insulin increased in non-diabetic rats receiving 62 mg/kg/body mass, compared with the non-diabetic group, without the extract. In cell cultures of pancreatic islets treated with STPZ (6 mg in 30 μL polyethylene glycol), a decrease in the liberation of the basal insulin culture with glucose (2 g/L) occurred. Besides, when added to the extract (6 mg in 30 μL polyethylene glycol) applied to cells with STPZ, more insulin liberation occurred. However, when STPZ was added simultaneously with the extracts (3, or 6, or 12 mg in 30 μL), insulin liberation was diminished in the three conditions, independently of the dose. However, when STPZ was added after 5 days of using the extracts, the insulin liberated from the pancreatic islets was superior to the cells of the control group, normal and similar to cells with the 6 mg extract, suggesting that the extracts have a protective action on pancreatic islets. The results are confirmed by an immune histochemical trial of the spleen, in which it was verified that the diameters and areas were larger in the groups treated with *C. papaya* extract, compared with the diabetic group [61].

Among the bioactive compounds of the major proportion in the chloroform extract are the steroids. In diabetes, changes occur in the structure and function of the absorption of intestinal glucose, e.g., an increase in glucose uptake that could cause postprandial hyperglycemia. Thus, the hypothesis is that the steroids hinder the hydrolysis of carbohydrates and the absorption of intestinal glucose by hydrolyzing enzymes limiting the levels of post prandial glucose [61–63].

It is known that diabetes mellitus is characterized by a deficiency in insulin secretion and by a low response of the organs in the action of insulin [64]. The compounds present in the *C. papaya* extract may be related to effects similar to those of insulin in glycemic metabolism, promoting glucose uptake in peripherical tissues or in the skeletal muscle and adipose tissues by a process of regeneration and revitalization of their main β-cells [60,65].

Another mechanism, which may be related to the effects of *C. papaya* on glycemic metabolism, may be the inhibition of important enzymes involved in the digestion of carbohydrates, such as α-amylase and α-glycosidase. Oboh et al. (2013) [66] demonstrated that the water extract of different parts of the green fruit of *C. papaya* was able to promote the inhibition of α-amylase and α-glycosidase in a dose-dependent way (0 to 2.0 mg/mL), and the combination of different parts of the green fruit, such as seeds, pulp and peel, in equal proportions had the best inhibitory effects on both enzymes. The α-amylase degrades complex carbohydrates in the diet into oligosaccharides and disaccharides, which are converted into monosaccharides by α-glycosidase. The liberated glucose is absorbed by the intestine, resulting in post prandial hyperglycemia. A higher inhibition of these enzymes

thereby occurs, and the rise of post prandial glucose from a carbohydrate-rich diet will be significantly diminished, slowing the process of hydrolysis and uptake of carbohydrates [67,68].

Oxidative stress is also one of the mechanisms conducive to the development and progression of diabetes mellitus, since an exacerbated increase in the production of free radicals occurs simultaneously with the decreased mechanisms of antioxidant defenses, which can result in the cell damage of organelles and enzymes, increased lipidic peroxidation and, consequently, the development of insulin resistance [69,70]. In this way, *C. papaya* was also able to present antioxidant activity [68]. Different parts of the green fruits of *C. papaya* inhibited the lipidic peroxidation induced by sodium nitroprusside in rat pancreatic cells in vitro [66]. Sodium nitroprusside is an anti-hypertensive drug, which causes cytotoxicity by the liberation of cyanide and/or NO. Under conditions of oxidative stress, NO, together with other ROSs, such as the radical superoxide, lead to the formation of the radical peroxynitrite (ONOO-), which is a potent oxidant agent that can harm most cell components, such as proteins, DNA and membrane phospholipids [71,72]. Thus, the study showed that the extract of the pulp with the peel of green fruits can have a strong inhibitory effect on the production of malondialdehyde (MDA) and a greater ability of NO radical scavenging than seeds. Such effects are attributed to the phenolic compounds and alkaloids present in the pulp, seed and peel extracts of *C. papaya*, which are biocomponents with a high antioxidant action in removing free radicals, catalyzing chelating metals, activating antioxidant enzymes, reducing the radicals of α-tocopherol and inhibiting oxidases [64,73].

Corroborating the antioxidant actions of *C. papaya*, Salla et al. (2016) [74] reported on the antioxidant activity of the methanolic and hexanic extracts at concentrations of 50, 100 and 250 μg/mL of *C. papaya* seed on HepG2 cells, the cell strain of the human hepatoma, which incurred an induction of oxidative stress by the application of hydrogen peroxide (H2O2) (500μM). The activity of the antioxidant enzyme superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) and levels of glutathione (GSH) were lower after the induction of oxidative stress by H2O2, and after the use of methanolic and hexanic extracts, the activity of SOD was restored, except with 50 μg/mL. The GSH levels increased with the concentration of 250 μg/mL methanolic extract and 100 and 250 μg/mL hexanic extract, and the CAT activity rose with the concentrations of 250 and 500 μg/mL, with GPx only at 250 μg/mL of methanolic extract. Besides, the highest concentrations of both extracts diminished cell viability, but this could be verified in higher proportion with the hexanic extract. The levels of flavonoids in the extracts were superior in the methanolic extract, compared with the hexanic, confirming that the antioxidant activity is related to the presence of these polyphenols.

Like glycemic metabolism, the bioactive compounds present in the *C. papaya* extracts can exert effects similar to insulin in the lipidic metabolism, as under normal conditions the insulin activates the lipoprotein lipase, hydrolyses triglycerides and inhibits the lipolysis process. Insulin deficiency, in turn, stimulates lipolysis in the adipose tissue, leading to hyperlipidemia and an accumulation of hepatic fat, decreasing the content of the enzyme lipoprotein lipase, which hydrolyses lipids, resulting in increased concentrations of serum triglycerides. Increased LDL-c levels occur because of the inhibition of the insulin action in the activity of the enzyme, β-hydroxy-β-methyl glutaryl CoA reductase (HMG-CoA reductase), which exerts an important role in cholesterol metabolism [53,66].

The action of the*C. papaya* extract on the enzyme, HMG-CoA reductase, is reported by Hasimun et al. (2018) [75], assessing specifically the actions in the lipidic metabolism of the ethanolic extract of *C. papaya* leaves (50, 100, 200 mg/kg/body mass) by gavage in Wistar rats, receiving 25% of D-fructose in drinking water for 21 days. The results showed an anti-hyperlipidemic activity of the extract at a dose of 200 mg/kg/body mass, leading to decreased total levels of cholesterol, triglycerides, and LDL-c and an increase in HDL-c. The mechanism involved is related to the inhibition of the enzyme, HMG-CoA reductase, activity in the liver, an enzyme with an important role in the synthesis of endogenous cholesterol, the inhibition of which is similar to the effects of drugs of the class of statins, such as simvastatin, used as a positive control. Besides, the phytochemical analyses revealed secondary metabolites, such as alkaloids, flavonoids, tannin, saponins, steroids/triterpenoids and quinones, suggesting that the flavonoids contained in the leaf extract, especially quercetin, could

be the responsible for exerting the same mechanism as that of the statins in inhibiting HMG CoA reductase [75–77].

Similar data in the lipidic metabolism of *C. papaya* extract were observed in albino Wistar rats, fed with hyperlipidic diet. The effects on dyslipidemia were observed, testing the water extract of papaya seed (200 and 300 mg/body mass/day) by gavage for 5 weeks, which led to a significant reduction of total cholesterol, triglycerides, and LDL-c and an increase in HDL-c in hypercholesterolemic animals. However, the antilipidemic effects of different extract concentrations were not superior to the group receiving the reference drug, simvastatin (1.8 mg/bodymass/day) [78].

Besides the effects on glycemia and lipidic series, *C. papaya* showed actions on the systemic arterial hypertension (SAH) in animal models. After evaluating the inhibitory action of the extracts on the activity of the angiotensin-converting enzyme (ACE) in vitro, the methanolic extract of *C. papaya* leaves was chosen for the study by Brasil et al. (2014) [79]. The methanolic extract (100 mg/body mass/twice a day) was given by gavage in spontaneously hypertense Wistar rats (SHR) for 30 days. Like the reference drug (enalapril 10 mg/body mass/day), an ACE inhibitor, the methanolic extract inhibited plasmatic ACE activity, enhanced cardiac hypertrophy and normalized baroreflex sensibility, suggesting the efficiency of this extract as an anti-hypertensive [79]. The systemic arterial pressure is controlled by both the renin-angiotensin system (RAS) and baroreflex. The latter is an important short-term reflex, which controls the responses of the heart beats [80]. The uncontrolled activation of RAS has an important role in the development of cardiac hypertrophy, the ACE inhibitors being important treatment options, since ACE is an important component of RAS, which leads to the formation of angiotensin II, the main vasoconstrictor of RAS, and to the reduction of baroreflex sensibility for rising blood pressure and sympathetic regulation [81,82]. In that study, the extract effects on arterial pressure could result from the presence of different bioactive compounds, especially flavonoids, e.g., ferulic acid, caffeic acid, gallic acid and quercetin, with a suggested action on ACE inhibition [79]. Previous studies demonstrated the presence of quercetin, luteolin and kaempferol in apple peel extract, which acted as inhibitors of ACE activity in vitro [83,84].

In a model of arterial hypertension induced by deoxycorticosterone acetate (DOCA, 15 mg·100 g<sup>−</sup>1) in Wistar rats, the crude extract of *C. papaya* fruit (20 mg/kg), besides not presenting toxic effects, was able to generate a fast drop of arterial blood pressure and heart rate, compared with normotensive rats, and had a more potent anti-hypertensive action than hydralazine (200 mg/100 g, intravenous), a vasodilator and anti-hypertensive agent [85]. Earlier studies revealed a higher activity in the synthesis of catecholamines, e.g., a higher activity of tyrosine hydroxylase in the adrenal glands of DOCA-induced hypertense rats and in rats with renal hypertension [86,87]. Therefore, the capacity of the extract to depress the arterial pressure and the heart rate may be caused by the reduction of levels of catecholamines, liberated in response to the extract [88].

While the reviewed studies hitherto were not performed in specific models of metabolic syndrome or obesity, the achieved results demonstrated that *C. papaya* has a therapeutic potential for various types of metabolic dysfunctions, such as diabetes mellitus type 1, leading to alterations in both glycemic and lipidic metabolism, oxidative stress and in models of arterial hypertension. After those studies on the actions of *C. papaya* in metabolism, further investigations on this plant in models of obesity and metabolic syndrome are needed, which would facilitate the search for new therapeutic approaches and a better understanding of the mechanisms of action in the metabolic dysfunctions associated with obesity.


**4.** Summary of effects of the use of*Carica papaya*L. against metabolic dysfunctions.

**Table**



#### **5. Conclusions**

This review evaluated the nutritional and phytochemical composition of *C. papaya* as well as the effects of the use of several types of extract from different parts of the plant. *C papaya* exhibits curative properties, such as improvements in hepatotoxicity and nephrotoxicity induced by drugs, antimicrobial, antimalarial, anti-parasitic, antitumor, anti-inflammatory actions and wound healing effects. In relation to the metabolic dysfunctions, *C. papaya* displays hypoglycemic, hypolipidemic and antihypertensive potential and demonstrates increased antioxidant activity in experimental models in vivo and in vitro. Therefore, further studies including researches on diet-induced and genetic obesity models in addition to the isolation of specific substances from different parts of *C. papaya* will be important for the development of novel natural products on the treatment and prevention of obesity and metabolic disturbances.

**Author Contributions:** L.F.S., A.P., F.M.A. and W.F.O.F.: assistance with structuring the review, writing, and literature review; A.C.I., B.L.S.d.E.S., R.d.C.A.G., P.A.H. and K.d.C.F.: assistance with structuring the review.

**Funding:** This research received no external funding.

**Acknowledgments:** We thank the graduate program in Health and Development in the Central-West Region of Brazil, Federal University of Mato Grosso do Sul-UFMS, for support.

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

#### **References**


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