Plant-Derived Substances for Prevention of Necrotising Enterocolitis: A Systematic Review of Animal Studies

Inflammation, oxidative injury, and gut dysbiosis play an important role in the pathogenesis of necrotising enterocolitis (NEC). Plant-derived substances have historically been used as therapeutic agents due to their anti-inflammatory, antioxidant, and antimicrobial properties. We aimed to review pre-clinical evidence for plant-derived substances in the prevention and treatment of NEC. A systematic review was conducted using the following databases: PubMed, EMBASE, EMCARE, MEDLINE and Cochrane Library (PROSPERO CRD42022365477). Randomized controlled trials (RCTs) and quasi-RCTs that evaluated a plant-derived substance as an intervention for NEC in an animal model of the illness and compared pre-stated outcomes (e.g., clinical severity, severity of intestinal injury, mortality, laboratory markers of inflammation and oxidative injury) were included. Sixteen studies (n = 610) were included in the systematic review. Ten of the sixteen included RCTs (Preterm rat pups: 15, Mice: 1) reported mortality and all reported NEC-related histology. Meta-analysis showed decreased mortality [12/134 vs. 27/135; RR: 0.48 (95% CI: 0.26 to 0.87); p = 0.02, 10 RCTs] and decreased NEC in the experimental group [24/126 vs. 55/79; RR: 0.34 (95% CI: 0.22 to 0.52); p < 0.001, 6 RCTs]. Markers of inflammation (n = 11) and oxidative stress (n = 13) improved in all the studies that have reported this outcome. There was no significant publication bias for the outcome of mortality. Plant-derived substances have the potential to reduce the incidence and severity of histologically diagnosed NEC and mortality in rodent models. These findings are helpful in guiding further pre-clinical studies towards developing a food supplement for the prevention of NEC in preterm infants.


Introduction
Necrotising enterocolitis (NEC) is a potentially devastating illness, particularly in extremely preterm (gestation < 28 weeks) infants [1,2].Overall, the incidence of NEC ≥ Stage II is reported to be 4 to 6% in very preterm infants, rising to 8 to 12% in extremely preterm infants with higher mortality (~20% vs. 45%) and morbidity, including long-term neurodevelopmental impairment [1][2][3][4][5].The complications associated with NEC, such as prolonged hospital stay and short bowel syndrome, explain the enormous socioeconomic burden of the illness, reported to be as high as USD 500 million to 1 billion per year in the USA [6].
Despite decades of research, the pathogenesis of NEC continues to be poorly understood.An interplay between prematurity, hypoxia-ischemia-reperfusion-free radical injury, gut dysbiosis, and formula feeding is considered important in the pathogenesis [1,2].A gestational age-dependent excessive pro-inflammatory response following interaction between Toll-like receptor-4 (TLR-4) and lipopolysaccharides (LPS) is currently considered an important trigger for gut injury in NEC in preterm infants involving oxidative stress [1,2,[7][8][9][10][11].Current strategies for the prevention of NEC include antenatal glucocorticoids, early preferential feeding with breast milk, standardised feeding protocols, probiotic supplementation, avoiding formula feeding and undue prolonged exposure to antibiotics, and acid-supressing agents [12][13][14].
The need for new strategies for prevention and treatment of NEC cannot be overemphasized considering the health burden associated with the illness.Plant-derived substances including herbs and spices may provide a novel option in this context based on their anti-inflammatory, antioxidant, and antimicrobial properties, and the fact that they have been used as therapeutic agents for centuries [15][16][17][18][19][20][21][22][23].Importantly, pre-clinical studies have shown significant benefits of ginger, curcumin, and Nigella sativa oil in reducing the severity of intestinal injury and markers of oxidative injury in preterm rat pup models of NEC [24][25][26][27][28]. Given these data, we aimed to review the evidence on plant-derived substances as a potentially novel option for the prevention and treatment of NEC in preterm infants.Our findings are expected to guide translational research in this field.

Materials and Methods
We used the animal intervention study (SYRCLE) protocol and the preferred reporting items for systematic reviews and meta-analyses (PRISMA) guidelines for conducting and reporting this systematic review, respectively [29,30].The protocol was registered on PROSPERO, the international prospective register of systematic reviews (CRD42022365477).

Study Selection
Eligibility and exclusion criteria: Pre-clinical randomized controlled trials (RCTs) and quasi-RCTs meeting the following criteria were eligible for inclusion: (1) Use of a validated animal model of NEC in preterm infants.(2) Evaluation of a plant-derived substance as an experimental intervention for NEC.(3) Comparison of pre-stated clinical and laboratory outcomes between the experimental and control group.Observational and in vitro studies, and studies that used unvalidated models, were excluded.
Outcomes: These included the following: (1) Clinical assessment of the severity of NEC.(2) Severity of gut injury assessed by histological grading.(3) Mortality from NEC. (4) Laboratory markers of inflammation and oxidative injury.(5) Other outcomes.

Data Extraction and Quality Assessment
Two authors independently screened the titles and abstracts to identify studies potentially eligible for inclusion in the review.Full-text articles of such studies were read by two reviewers to confirm their eligibility for inclusion.A standardised piloted form was used to extract data.The incidence of various clinical outcomes of interest in the experimental vs. control group was recorded.We used risk ratios (RR) and 95% confidence intervals (CI) for dichotomous outcomes.All authors were contacted for additional information.The quality of the RCTs was assessed using the SYRCLE risk of bias (ROB) tool [29].Two authors independently assessed the ROB in the domains of random number generation, allocation concealment, random housing of animals, blinding of intervention and outcome assessors, selective reporting, and other potential sources of bias.For each domain, the risk was assessed as low, high, or unclear The certainty of evidence (COE) was assessed using the GRADE methodology and classified into one of the four categories: high, moderate, low and very low [31].In case of discrepancies, group discussions involving all reviewers were held to reach consensus.

Data Synthesis
We conducted meta-analysis using the Stata 16.0 software (StataCorp.2019.Stata Statistical Software: Release 16.College Station, TX, USA: StataCorp LLC).We used the DerSimonian and Laird random-effects model (REM) for meta-analysis since heterogeneity was expected.We used raw numbers to calculate the RRs for pooling if the included studies did not provide this information.For dichotomous outcomes, the pooled effect estimates were presented as pooled RRs with 95% CIs.Qualitative synthesis and diagrammatic representation were provided for studies where meta-analysis was not possible.We assessed statistical heterogeneity by visual inspection of the forest plots and quantified this using the I 2 statistic.The I 2 results were interpreted as follows: 0% to 40%: heterogeneity might not be important; 30% to 60%: may represent moderate heterogeneity; 50% to 90%: may represent substantial heterogeneity; 75% to 100%: considerable heterogeneity [32].We assessed publication bias (small study effects) using visual inspection of the funnel plots, Egger's test, Begg's test, and Harbord's test if ≥10 studies were included for any individual outcome.

Results
A PRISMA flow chart of screening and selection results is shown in Figure 1.The initial search identified 544 articles of which 16 studies [25,27,28,[33][34][35][36][37][38][39][40][41][42][43][44][45] (n = 610) were included after application of the selection criteria.Two studies were excluded because they evaluated a genetically engineered analogue of plant-derived substance [26,46].All included studies were RCTs that evaluated a plant-derived substance in an animal model of NEC (Table 1).The ROB was generally high for the included studies.Majority of the included studies carried unclear or high ROB for randomization or reporting (Table 2).
Publication bias: Publication bias was assessed only for the outcomes of mortality since it had ≥10 studies in the meta-analysis.Visual inspection of the funnel plot (Figure 4) and the results of Begg's test, Egger's test, and Harbord's test suggested that publication bias was unlikely for mortality (Egger's test p value: 0.0.86,Begg's test p value: 0.36, and Harbord's test p value: 0.36).Certainty of evidence: The level of evidence was deemed low when considering small sample size, high ROB of the included studies, and significant heterogeneity.

Discussion
Our systematic review showed that supplementation with plant-derived products significantly reduced mortality and severity of histologically diagnosed NEC in rodent models of illness.Findings for NEC were consistent across studies despite the heterogeneity of products, dose, route of administration, and duration of supplementation.
Overall, our findings suggest that as a class of intervention, plant-derived substances may have shared pathways of benefit (e.g., anti-inflammatory and antioxidant properties) for reducing the incidence and severity of NEC.This hypothesis is supported by the histopathological findings of reduced severity of gut injury, along with decreased expression of various pro-inflammatory mediators, reduced total oxidant score, oxidative stress index, and increased total antioxidant score.The histopathological findings correlate well with the current understanding of the pathogenesis of NEC that involves an exaggerated proinflammatory response, decreased anti-inflammatory response, reactive oxygen species, and oxidative stress [1,2,[7][8][9][10][11]47]. Targeting these pathways, as plant-derived substances assessed in the included studies appear to do so, could be fundamental in modifying the disease process.
The benefits of plant-derived substances relate to their phytochemical content which increase the quantity and diversity of gut microbiota, reduce inflammation and oxidative stress, enhance immune function, regulate gene expression and intracellular signalling, and reduce DNA damage [48,49].Evidence supports their role in the prevention of NEC through trophic effects on intestinal mucosa and increased secretion of regulatory peptides and digestive enzymes resulting in accelerated intestinal maturation [50].The discovery of the anti-inflammatory and anti-oxidant properties of plant-derived substances is not a new finding.Based on these properties, such products have been used for centuries, to promote health and treat various conditions including communicable diseases, cardiovascular disease, inflammatory conditions, and cancers [15,16,51,52].Our findings open a new frontier for developing novel therapeutic agents using plant-derived substances for the prevention and treatment of NEC.
A comprehensive review of all ingredients included in our review is beyond the scope of this report.Evidence suggests that curcumin, ginger and fennel (all considered generally safe by the FDA, USA (https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/CFRSearch.cfm?fr=582.10&SearchTerm=fennel,accessed on 12 June 2023)) warrant further evaluation in high quality pre-clinical studies.Safe use of ginger and curcumin in pregnant or lactating women and in the paediatric age group is reassuring [53][54][55][56][57][58].Comprehensive search for the evidence on the safety and efficacy, and independent assessment of the quality of a plant-derived substance/product is critical if it is to be developed for use in preterm infants.
The limitations of our review need to be acknowledged.These include the small sample size of the included individual studies, high ROB due to the unclear method for allocation concealment, sub-optimal blinding in majority of the included studies, lack of details of registration of the study protocol on an animal trial registry, and heterogeneity in the methods used for inducing NEC and evaluated plant-derived substances.The possibility exists of shared mechanisms of benefits (e.g., anti-inflammatory effect, and protection from free radicle injury) of plant-derived substances as a class of intervention.We hence conducted a meta-analysis for the outcomes of NEC and mortality.However, future research should focus separately on each plant-derived substance as their effects could be specific to the plant/ingredient.
To our knowledge, this is the first comprehensive systematic review and meta-analysis in this field with robust methodology using SYRCLE's ROB tool for animal studies.Despite the limitations, we believe that our findings add meaningful data in an important field of research in neonatal medicine, considering the reasonably large total sample size, use of the validated preterm rat pup model of NEC, and consistent significant benefits for mortality and NEC across studies with narrow confidence intervals and low p values.
Our findings are beneficial in designing and conducting robust, definitive, and adequately powered RCTs in validated models of NEC including preterm rat pups to ascertain the safety and efficacy of the plant-derived substances assessed in the studies included in our review.The evidence provided in our review is helpful in selecting the most appropriate plant-derived substances for further evaluation in pre-clinical studies.A note of caution is warranted in selection of products for further translational research considering that the safety and efficacy of plant-derived substances cannot be ensured given the poor regulatory control over the herbal medicine and food supplement industry [59,60].Certain plants used for medicinal purposes have been shown to be harmful [59,60].
It is important to note that results from animal models may not translate into clinical benefits as the effects of an intervention could be species-specific with significant differences in pharmacokinetics of therapeutic agents between humans and animal species [61][62][63][64].Furthermore, different exposures (e.g., hypoxia, hypothermia, LPS, formula feeding, acetic acid enema) inducing the illness in laboratory animals may not result in NEC as it occurs in its complex and multifactorial nature in human preterm infants [61][62][63].

Conclusions
In summary, the results of our systematic review indicate the potential of plant-derived substances as a novel option in the prevention and treatment of NEC in preterm infants.Rigorous evaluation of the most suitable agent/s selected from the studies included in our review is needed for further progress in this field.

Figure 1 .
Figure 1.PRISMA flow chart for study selection.

Figure 4 .
Figure 4. Funnel plot for the outcome of mortality.

Table 1 .
Characteristics of the included studies.