Effects of Moringa oleifera Leaf Extract on Liver Histopathology: A Systematic Review

Introduction Moringa leaves (Moringa oleifera), which are members of the Moringaceae family, are one of the herbal plants that are widely known in Indonesia. Phytochemical contents of moringa leaf, such as flavonoid, quercetin, and phenolic acid, are believed to have an effect on improvement of NAFLD. Therefore, moringa leaf is considered as one the herbal plants that can be used as supplementation in the form of adjuvant therapy to NAFLD. The study objective of our research is to review the effect of giving moringa leaf to the liver, especially the histopathologic features. This study will be conducted on literature review research design, more specifically in the form of a systematic review. Research Method. Five major electronic web databases, including PubMed, Cochrane Library, Google Scholar, Scopus, and ScienceDirect, were used in identifying literature from 2014 to 2023. Results From a comprehensive analysis of 13 relevant literature sources, we elucidate the impact of Moringa oleifera leaf extract on liver histopathology, glucose, and lipid metabolism. Furthermore, we provide insights into its safety profile concerning human health. Conclusion The phytochemical content of Moringa oleifera leaf extract had shown a significant benefit in plant medicinal sector. From the research that had been done, Moringa oleifera leaf extract contributes to give significant improvement on liver histopathological features, glucose, and lipid metabolism on animal sample model.


Introduction
Moringa leaves (Moringa oleifera), which are members of the Moringaceae family, are one of the herbal plants that are widely known in Indonesia [1,2].Every part of the moringa leaf is a storehouse of important nutrients and antinutrients [3][4][5].Terefore, moringa leaves are also known as "miracle trees" or magical plants because of their various benefts, including medicinal plants, cosmetics, and food ingredients [1].Phytochemical contents of moringa leaf, such as favonoid, quercetin, and phenolic acid, are believed to have an efect on improvement of NAFLD [6][7][8].
Metabolic syndrome encompasses a group of metabolic irregularities associated with hypertension, central obesity, insulin resistance, and atherogenic dyslipidemia and is closely linked to a heightened likelihood of developing diabetes as well as cardiovascular diseases, both atherogenic and non-atherogenic in nature [9].One of the important risk factors for metabolic syndrome is lack of physical activity [10].Several studies have been conducted using accelerometric methods to examine the association between sedentary behaviour and the metabolic syndrome [11].Tese studies have shown that increasing the duration and percentage of time spent in sedentary behaviour is associated with increased metabolic risk in adults [11].One of the concerning manifestations of metabolic syndrome in the liver is non-alcoholic fatty liver disease [12].Research indicates a robust connection between NAFLD and the characteristics of metabolic syndrome.Insulin resistance serves as a pivotal factor contributing to both NAFLD and metabolic syndrome [13].Evidence from clinical trials, experimental investigations, and population-based studies suggests that NAFLD could potentially manifest in the liver as a representation of metabolic syndrome [13].
Non-alcoholic fatty liver disease (NAFLD) is identifed by liver fat accumulation, or in medical term called steatosis, alongside one of the three conditions: excessive weight or obesity, type 2 diabetes mellitus (T2DM), or being lean or of average weight with indications of metabolic disruption [14].Non-alcoholic fatty liver disease (NAFLD) is the liver expression of a complex disorder that afects multiple systems [15].NAFLD as hepatic manifestation of metabolic syndrome is also associated with lack of physical activity as one of the concerning risk factors [16].Epidemiological studies reveal that NAFLD global prevalence in 2023 is 30% and still increasing in upcoming years [17].It is considerably associated to increasing in prevalence of sedentary lifestyle in human population [18].Tis is supported by the results of a study conducted in South Korea, where there was an increase in the frequency of NAFLD based on the length of time a population had been sitting [19].
Lifestyle modifcation is one of the most recommended ways for patients with NAFLD or to prevent NAFLD itself.Available evidence suggests that as little as 3% to 5% weight loss is needed to improve simple hepatic steatosis [20][21][22][23].In addition, pharmacological therapy can also be given to individuals who have NAFLD [24].Te goals for pharmacological therapy of NAFLD should target the fat accumulation that occurs and the injury and fbrosis that are consequences of NAFLD [25].Another way that can be done as a management or prevention of NAFLD is supplementation in the form of adjuvants.Many adjuvants are available in the market, one of which is herbal plants.Herbal plants that are believed to have an efect on NAFLD are Moringa oleifera leaves [26].
Until now, research and clinical trials on moringa leaves have begun to develop and are being intensively carried out.However, knowledge about the utilization of moringa leaf in Indonesia is still low [27].Terefore, we are interested in conducting this review on the efect of giving moringa leaf to the liver.Tis study will be conducted on literature review research design, more specifcally in the form of a systematic review.Tis review was done to see the efect of the nutrient content and active substances in moringa leaf on tissue structure and liver function.

Research Method
Tis study is conducted on literature review research design, more specifcally in the form of systematic review.Tis review identifes the efects of Moringa oleifera leaf extract on liver structure and function in animal sample model.We used the PICO method approach to generate the clinical question for designing this study.Te population/patient we use for this study is NAFLD patient.We use Moringa oleifera leaf extract as the intervention to the population/patient.Te intervention was compared with standard diet control group.Lastly, we use liver histopathology as the outcome analysis.
Five major web electronic databases, including PubMed, Scopus, Google Scholar, Cochrane, and ScienceDirect, were used for identifying the literature that provides explanation on efects of Moringa oleifera leaf extract on liver histopathology.Search of the literature was conducted through a thorough searching process by using some concepts as Moringa oleifera, leaf extract, liver histopathology, rat, mice, and guinea pigs.Every concept was searched and combined using Boolean Operators "AND."Other similar terms of the concepts also were searched and combined using Boolean Operators "OR."Research conducted from 2014 to 2023 was encompassed in the analysis.Te pertinent research papers were gathered and will be selected through thorough selection process.Before the identifed research papers were selected, the research articles identifed as duplicates were eliminated.
Te selection process is conducted in two steps.Te initial step involved screening the titles and abstracts of the articles and studies identifed from the search results.Te title and abstract were screened by matching the criteria of the inclusion.Inclusion criteria used in the selection process were papers that explained the efects of Moringa oleifera on liver histopathology.Also, we only included experimental studies that used rat, mice, or guinea pig as the animal sample model.Other research designs, such as review, comment, and letter, were excluded.Experimental studies that used animal sample model besides rat, mice, or guinea pig were also excluded.We also verifed that the chosen articles and studies were indexed in the Scimago Journal and Country Rank (SJR) and Science and Technology Index (SINTA) for journal publications.Two reviewers (MFR and TN) independently screened the titles and abstracts in duplicate with >80% agreement.In cases where screening results were conficting, a third reviewer (PT) was involved to resolve the discrepancies.
Te next selection steps were to screen the full text of the articles that passed the frst selection process.In the full-text screening, we carefully reviewed the articles to meet the right criteria and outcome related to the efects of Moringa oleifera leaf extract on liver histopathology.Te same two reviewers (MFR and TH) did the full-text screening independently and in duplicate with >80% agreement.Te same third reviewer (PT) also did take part to resolve some conficts.
To assess the quality of the literature that included, we used Joanna Briggs Institute (JBI) Critical Appraisal Checklist [28].Te quasi-experimental checklist type was used to evaluate the quality of the literature [28].Te studies were considered of good quality if they obtained a minimum of 60% "YES" responses across all questions and of lower quality if they did not meet this threshold.Te critical appraisal was done by the same two reviewers (MFR and TH) independently and in duplicate.
Two reviewers (MFR and TH) extracted data independently and in duplicate using a standardized form designed for this study.Several data provided by the articles that were eligible were collected.We retrieved the following data: research objective, study design, target groups, and major fndings.Te data were compiled using Microsoft Excel and arranged in the form of table.
Various varieties of Moringa oleifera leaf extracts, model experiments, phytochemical content, and outcomes were explained and compared.Besides liver histopathology, we also explain the efects of Moringa oleifera leaf extract on glucose and lipid metabolism.In addition, we also provide explanation on safety of Moringa oleifera leaf extract usage, including lethal dosage and toxicological properties.

Result
3.1.Studies Included in the Review.Six hundred twentyseven studies were identifed through literature search in 5 databases.We found 0 studies on PubMed, 0 studies on Cochrane Library, 70 studies on Scopus, 26 studies on ScienceDirect, and 531 studies on Google Scholar.Before screening, we deleted 129 studies identifed as duplicate.After removal of duplicate studies, we screened the title and abstract of 498 studies by matching the inclusion and exclusion criteria that we used.Ten, 435 studies were excluded and 63 studies remained.Studies that passed the frst selection then went through the full-text screening.After that, 50 studies were excluded and 13 studies remained.Tese 13 studies have met our inclusion criteria, so we used these included studies for this literature review research.

Data
Collected from the Included Studies.All included studies are experimental studies, specifcally the included literatures are a randomized control trial in animal sample model.All studies explained the efects of Moringa oleifera leaf extract on liver histopathology.Te principle fndings and all collected data from the included literature were summarized in Table 1.
Te included studies were conducted by experts from Indonesia, Mexico, Egypt, Nigeria, Iran, Saudi Arabia, South Korea, Pakistan, and Libya.
In addition, some references, besides included literature, were chosen to provide the efects of Moringa oleifera leaf extract on glucose and lipid metabolism.Glucose and lipid metabolism were also illustrated due to their involvement in the pathogenesis and pathophysiology of liver disease, specifcally non-alcoholic fatty liver disease.Te phytochemical contents of Moringa oleifera are provided in the form of Table 2.In addition, we also provide the safety of Moringa oleifera leaf extract usage by explaining its lethal dosage.Figure 1 shows the fow diagram of literature selection process from the electronic databases.

Phytochemical Content of Moringa oleifera Leaf Extract.
Te phytochemical composition of Moringa oleifera leaf primarily comprises phenolic compounds, which are essential plant-derived micronutrients [1].Phenolic compounds are a group of compounds characterized by hydroxyl groups directly attached to aromatic structures, encompassing phenolic acids, favonoids, xanthones, quinones, coumarins, tannins, and lignans [2].Phenolic compounds play signifcant roles in safeguarding plants against physical damage, ultraviolet radiation, oxidative stress, and similar factors.Currently, there is growing interest in uncovering, extracting, and enhancing phytochemicals due to their potential to serve as alternatives to synthetic drugs with fewer side efects.Unquestionably, dried Moringa oleifera leaves serve as signifcant natural reservoirs of phenolic compounds [3].
Tis fnding confrmed the phenolic compound stated in the included literature.Some of the included literature also conducted an analysis on phytochemical component in the Moringa oleifera.Te analysis was conducted to reassuring the presence of the phytochemical compound in the Moringa oleifera leaf extract.Most of the articles that conducted analysis on phytochemical content stated that they identifed phenolic acid and quercetin as the key fndings for the phytochemical content of Moringa oleifera leaf extract [29][30][31][32][33][34][35][36][37][38][39][40][41].

Efects of Moringa oleifera Leaf Extract on Glucose
Metabolism.As previously noted, a variety of polyphenols are present in Moringa oleifera.Many compounds contained in moringa leaf are proven to have involvement in the metabolism of glucose homeostasis [46].Among the most noteworthy are favonoids like quercetin and kaempferol, as well as phenolic acids such as chlorogenic acid and caffeoylquinic acid [51].Tese compounds appear to possess antihyperglycemic attributes, acting as competitive inhibitors of sodium-glucose linked transporter type 1 (SGLT1) in the mucosa of the small intestine (specifcally, the duodenum and jejunum).Tis action leads to a reduction in the absorption of glucose in the intestines [52].However, it is important to note that glucose absorption involves other mechanisms, including the involvement of glucose transporter 2 (GLUT2), which can be infuenced by the presence of circulating glucose, directing it towards the basolateral membrane of the small intestine [53].
Moringa oleifera has been investigated for its potential in managing glucose metabolism by contributing to the reduction of glucose levels.One suggested mechanism involves quercetin, which can function as an inhibitor of GLUT2 at the apical surface [54].It is important to note that this infuence is specifc to GLUT2 and does not afect GLUT5 or SGLT1 [55].Moreover, quercetin has demonstrated the ability to activate adenosine monophosphateactivated protein kinase (AMPK), thereby enhancing glucose uptake through the stimulation of GLUT4 in skeletal muscle.Additionally, it plays a role in diminishing glucose production by suppressing the activity of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) in the liver [56].
Te aqueous leaf extract of Moringa oleifera has demonstrated the ability to hinder the activity of α-glucosidase, pancreatic α-amylase, and intestinal sucrose, contributing to its antihyperglycemic efects [57].Tese inhibitory outcomes are attributed to the presence of phenols, favonoids, and tannins in Moringa oleifera.By delaying the digestion of carbohydrates through enzyme inhibition, there is a consequent reduction in post-meal hyperglycemia and levels of hemoglobin A1C (HbA1C).Te inhibitory impact of favonoids like quercetin and kaempferol can be explained biochemically by their increased number of hydroxyl groups 8

Journal of Nutrition and Metabolism
Journal of Nutrition and Metabolism on the B ring and the presence of a 2,3-double bond [58].Moreover, these compounds have undergone scrutiny for their protective and regenerative efects on pancreatic betacells, leading to enhanced insulin production and release [59].Quercetin, for instance, stimulates insulin secretion by activating the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway and simultaneously shields pancreatic beta-cells against oxidative harm [5].Tis mechanism of action of polyphenols contained in Moringa oleifera leaf extract is in parallel with the fndings of the included literature.Some studies showed that there is signifcant reduction of blood glucose level and improvement of insulin sensitivity measured in group of animals treated by Moringa oleifera leaf extract [29,33,36,37,49].From the studies included, we conclude that dosage of Moringa oleifera leaf extract of more than 200 mg/kg body weight showed more signifcant result of glucose and insulin profle than the lower dosage [29,33,36,37,49].

Efects of Moringa oleifera Leaf Extract on Lipid
Metabolism.Phenolic compounds, as well as favonoids, have an important role in lipid regulation [49,60].
Terefore, Moringa oleifera has also been considered as a potential hypolipidemic agent.An aqueous extract derived from Moringa oleifera leaf has been reported to possess lipidlowering attributes by diminishing the creation of cholesterol micelles and inhibiting enzymes like pancreatic lipase and pancreatic cholesterol esterase, as well as impeding bile acid binding [57].Furthermore, an increase in the expression of peroxisome proliferator-activated receptors (PPARs) α1 (PPARα1) and c (PPARc) has been observed in rats administered Moringa oleifera seed powder [61].PPARs play a pivotal role in lipid metabolism, ketogenesis, and cellular energy balance, and they are present in various organs, including the liver, brain, muscles, and heart, among others.Additionally, PPARs are implicated in processes such as infammation, immunity, and glucose regulation [62].Another research demonstrated that administering Moringa oleifera leaf extract during the process of adipogenic differentiation leads to a decrease in infammation and lipid buildup, while also triggering thermogenesis through the activation of key proteins such as uncoupling protein 1 (UCP1), sirtuin 1 (SIRT1), peroxisome proliferator-activated receptor alpha (PPARα), and coactivator 1 alpha (PGC1α).Furthermore, Moringa oleifera Lam.induces the expression of heme oxygenase-1 (HO-1), a well-recognized protective and antioxidant enzyme [63].
Tose fndings on hypolipidemic efects of Moringa oleifera are in parallel with the outcomes of the included literature.Most of the studies provide explanation of the resulting efects of Moringa oleifera leaf extract on lipid profle.Te collected data of included literatureexplained that Moringa oleifera leaf extract provided signifcant reduction of triglyceride, LDL-C, VLDL-C, and cholesterol in group fed by Moringa oleifera leaf extract [29,33,35,36,49].Besides that, included literature also stated that there is an improvement of HDL-C in lipid profle of group fed by Moringa oleifera leaf extract [36].
By now, it is recognized that the consumption of a highfat diet stimulates the generation of proinfammatory cytokines like IL1B and TNFA, which are activated via the NFkB pathway [64,65].Tis dietary pattern also results in an elevation of mitochondrial ROS production.Tese free radicals contribute to the peroxidation of lipids on cellular and organelle membranes, leading to the formation of malondialdehyde (MDA), which serves as an indicative marker of oxidative stress [66].Choi and Das illustrated an increase in lipid peroxidation products such as MDA and 4HNE in a model of NAFLD, attributable to the generation of reactive oxygen species [67,68].Some studies revealed that the application of moringa results in a decline in the generation of MDA, attributed to a reduction in ROS production [49].Tis fnding is in parallel with some included studies that also show signifcant reduction of lipid peroxidation in liver characterized by reduction of MDA level in group fed by Moringa oleifera leaf extract [29,30,34,38].
From the studies included, we conclude that dosage of Moringa oleifera leaf extract of more than 200 mg/kg body weight showed more signifcant result on lipid profle and MDA level than the lower dosage [29,30,34,38].

Efects of Moringa oleifera Leaf Extract on Liver
Histopathology.Several hypotheses have been proposed to explain the mechanisms of liver injury leading to liver steatosis.Tese include liver injury caused by hepatotoxins, damage from a diet high in fat, and liver injury related to metabolic disorders [69][70][71].All those etiologies may trigger liver damage by means of oxidative stress, infammation, fbrogenesis, and liver necrosis.Sudden damage to liver cells disrupts their transport function and the permeability of their membranes, causing the release of marker enzymes [72].Te internal processes of damage within hepatocytes involve the creation of reactive metabolites, reduction of glutathione levels, and protein alkylation, particularly affecting mitochondrial proteins [73].Tese initial actions prompt the opening of the mitochondrial membrane, resulting in permeability transition.Te deterioration of the mitochondrial membrane permeability transition occurs prior to the failure of the cell's outer membrane, leading to cell swelling and the release of cellular contents.Tis ultimately culminates in cell death through a process known as oncotic necrosis [74].
Te evaluation of liver damage was gauged through the measurement of serum levels of ALT and AST, which are widely recognized as prevalent indicators of liver harm.When the integrity of the liver cell membrane is compromised, several enzymes that typically reside within the cell's cytosol are discharged into the bloodstream.Te concentration of these enzymes in the serum provides a valuable quantitative gauge of the magnitude and nature of hepatocellular injury [71].
Liver injury, in the term of NAFLD, is often characterized by macrovesicular steatosis, ballooning degeneration of hepatocytes, scattered (mainly lobular) infammation and apoptotic bodies, and Mallory-Denk bodies (MDBs).Fibrosis also often presents alongside the other histological features [75].
Te included literature had revealed that there is signifcant improvement in the histopathological analysis of the liver tissue conducted on animal sample treated by Moringa oleifera leaf extract.All studies included explained that after administration of Moringa oleifera leaf extract, there is signifcant reduction of fbrosis, hepatic cells necrosis, lipid accumulation, infammatory cells infltration, hepatocellular degeneration, vesicular congestion, and sinusoidal dilatation.Various dosages of Moringa oleifera leaf extract had been analyzed.Higher dosage of Moringa oleifera extract showed more reduction on the histopathological feature of the liver tissue, while lower dosage did not give any notable improvement on the histopathological feature of the liver tissue [29][30][31][32][33][34][35][36][37][38][39][40][41]49].Some studies included also measured liver injury biomarkers as other fndings besides liver histopathological features.Tese studies stated that there is signifcant reduction on the AST, ALT, ALP, SGOT, and SGPT in the group of animals treated by Moringa oleifera leaf extract [29-31, 33-36, 40, 41, 49].
4.5.Safety of Moringa oleifera Leaf Extract.Earlier investigations were carried out on animal models to analyze the oral toxicity (LD50) of Moringa oleifera leaf extract [76].Te fndings indicated that the aqueous leaf extract of Moringa oleifera did not result in any fatalities even at the highest administered dose of 6400 mg/kg body weight [77].A study conducted by Fouad et al. [78] on acute oral toxicity reported that Moringa oleifera leaf extract demonstrated non-lethal efects on animals at a dose of 2000 mg/kg body weight and mentioned that animals may display some adverse changes at doses beyond this level.Moreover, Diallo et al. [79] documented that the aqueous leaf extract of Moringa oleifera was safe even at dosages as high as 5000 mg/ kg body weight.
Tese fndings suggest that the aqueous leaf extract of Moringa oleifera is safe for oral consumption, displaying no lethal efects during acute administration.It is noteworthy that a dose of 2 g/kg body weight was identifed as the threshold for medicinal plant toxicity in acute oral toxicity studies [80].However, this safety assertion might not hold true for medicinal plants consumed over an extended period.Slight lethargy was observed in animals receiving doses above 1600 mg/kg body weight during acute administration, which aligns with the observations of Adedapo et al., who noted toxic changes in animals above 2000 mg/kg body weight.Te LD50 from an acute oral intraperitoneal toxicity study for Moringa oleifera leaf extract was determined to be 1585 mg/kg body weight.
Previous study conducted by Kushwaha et al. [81] documented the adverse outcomes of Moringa oleifera leaf extract in studies involving human subjects.In this research, a group of 30 post-menopausal women was provided a daily supplementation of 7 g of Moringa oleifera leaf powder for a duration of 12 weeks, and their results were compared with those of a control group.Te study revealed a rise in antioxidant biomarkers that contributed to hypoglycemic and hypolipidemic efects, all without inducing any harmful efects.

Conclusion
Te phytochemical content of Moringa oleifera leaf extract had shown a signifcant beneft in plant medicinal sector.From the research that had been done, Moringa oleifera leaf extract contributes to give signifcant improvement on liver histopathological features, glucose, and lipid metabolism on animal sample model.In the coming times, further investigation into this medicinal plant could ofer promising prospects regarding its efectiveness and safety as a therapeutic solution for NAFLD.

Limitation. Due to the various types of extracts
employed in studies, it becomes imperative to determine if specifc phytochemicals within Moringa oleifera leaf extract are present in each variant.Consequently, additional investigations are warranted to discern the most potent type of Moringa oleifera leaf extract and its optimal bioavailability.