Anti-hyperglycaemic Effect of Aegle marmelos (L.) in Animal Experiments-PRISMA based Systematic Review and Meta-analysis

Aegle marmelos is a folklore medicine which is widely consumed as an anti-hyperglycaemic drug, its properties are known since prehistoric times. Plenty of cell line as well as animal research have reported the anti-hyperglycaemic activity of the drug. Even though quality researches are necessary to fulfil further human studies and marketing, no quality assessments for these studies have been undertaken till date. The aim was to critically analyse the quality standards and evaluate the anti-hyperglycaemic potential of A. marmeloes in rodent model researches. The search strategy was done using the key words ‘Aegle marmelos, along with ‘Diabetes or Hyperglycaemia,’ in different databases like PubMed (n = 45), Google Scholar (n = 43) and Science Direct (n = 46). However, the filter used during the search was ‘Animal experimentation’. All animal experimentations collected with the keyword search was analysed for the inclusion criteria like serum glucose estimation as primary outcome, timeline of publication 1997 to 2017 in English and treatments containing any part and any form of A. marmelos in single or in combination. Based on the inclusion criteria, 11 articles were selected for the review. Data extraction underwent a blind procedure independently by two reviewers. If full texts were not available, communicating the authors was also tried and all the four authors responded. All the studies selected after screening were given unique codes, to ensure blind review. Result observed indicated higher reduction of serum glucose levels in hyperglycaemia-induced rodent models. After administering the drug, significant changes were observed in total cholesterol as well as triglycerides. Rest of the LDL, HDL, SOD and serum insulin were an in-significant find. Sample size calculation was not mentioned in any of the studies. Only 9% of the studies reported on blinding and 18% reported on randomisation. This might have affected the internal validity of the studies. It can be concluded that A. marmelos is effective in lowering the serum glucose levels in experimental hyperglycaemia. Further research has to be conducted to explore its target specific mode of action. This may lead to the development of an effective anti-hyperglycaemic drug with minimal side effects. DOI:10.18311/jnr/2019/23676


Introduction
Pre-senile diabetes mellitus is the primary cause of a majority of the deaths in India. Diabetes will be the seventh pre-eminent cause of death in 2030 as per WHO. Its prevalence rate has augmented from 108 million to 421 million in 30 years. The occurrence of diabetes among adults above 18 yrs has also risen to about 3.7% in 30 years. The dispersion of diabetes is high in developing countries 1 . Medicinal herbs are the plausible source to aid treating diabetes in health care system all over the world. Ayurveda, the ancient Indian system of medicine elaborates the use of plants in various ailments. Chemical constituents in plants vary depending on the species, the plant part used, the storage measures use, the harvesting time, as well as the tropo-geographical area etc. The finished products may also contain constituents in varied amounts in different manufacturing batches. This variability accounts for the difference in pharmacological action of the herbal medicine 2 . It is the time to expound and conduct a pragmatic herbal research as there is enormous and emergent use of the natural products. It is worth procuring an unswerving data for the health care system 3 .
Aegle marmelos is extensively consumed as an antihyperglycaemic drug and its properties are known since prehistoric times. Bark juice and leaves of Aegle marmelos are said to hold anti-hyperglycaemic property as mentioned in classical texts of Ayurveda 4 . Effect and quality of these experimental studies have to be analysed for the maintenance of uniform standards. This meta-analysis is the first of its kind conducted in Ayurveda. Various pharmacological activities of the herb were researched pre-clinically. Analysis of these studies in terms of risk of bias and heterogeneity is essential for further clinical and marketing researches. Aim of this review was to analyse the effects and quality assessment in terms of risk of bias and calculation of heterogeneity of pre-clinical anti-hyperglycaemic activity of A. marmelos.

Materials and Methods
Animal experiments commenting on the antihyperglycaemic action of A. marmelos published from 1997 October -2017 October, were searched using the key words ' Aegle marmelos' and 'Hyperglycaemia' , 'Aegle marmelos' with 'diabetes' in databases like PubMed, Science direct and Google scholar. Experimental studies that involved rodent animal models were considered for this review. The filter used while searching was 'animal experiments' . Published researches in English language were included in the review. Those studies that contained serum glucose estimation as outcome measures were considered. The title may or may not contain the word 'hyperglycaemia' or 'diabetes' . Among the selected studies, those studies that did not include serum glucose estimation as outcome measure or sub group analysis were excluded. Available animal publications of A. marmelos were searched irrespective of parts, dose, route, and duration of administration 5 . In the current study, if the parameter concerned was described in three or more studies, meta-analysis of that activity was performed. Serum glucose was taken as a primary parameter and serum insulin, total cholesterol, triglyceride, LDL, HDL and SOD were used as secondary parameters. All the study characteristics like inducing agent, control drug, number of groups described were taken into consideration 6 . Random statistic model was used in the meta-analysis. Heterogeneity was assessed using I 2 . Sources of heterogeneity were compared between studies. The data was analysed using MICROSOFT EXCEL-2O15. Statistical significance was defined in all studies where the P-value is < 0.05 7 .

Article Selection
Based on the inclusion criteria, 11 articles were selected for the review. The search scheme and portrayals of the studies are briefed in Figure 1 and Table 1 respectively.

ROB Evaluation
The ROB and quality of the included studies were evaluated individually. ROB was analysed with SYRCLE' s Tool 8 . Possible sources of risk of bias were not reported in   majority of the studies. While assessing the quality of the studies, the process of blinding and sample size calculation were not explained in most of the studies (80%). There were 60% of unclear risk of bias as essential parameters Key to abbreviation-#=unclear risk of bias, L-low risk bias, H=high risk of bias, y=yes, N=no were not mentioned, this includes reporting of withdrawal symptoms, which constitutes attrition bias. The results of the ROB evaluation are given in Figure 2 , 3 and

Data Blend
In meta-analysis, mean differences in each studies were used in all calculations. Individual and overall effect sizes were calculated with random effect model of Cohen's D. Cohens' D is a simple and effective measure for estimation of effect sizes individually and as a whole. Heterogeneity I 2 were also determined. Subgroup analysis was accomplished. Chi-square values were assessed with degrees of freedom, k-1 where k denotes the number of total studies included. For each outcome, inverse variance, significance levels, Q value and 95% CI were also analysed.

Synthesis of the Result
Primary outcome: Serum glucose levels

Outcome of A. marmelos on Serum Insulin
In five studies, serum insulin was being used as an outcome measure. There were no significant changes in the serum insulin after administration of the drug.

Outcome of A. marmelos on SOD
Rodents might develop stress induced cell damage as they are exposed to different chemicals, foods, care takers and environment. In order to distinguish the cause of the cell damage in the context, SOD evaluation is important. Administration of A. marmelos showed non-significant results of decreased SOD activity (Effect size, Cohens d = .40; inverse variance (IV) =50, 95% Confidence interval (CI)=0.13-0.67; I2=51.6%, P < 0.05 level even though it was significant in individual studies (Table 10, Figure 10) 15,18,19 .

Active constituents of A. marmelos
Different parts of the A. marmelos possess different chemical constituents.

Fruits
A. marmelos is an herb that is described to hold a variety of sterols, alkaloids, coumarins and essential oils. Alkaloids present were aegeline and marmeline, which were identified as N-2-hydroxy-2-ethyl cinnamide and beta sitosterol identified as N-2-hydroxy-2-ethyl cinnamide. Coumarins present were imperatorin, xanthotoxol and alloimperatorin 20 .

Seeds
In addition to this, a few new compounds like plumbagin, imperatorin, b-sitosterol, b-sitosterol glucoside, stigmasterol, vanillin and salicin were identified from seeds of Aegle marmelos Correa 23 .

Limitations
Sample size calculation was not mentioned by any of the studies. Only 9% of the studies reported on blinding and 18% reported on randomisation. This might have affected the internal validity of the studies. External validity is mainly through biological differences of study subjects, pathological differences occurring during induction, animal species and sexual variation. It was not possible to rule out the risk of publication bias such as overestimation of the result based on available data.

Discussion
This meta-analysis using 11 animal research studies found that anti-hyperglycemic potential of A. marmelos, was through reduction in serum glucose, serum insulin; antiobesity effect through change in total cholesterol, LDL, HDL and oxidation potential through enzyme SOD are analysed. Values were taken from the group containing drugs in 2:2:1, in which A. marmelos constitutes 2 parts. This group showed significant reduction of serum glucose compared to others. Serum insulin was a parameter included in five of the selected studies. Active principles present in A. marmelos might be accountable for its favourable actions such as Aegeline, Marmeline, Halfordinol, Quercetin, Rutin and Umbelliferone. In addition, the same has showed significant antihyperglycaemic action in animals. Quercetin and rutin are two active ingredients found in A. marmelos, which were found to be possessing anti-hyperglycaemic action of A. marmelos. Other compounds having effect on insulin secretion were ferulic acid, ellagic acid, and eugenol. Umbelliferone could reverse plasma glucose and some action on serum insulin. Halfordinol from leaves have potent effect on hyperglycaemia. It augments insulin secretion by inhibition of alpha amylase enzymes. All the animal studies reported showed significant improvement except in glucose insulin, cholesterol and SOD. The variation might be, owing to not considering the outcomes of the study in terms of the effective size individually. This meta-analysis revealed that A. marmelos significantly reduces the serum glucose levels and total cholesterol levels in experimental rats. Nevertheless, high variability was observed in the estimation of serum glucose (I 2 =86%).
In this meta-analysis, the number of sub-group analysis was limited, so the expected outcome measure was not available. Total of 11 studies were included in the analysis which used serum glucose as one of the parameter. Two different chemicals, streptozotocin and alloxan were found in the studies as inducing agents. Glibenclamide, metformin and glipizide were the drugs included as diabetic controls. Allocated study groups were in a range of 2-8 groups, which contained test drug in different concentrations like-non-diabetic control and diabetic control in all the studies. Number of animals present in each group varied from 6-8. If more than one concentration of test drug was included in the study, the mean differences and standard deviations of maximum concentration of the test drug was taken for analysis. This might be the reason behind validity issues and unsolved crises in the study. After analysing the sub-groups, it was found that A. marmelos possess superior effect than the control drug, glibenclamide in lowering the serum glucose levels. The authenticity of the outcome was lowered due to the fewer number of subjects in each group and other differences in experimental designs.
Some of the detected advantageous effects of A. marmelos were accountable to its anti-oxidant effect. The induced stress might have produced profuse amounts of reactive oxygen species in animals. However, SOD findings in the present meta-analysis has showed non-significant results. This result might be due to the variations in external validity. High variability observed in some of the parameters might be due to the differences in sample size, designs, inducing agents, dose, number of groups present and the part used. The same study can be repeated with sophisticated and stringent study protocols.

Conclusion
Antihyperglycaemic potential of A. marmelos might be accountable for the additive or synergistic mechanisms of active constituents. Umbelliferone can reverse plasma glucose and halfordinol has potent effect on hyperglycaemia. It augments insulin secretion by inhibition of alpha amylase enzymes. Total cholesterol and triglycerides were significantly lowered from the administration of A. marmelos, while LDL and HDL values were found not to be significant. To develop valid clinical data, pre-clinical animal researches are very essential, provided experiments are screened for checking its adherence to rules and regulations.