Abstract

Type II diabetes mellitus is one of the most common public health problems worldwide. Its increasing prevalence in several countries and the difficult metabolic control of individuals with the disease justify studying strategies for primary prevention. The population has sought alternative and cheaper ways to treat the disease, including the use of plants considered medicinal by the population. In this study, we carried out a systematic review on the applicability of isolates and fractions of plant extracts in animal models in type II diabetes. A literature search was performed in MEDLINE/PubMed and Scopus databases. Studies using other experimental animals (horses, rabbits, and monkeys) and humans as well as articles in Chinese, German, and Russian were excluded. We assessed the quality of the studies included by using the criteria described in the ARRIVE guidelines. In general, the animals that received fractions or isolates presented reduced blood glucose levels, normalization of body weight and plasma insulin levels, and reduced total triglycerides and cholesterol. In addition, we observed wide variation among the analyzed parameters, which hindered comparison between the studies found. In further studies, standardized reports and experimental design would help to establish comparable study groups and advance the overall knowledge, thus facilitating translatability from animal data to human clinical conditions.

1. Introduction

Diabetes mellitus (DM) is a syndrome caused by changes in the metabolism of carbohydrates, lipids, and proteins and may occur in two different forms: type I (10%) and type II (90%) [1]. Type I diabetes results from body inability to produce insulin [2], while type II may be caused by a failure in the production and secretion of insulin by the pancreas, due to insufficient production or a problem in the beta-cell receptors, thus decreasing the sensitivity of the target tissue to the metabolic effect of this hormone. This decreasing sensitivity is known as insulin resistance [3]. Patients with this disease present polydipsia (excessive thirst), polyuria (urine production over 2.5 liters/day), polyphagia (excessive avidity for food), and delayed wound healing [4]. Besides, poor glucose metabolism, reduced insulin signaling, excessive release of free fatty acids, and interleukin-6 are changes also considered important for analysis in clinical and preclinical studies [5].

According to the World Health Organization (2012), diabetes mellitus accounts for 3.5% of the noncommunicable diseases in the world. The International Diabetes Federation (IDF) [6] estimates that, by 2013, more than 382 million people around the world had been affected by the disease, and the figures are increasing in all countries. Portugal, for example, is the second country in Europe with the highest prevalence of diabetes mellitus: 12.7 people with DM per 100 inhabitants in 2011 [7].

In Brazil, according to estimates, there are more than 12 million people with type II diabetes mellitus (DM2) [8], mainly people who are over 40 years of age and obese. However, recent studies have shown a considerable increase in the number of children and teenagers with the disease, which may be associated with bad eating habits and lack of physical activity. This leads to increasing rates of obesity, which is considered a risk condition for the development of type II diabetes [9].

The population has sought alternative and cheaper treatments for the disease, including the use of plants considered medicinal by the population. In several countries, such as Japan, China, and India, the use of medicinal plants and their derivatives is increasing, since they are considered simple, cheap, and effective treatment alternatives [10, 11]. There are some reports in the literature about the benefits of different herbal treatments on several metabolic changes caused by type II diabetes. The studies generally show positive effects of plant extracts on the normalization of body weight and decrease of glucose levels, total cholesterol, and triglycerides [12].

Therefore, the use of phytotherapy has opened a new perspective for the management and treatment of metabolic diseases, such type II diabetes, since it is an affordable treatment [13]. However, the use of most of these plants has not been investigated [14, 15]. Research on plant extracts should be conducted to ensure that they are effective and safe for the population [16]. Due to these factors and the high number of type II diabetes carriers, the demand for less expensive therapies may significantly benefit population health [11].

In vivo and in vitro studies are commonly used in biomonitoring research of plant extracts, aiming to identify their biological activity. The fractions are metabolites obtained by fractioning plant extracts, which provides a more specific analysis of the plant active principle [17]. These studies are important for the treatment of chronic diseases, including diabetes, since the treatment or control of these diseases is expensive and the number of affected people has increased considerably. Besides the fractions, the isolates obtained from plants have also played an important role in the treatment of metabolic disorders. After isolating a particular component of the crude extract, it is possible to ensure that the effects on the tissue are caused only by a specific constituent of the extract. This makes the molecule more attractive to the drug market, which aims to develop drugs from plants or other materials.

A systematic review is based on predetermined criteria and consistent scientific evidence. It aims to collaborate with research selection and/or tools for the development of products based on original information [18], with well-defined criteria selection, to ensure the quality of the summarized studies and their reproducibility. Moreover, a conclusion providing new information based on filtered content is necessary [19]. Generally, due to its rigorous methods to identify, select, collect, and analyze data, this kind of study provides the highest level of scientific evidence. Therefore, our study aimed to make a descriptive and critical analysis of studies on the activity of plant fractions and isolates in the treatment of type II diabetes in animal models.

2. Material and Methods

2.1. Selection of Papers

The papers analyzed in this review were selected from two electronic databases, PubMed and Scopus, accessed on September 3, 2015, using the search filters: “animal model”, “plant extract”, and “diabetes mellitus type II”. These filters have been developed for the search on PubMed, according to the Medical Subject Headings (MeSH terms), used for a more efficient indexing of publications on the subject under study [20]. In order to expand the search, MeSH terms were combined with the title and abstract (TIAB). A standard filter was used [21] to identify all studies with animals in PubMed. The terms used to search on PubMed were adapted for the selection of Scopus publications, and the “animal model” filter was provided by the site itself (Supplemental Data 1 in Supplementary Material available online at http://dx.doi.org/10.1155/2016/3537163).

The Prism guideline was used to develop this review [22]. After the papers were collected from the two electronic databases, the duplicates were excluded by comparing the title, author, year, and country. A screening was performed for title and abstract, guided by the eligibility criteria: in vivo experimental studies; studies using rats or diabetic mice; use of fractions or isolates of noncommercial plants; treatment of the main symptoms of type II diabetes; studies written in English or Portuguese.

Next, all selected papers were obtained in full for a second screening, when all of them were examined to select those that met the criteria for the inclusion in the systematic review. Those unavailable on the internet were requested from their respective authors. When they did not respond, the studies were excluded. The entire search process, exclusion, and the number of selected papers were described in detail in the PRISMA Guideline (Figure 1).

2.2. Qualitative Characteristics of Publications

After screening, the papers were reviewed. Table 2 shows the description of the main characteristics of the studies. The following parameters were assessed: (1) publication features: author, year, and country; (2) experimental features: animal model, species, sample number, sex, weight, age, type of caging used, number of animals per cage, number of experimental groups and number of animals in each group, if randomization was made, and control groups; (3) treatment features: plant species used, name of the fraction or isolate, dose, route of administration, and treatment duration; (4) diabetes induction: drug used, dose, route of administration, and testing to prove diabetes occurrence (Table 1).

2.3. ARRIVE (Bias Analyses)

The detailed reports of experiments are crucial in the review process, so that they can be validated and used as a source of information for further research. However, many studies do not bring relevant or concise information, which leads to the realization of redundant and duplicated experiments [23]. Therefore, guidelines were developed for animal research reports, such as the ARRIVE guideline, based on the CONSORT Statement. The ARRIVE guidance is a list of 20 items that describe the minimum information that all scientific publications reporting research using animals must include, aiming at high quality reports and critical and accurate review of what was performed and found [24]. Thus, based on these fundamentals and the objective of this study, a table displaying the most relevant and applicable items from the ARRIVE was developed for a critical evaluation of the studies included in this review (Table 2). The authors assessed the quality, integrity, and transparency of each publication. Divergent opinions were resolved by consensus.

3. Results

3.1. Prism

The search conducted in this study found a total of 1,067 papers, out of which 571 were found in PubMed and 495 in Scopus. Out of this total, 449 papers were duplicates; thus 618 studies remained. Then, a title and abstract screening was performed, guided by the eligibility criteria listed above. In this respect, 574 studies were excluded due to inadequate research topic. Among the excluded studies, we can highlight those on the crude extract of the plant (200), studies in languages other than English and Portuguese (63), secondary studies, literature reviews, editorials, comments (60), in vitro studies (54), and studies in which alcohol was administered in the diet (35). Next, 39 studies were selected and their reference lists were screened to identify additional relevant studies missed in the initial search strategy. Thus, all the studies that met the eligibility criteria were included in the review, taking into account the use of fractions and isolates from noncommercial plants in the treatment of type II diabetes in animal models of rats and mice. All search process is shown in Figure 1.

3.2. Qualitative Results

With respect to papers reporting treatments with plant fractions (), the years of publication ranged from 2001 to 2015. Most studies used rats (60.9%) and mice (39.1%). The sample size varied greatly; some studies used 14 animals and others, 98 animals, while 36.4% of the publications did not report such data. Most studies used male animals, but 2 papers reported the use of both sexes, and 21.7% of the studies did not provide this information. The age of the animals ranged from 3 to 14 weeks and 56.5% of the studies did not report these data. The weight of the animals was not reported in 21.74% of the studies. Only 26.1% of the papers reported if randomization was applied in the experimental groups. Fractions of the extracts were administered orally in 91.29% of the studies and the treatment duration ranged from 7 days to 10 weeks. Regarding the drug used to induce type II diabetes, 60.86% of the studies used streptozotocin; 8.69%, alloxan; and 4.35%, PX-407 (Table 1). China (34.9%) and India (26%) are the countries with the largest number of publications on this subject. Around 47.8% of the studies used a control group. Metformin (30.5%) and glibenclamide (21.7%) were the most commonly used drugs (Figure 2).

The studies that used plant isolates in the treatment of diabetes () were carried out from 1998 to 2014. Mice (56.25%), rats (37.50%), and both (6.25%) were the species used in the experiments. The sample size ranged from 12 to 102 animals, and 37.50% of the studies did not report such information. Most studies used male animals (87.50%) and 12.50% used both sexes. The age of the animals ranged from 7 to 26 weeks. The weight of the animals was not reported in 75% of the papers and the strains used were not provided in 31.25% of the studies, while 62.50% of the papers did not report animal randomization. The treatment was administered orally in all analyzed studies (100%). Diabetes was induced with the use of streptozotocin (56.3% of the studies), PEG300 (6.25%), and insulin solution (12.5%) (Table 2). Japan (23.5%) stands out among the countries that have developed studies in the area, followed by India and Taiwan (17.64%) (Figure 3).

The main results for plant fractions and isolates in the treatment of type II diabetes are shown in Figure 4. The main findings were (A) reduced blood glucose levels in isolate treatments (25, 27, 30, 31, 32, 33, 34, 35, 36, 37, 39, and 40) and fractions (17, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 56, 57, 58, 59, 60, 61, and 62); (B) normalization of body weight in studies using fractions (17, 43, 44, 49, 50, 52, 55, 57, and 59) while only two studies evaluated this parameter in isolates (27 and 34); (C) normalization of plasma insulin levels during treatment with fractions (17, 45, 47, 48, 49, 50, 51, 53, 55, 58, 60, and 61) and treatment with isolates (25, 26, 27, 28, 29, 30, 34, 36, and 38); (D) reduced total triglycerides and cholesterol in studies using fractions (17, 42, 45, 44, 46, 48, 52, 54, and 59) and isolates (31, 35, 38, and 40); (E) increased glycogen synthesis in studies using fractions (45, 51, and 60) and isolates (27, 29, and 35). Decreased water and food intake has been described in only one isolate (35) and four fractions (41, 51, 54, and 60). The normalization of glycosylated hemoglobin was observed in seven fractions (44, 49, 52, 54, 56, 60, and 61) and one isolate (27) (Figure 4).

3.3. ARRIVE (Bias Analysis)

The ARRIVE guidelines were used to assess the quality of the papers under analysis (Table 1). After reading and performing critical analyses, the researchers observed that 84.61% of the studies had exact title and concise description. Abstracts describing the purpose, methods, main results, and conclusions were found in 92.30% of the studies. Primary and secondary objectives were clearly stated by 82.05% of the studies, while 92.30% reported in the methodology description that they had obtained permission from the ethics committee for performing the research; on the other hand, experimental information about controlled or blind study was observed in only 15.38%. The animal species were cited in 89.74% of the papers, while weight and sex were described in only 56.41% of the studies. It was observed that 46.15% of the publications reported genetic changes in animals, while lodgment and environmental conditions (light/dark cycle, temperature, and water) were reported in 35.89% and 84.61% of the studies, respectively. Regarding the sample size, 56,41% reported the total number of used animals, but only 5.12% explained the reason for choosing such numbers, and 28.20% of the authors reported the use of randomization. It was observed that 87.17% of the studies specified each statistical analysis method. Only 7.69% of the papers reported the occurrence of animal mortality during the experiment. Among the evaluated discussions, 89.74% interpreted the results taking into account the objectives and hypotheses of the study, current theory, and relevant publications. Only 30.76% commented about the limitations of the studies. Comments on the importance of applying the results to human biology were found in 56.41% of the studies.

4. Discussion

This review aimed to describe the main findings in literature on the effects of fractions and isolates obtained from plant extracts on the treatment of type II diabetes in murine models. We believe that the information obtained may help and provide guidance to researchers about the best animal models, drugs, and most used doses in disease induction. Besides, it will guide further research on the most common and important parameters to describe the best results for controlling the metabolic changes caused by the disease. Studies that tested crude plant extracts were not included in this review, due to their wide variability. Studies that obtained fractions and isolates commercially were also excluded. Although species differences prevent the direct extrapolation to clinical applications in humans, the current findings strongly point to the need for a more controlled preclinical research in animals and then in humans, mainly in relation to the doses of fractions and isolates and the most used plant species.

The present review showed that isolates or fractions of plants had positive effects on diabetes treatment and reduced various animal blood and tissue parameters that had been changed by the disease. This study also highlights important issues related to the quality of the models and protocols, drugs and doses used in the study for inducing the disease, the most commonly used administration routes, and main tests used for disorder confirmation. Systematic review studies are focused on the assessment of the quality of the reviewed studies, using acknowledged scales and protocols. Although these scales have not been formally developed for experimental model studies, the assessment of the quality of the reviewed studies considered the items normally included in scales for randomized clinical studies. Therefore, we used the ARRIVE platform for work quality analysis and observed that most studies did not provide many details about the materials and methods used, which prevents the replication of some studies. There were no reports of the number of animals used, age, weight, and even the presence of randomization to reduce bias in the selection of the animals and assessment of the results in many studies. These findings corroborate the need for guidelines to describe the required information for all scientific publications that use animals as experimental models [24]. In this study, out of the 618 articles analyzed, 39 were selected according to the eligibility criteria and the proposed objective. The PRISMA recommendations were used to guide the development of this systematic review and improve the visualization of the steps of an effective search [22].

Most animals studied were male, since males suffer less hormonal fluctuation and hence less change in behavior compared to females [62]. The number of studies with rats () and mice () was very close. However, is it possible to detect the increasing use of mice in preclinical experiments, due to the genetic similarities between this species and humans. According to Machado and Zatti [63], about 99% of human genes have been mapped in mouse, which allows the association between them. Moreover, it must be taken into account that the small size of these animals reduces the costs of the experiment and makes it easy to handle and perform a great number of procedures. There was wide variation in the age of the animals. The youngest animals were 3 weeks old and the oldest, 26 weeks. In addition, many studies did not provide such information (). The weight of the animals ranged on average from 25.5 g in mice to 61 g in rats. The authors attribute this great variability to the discrepancies in the age of the animals. Besides, the variable weight was not reported in 38.4% of the studies. The number of studies that did not describe variables such as age and weight is worrying, since these characteristics are important for further replication of the studies and elaboration of extensive reports on the procedures adopted [64].

Streptozotocin, either combined or not with another drug, was the main drug selected for type II diabetes induction in animals. Streptozotocin is a large spectrum antibiotic, used as a diabetogenic agent in experimental animals [65]. This action is mediated by the destruction of beta cells in the pancreas, which leads to insulin deficiency and also occurs in human type II diabetes in relation to metabolic characteristics [66]. Wide variation was observed in the streptozotocin dose used to induce diabetes, from 20 mg/kg to 137 mg/kg. The analysis of works with drug-induced diabetes mainly requires the establishment of the most appropriate dose and the correct administration time, since these variables may reduce the time and costs of the experiment. In this review, after the analysis of the work, no consensus was found for the best dose and timing for drug application. Metformin was the main drug selected for the control group, due to its relevant clinical use, favorable toxicity profile, and safety. Besides, it is well tolerated during treatment [67].

Although many drugs have been used for diabetes treatment, some of them are expensive and inefficient and cause severe side effects. Thus, there is a growing interest from researchers and pharmaceutical companies in the development of alternative drugs, such as medicinal plants, for diabetes treatment [6870]. However, further studies should be conducted, since some plants associated with diabetes mellitus treatment are considered toxic and may cause various tissue lesions [71]. The present study reports several plant species that have been used to obtain both fractions and isolates. Most studies were carried out in China, India, and Japan. These countries have shown great interest in the development of drugs from plant extracts, due to their great flora diversity. Japan has always had an interest in the development of new technologies. Phytotherapy is promising for health care in many ways. The analysis of the results of the studies revealed that most authors reported decreased blood glucose in treated diabetic animals () and normalized plasma insulin levels (). Postprandial hyperglycemia is a common pathogenesis of type II diabetes induced by insulin resistance, as well as the partial destruction of pancreas β cells [7275]. The effective control of blood glucose and insulin level is a key step in preventing or reversing diabetic complications and improving the life quality of patients [76]. Hyperlipidemia is another complication caused by diabetes, characterized by high cholesterol and triglyceride levels and lipoprotein composition changes [77]. These data were analyzed in this study, due to their relevance. Thirteen studies reported decreased total cholesterol and triglyceride levels in animals treated with plant derived medicine.

Besides, polydipsia, polyphagia, and changes in weight (weight loss or gain) are common occurrences in patients with diabetes [78]. Weight loss is usually observed when the disease is acquired and can be associated with dehydration and catabolism of fat tissue or protein degradation and consequent muscle mass loss. According to this review, many studies reported increased animal water and food intake, as well as weight normalization. These data can be justified by the increased glucose and insulin uptake and decreased secretion of blood glucose, which indicate improved animal glycemic control [49]. These improved results in the body weight of diabetic animals are consistent and were reported by some studies using medicinal plants with potential antidiabetic effects [79, 80]. The increased glycogen synthesis was also analyzed, since the liver metabolism of this substance regulates glucose blood level [50]. In addition, some studies reported the normalization of glycated hemoglobin levels, which is important to assess diabetes control levels, since its dosage directly reflects the average blood glucose levels, from two to three months prior to the collection of the biological material [81].

5. Conclusion

The results of this study demonstrate that plant fractions and isolates improve the main physiological and morphological changes caused by type II diabetes and decrease food and water intake, total cholesterol, triglycerides, and glucose, thus normalizing body weight and blood insulin levels. However, serious methodological problems were found in many studies, including errors in the details of the procedures performed, which prevents the understanding of some studies and hinders the use of the data found in animals for studies on human clinical condition. Therefore, the improvements in research reports on preclinical studies require a collective effort from authors, journal editors, reviewers, and funding agencies to ensure that the papers will allow other researchers to reproduce the study.

Competing Interests

The authors declare that there are no competing interests regarding the publication of this paper.

Acknowledgments

The authors are thankful to the “Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG)” for the financial support (FAPEMIG Approval Register PPM-00868-15), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and “Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).”

Supplementary Materials

Descriptors used for advanced search in PubMed and Scopus.

  1. Supplementary Material
  2. Supplementary Material