INTRODUCTION
Among various life-threatening metabolic diseases, diabetes mellitus is taken into account as one of the major causes of substantial increment of health issues worldwide [1]. The result of the International Diabetes Federation (IDF) survey in 2019 revealed that 463 million adult people are suffering from diabetes mellitus globally [2]. Without any proper efforts to explore the ways of managing diabetes progression and its related detrimental outcomes, it is estimated that by 2045 more than 700 million people will live with diabetes [2]. Importantly, the progression of chronic hyperglycemia in people with diabetes has been shown to be closely associated with diverse subsequent deleterious effects including kidney failure, liver diseases, cardiovascular problems, and neurological issues [3–5].
Nowadays many synthetic drugs have been widely used to treat people with diabetic neuropathy and balance disorder in common medical practices [6,7]. Unfortunately, diverse unwanted side effects are also frequently observed. For instance, the neuropathy drugs in the groups of anticonvulsants and antidepressants have been reported to cause water retention, body weight gain, and psychological issues including mood and behavior changes [8]. Other drugs also promote drowsiness, dizziness, and failure to concentrate [9]. Moreover, it is also indicated that diabetic neuropathy drugs cause gastrointestinal problems such as constipation, nausea, diarrhea, and vomiting [7,10] while increasing the risk of having cardiovascular diseases [11]. In addition, the cost of the medications has recently soared [12]. Therefore, the explorative efforts to find and formulate natural-based alternative medicines, with lesser unwanted outcomes while highly effective and affordable, are urgently needed.
Among diverse potent bioresources for natural-based medicines, plants in the family of Vitaceae namely wild vine (Vitis gracilis) are well known for their benefits against diseases [13]. Local people in north Sumatra (Karo tribe, Indonesia) usually consume its leaves in the form of decoction as a vitality enhancer [14]. Previous investigations also revealed that V. gracilis leaf extract contained terpenoids, flavonoids, alkaloids, glycosides, tannins, and saponins [13,14]. Another study in animal models also found that V. gracilis leaves could prevent inflammation and apoptosis in the gastrocnemius muscle cells [13]. A previous study showed that V. gracilis is capable of counteracting apoptosis in the alveolar cells [15]. Moreover, V. gracilis leaf extracts have been shown to protect hippocampal cells from apoptosis and inflammation caused by intense swimming activity [16]. In addition, it has been indicated that the nano herbal prepared from V. gracilis leaf extract was capable of protecting lung cells against apoptosis caused by maximum swimming exercise [17]. However, to date, the scientific evidence supporting the beneficial effects of V. gracilis in counteracting sensory and motoric disorders, oxidative stress, and brain degeneration due to diabetes mellitus remains limited. Thus, this present study was conducted to provide proof regarding the effects of V. gracilis, in the form of leaf decoction, in managing hyperglycemia and the impairments of sensory response and motoric balance as well as degeneration of cerebellar tissue and accumulation of oxidative stress in alloxan-induced diabetic mice as animal models.
MATERIALS AND METHODS
Materials
High-grade reagents and chemicals namely phosphate-buffered saline (PBS, pH 7.4), neutral-buffered formalin (NBF), xylene, ethanol, and alloxan monohydrate were purchased from Sigma-Aldrich (Merck KGaA, Germany). Physiological saline (NaCl 0.9%) was sourced from Otsuka (Japan). The hematoxylin-eosin staining kit and paraffin were purchased from Vector Laboratories (USA). The lipid peroxidation assay kit was obtained from Abcam (Abcam Inc., UK). The plant sample (V. gracilis) was collected from a tropical forest area in Langkat (North Sumatra, Indonesia). Male mice of the BALB/c strain were purchased from the Baso Veterinary Center (Bukittinggi, West Sumatra, Indonesia). The rodent-specific chow diet (RATBIO) was acquired from Citra InaFeed (Jakarta, Indonesia).
Sample collection and preparation of V. gracilis leaf decoction
The fresh leaves of V. gracilis (Fig. 1) were collected from the forest area in Langkat, North Sumatra Province, Indonesia (during September 2022). The species identity of the plant was verified by a botanist in Biology Department Faculty of Mathematics and Natural Sciences Andalas University and a voucher specimen was deposited in the Herbarium Andalas (Andalas University). The procedures of decoction preparation were performed as previously described [18] with several modifications. Briefly, 1 kg of fresh samples were first washed using distilled water five times before being sliced and subsequently shade dried at room temperature (25°C–26°C ) for 5 days to achieve a constant weight. Thereafter, the samples were assigned to three different portions (25 g, 50 g, and 100 g) before being soaked into 1,000 ml distilled water to achieve the concentration of decoction to be 25 g/l, 50 g/l, and 100 g/l, respectively. Afterward, the samples were boiled at 100°C for 25 minutes and filtered using Whatman filter paper. Next, the decoction was cooled and kept in a dark bottle before being used for the treatments.
 | Figure 1. The leaves of wild vine (V. gracilis). [Click here to view] |
Provision of animal models
All procedures for animal use and experimental treatments in this study have been approved by the Research Ethics Committee of Andalas University (No.5210/2022). This study used adult BALB/c strain male mice (n = 25; 2 months old; body weight 25–26 g) purchased from the Baso Veterinary Center (Bukittinggi West Sumatra). The acclimatization of the mice was conducted for a week in the animal room at the Biology Department of Andalas University with regulated environmental conditions (temperature 25°C–26°C; humidity 67%–68%; illumination 12 hours dark and 12 hours light). Each individual mouse was reared in a single cage and fed with a chow diet special for rodents (RATBIO, Citra InaFeed Jakarta) ad libitum while having free access to a water bottle filled with tap water. After the acclimatization period, 20 mice were randomly chosen to be alloxan-induced diabetic models, while other five mice were assigned as a control group (healthy; non-diabetic model). The alloxan-induced diabetic models were prepared by injecting alloxan monohydrate (Sigma Aldrich, Germany; a single dose of 150 mg/kg body weight) intraperitoneally [19]. Five days post injection, blood glucose levels were measured using a glucometer (AGM Dr Glucose 2100) by collecting blood samples from the tail vein. Mice with blood glucose levels of 250 mg/dl or higher were considered as diabetic. Accordingly, all 20 mice assigned for alloxan-induced diabetic models met the criteria of diabetes melltius, thus, eligible to be used in the experiments. Eventually, the mice were randomly divided into five different groups as follows:
Group 1: non-diabetic mice (healthy; non-DM)
Group 2: diabetic mice without any treatment (DM)
Group 3: diabetic mice treated with 25 g/l of V. gracilis decoction (DM + 25 g/l VgD)
Group 4: diabetic mice treated with 25 g/l of V. gracilis decoction (DM + 50 g/l VgD)
Group 5: diabetic mice treated with 25 g/l of V. gracilis decoction (DM + 100 g/l VgD).
In this experimental study, the non-diabetic group (healthy; non-DM) was assigned as the standard (control) group. Consequently, the justification of the decoction’s efficacy depended exclusively on comparisons with healthy mice, and not with diabetic mice treated with standard drug.
The animal treatments with decoction were immediately carried out upon confirmation of diabetic status. The decoction was orally given to the mice (0.5 ml/individual for each dose) on a daily basis in the morning (08.00–09.00) continuously for 30 days. The non-diabetic mice were also given distilled water (0.5 ml/individual) orally. The dose levels and the administration procedures of decoction were decided based on the previous study [20].
Blood glucose, body weight, water, and food intake measurements
The levels of random blood glucose were monitored weekly (08.00–09.00 a.m.) during the treatment by collecting blood samples from the vein tails before being measured using a glucometer. In addition, the fasting blood glucose levels were examined at the end of treatment under an 18-hour fasting condition. Moreover, the body weights of mice were monitored every 2 days, and food and water intakes were measured on a daily basis.
Assessment of sensory response by a hot plate test
The sensory response of mice was evaluated using a hot plate test performed at the end of treatment by following the procedure as described previously elsewhere [21]. Shortly, immediately after the treatments were ceased, each mouse was tested by putting its limbs on a hot plate (with a sustained temperature of 50°C). The latency time of the mouse responding to the hot sensation was recorded using a timer.
Assessment of motoric function by a balance beam test
A day after the hot plate test, mice were subsequently prepared for a balance beam test. The procedures of the test were as described previously [22]. Shortly, each mouse was positioned on a balance beam with a 40 cm length. The time spent by the mouse to completely cross the beam from end to end was recorded using a camera (Olympus TG6 Tough).
Determination of malondialdehyde (MDA) level in the brain tissue
Immediately after completing the other tests, mice were euthanized by cervical vertebrae dislocation. Subsequently, the head was dissected, and the brain was collected. For the purpose of measuring MDA levels, 0.3 g of brain tissue was sampled and homogenized in a PBS solution using a tissue homogenizer (Omni International). The homogenate was then used for MDA measurement, employing a lipid peroxidation assay kit (Abcam Ab118970, Abcam). The measurement procedures were carried out following the manufacturer’s instructions. Sample absorbance was determined using a UV-Vis Spectrophotometer (Biorad Lab) at a wavelength of 532 nm.
Histopathological examination of the cerebellum
Histopathological observations of cerebellum tissue were conducted using histological slides stained with hematoxylin-eosin (HE). First, the cerebellum was collected, immediately washed with physiological saline, and then fixed in 10% NBF overnight. The tissue was subsequently dehydrated using graded ethanol (70%–100%) and cleared with xylene. The sample was then embedded in paraffin and sectioned using a rotary microtome to a thickness of 5 µm. Afterward, the tissue was stained with HE and mounted under a cover glass. Microscopic observations were conducted using a light microscope equipped with an Olympus CX-33 camera. Five distinct slices of cerebellar tissue were prepared for each mouse, and the examinations involved five different view fields for each slice. Counting of the Purkinje cells and measurements of the molecular and granular layers of the cerebellar tissues were undertaken using the ImageJ software (NIH, USA).
Statistical analysis
The data were first subjected to tests for homogeneity and distribution. Since our data were homogeneous and normally distributed, they were subsequently analyzed using an analysis of variance, followed by Duncan’s New Multiple Range Test. A p-value of < 0.05 was considered significant. The analysis was performed using IBM SPSS Statistics Base version 22.0 for Windows.
RESULTS AND DISCUSSION
Effect of V. gracilis leaf decoction on blood glucose profile
To assess the blood glucose profile, the random blood glucose, fasting blood glucose levels, and insulin tolerance were determined. As shown in Figure 2A, 5 days after alloxan injection (0 week), blood glucose levels were markedly soared to the hyperglycemic state. This state was sustained until the end of the experiment (30 days). In contrast, non-diabetic mice (non-DM group) exhibited normal blood glucose levels during all the time points of measurements. There were no statistical differences in blood glucose levels of mice in the DM group as compared with those treated with various doses of V. gracilis leaf decoction (p > 0.05). However, the blood glucose values in the non-DM group were substantially lower at every time point of measurement as compared with all other groups (p < 0.01). Furthermore, fasting blood glucose levels (Fig. 2B) showed a noticeable increment in the DM group that was significantly different from the non-DM group (p < 0.05). There was a tendency for lower fasting blood glucose levels in decoction-treated groups as compared with the DM group. However, statistical analysis revealed that there were no significant differences in fasting blood glucose values in the DM group as compared with decoction-treated groups (p > 0.05). The fasting blood glucose levels were substantially higher in mice treated with V. gracilis decoction (at all doses) as compared with the non-DM group (p > 0.05). In addition, ITT conducted at the end of treatment (Fig. 2C) indicated that upon insulin injection, blood glucose levels in the DM group remained higher and became substantially different as compared with the non-DM group (p < 0.01). Similarly, blood glucose values of mice treated with V. gracilis decoction at all doses were also sustained higher than non-DM group (p < 0.05). The AUC values (Fig. 2D) also showed that those in the DM group and decoction-treated groups had significantly higher AUC of ITT values as compared with the non-DM group (p < 0.05).
 | Figure 2. Effect of V. gracilis leaf decoction on blood glucose profile in alloxan-induced diabetic mice. (A) Random blood glucose levels measured weekly, (B) fasting blood glucose measured at the end of treatment, (C) blood glucose levels in insulin tolerance test (ITT), and (D) area under the curve of blood glucose levels in ITT. **) in A and C indicate statistical significance between the non-DM group compared to all other groups (p < 0.01); different lower case letters above the bars in B and D indicate statistically significant differences (a vs. b indicates significant difference at p < 0.05). Non-DM (non-diabetes mellitus), DM (Diabetes mellitus), VgD (V. gracilis decoction). n = 5 for each group. [Click here to view] |
Effect of V. gracilis leaf decoction on body weight, food, and water intake
The results of monitoring on body weight (Fig. 3A) indicated that mice in the DM group exhibited lesser body weight increment from the beginning until the end of the experiment. Otherwise, mice in a non-DM group showed a significant increase in their body weight and became significantly higher at any time points of measurement as compared with the DM group (p < 0.01). Moreover, mice treated with V. gracilis leaf decoction at all doses of treatment exhibited comparable body weight with the DM group (p > 0.05). In addition, measurements of daily food intake (Fig. 3B) found that there was no significant difference among all groups of treatment (p > 0.05). However, daily water intake measurements (Fig. 3C) revealed that mice in the DM group had a higher water intake as compared with non-DM (p < 0.01). Similarly, mice treated with V. gracilis leaf decoction exhibited a substantial increment of water intake as compared with those in the non-DM group (p < 0.01).
 | Figure 3. Effect of V. gracilis leaf decoction on body weight evolution, food and water intakes in alloxan-induced diabetic mice. (A) Body weight as measured every 2 days, (B) daily food intake, and (C) daily water intake. **) in A indicates statistical significance between the non-DM group compared to all other groups (p < 0.01); different lower-case letters in A and C indicate statistical significance (a vs. b indicates a significant difference at p < 0.05). [Click here to view] |
Effect of V. gracilis decoction on sensory response and motoric balance
To evaluate sensory response and motoric balance, mice were tested using a hot plate test and a balance beam test. In a hot plate test, those in the DM group exhibited delayed latency response to hot sensation that was significantly longer as compared with the non-DM group (p < 0.05; Fig. 4A). Otherwise, mice treated with V. gracilis leaf decoction at all doses of treatment had substantial decrement in latency response against hot stimulation as compared with DM group (p < 0.05). Moreover, response latency in decoction-treated groups was comparable with those in non-DM group (p > 0.05). Furthermore, the result of the balance beam test (Fig. 4B) demonstrated that mice in the DM group had a markedly slower time in completing the balance beam test (8 times slower) than those in the non-DM group (p < 0.01). However, mice treated with V. gracilis decoction showed improvement as indicated by a significantly faster time of balance beam completion as compared with the DM group (p < 0.01). Those treated with decoction at a dose of 50 g/l had comparable time completion with non-DM, while mice received 25 and 100 g/l decoction had statistically longer time of balance completion than non-DM (p < 0.05).
 | Figure 4. Effect of V. gracilis leaf decoction on sensory and motoric disorders in alloxan-induced diabetic mice. (A) Response latency of mice in hot plate test. (B) Time completion of mice in balance beam test. Different lower case letters above the bars represent statistical significance (p < 0.05). [Click here to view] |
Effect of V. gracilis decoction on the oxidative stress and histopathology of the cerebellum
An examination of oxidative stress in the brain tissue (Fig. 5) found that the DM group had substantially higher MDA (a marker of oxidative stress) levels than the non-DM group (p < 0.05). However, treatment with V. gracilis leaf decoction at all doses caused a significant reduction of MDA (p < 0.05). Moreover, those treated with 100 g/l decoction had an apparent reduction of MDA to be substantially lower than non-DM mice (p < 0.05). A microscopic examination revealed the apparent alterations in the cerebellum of mice in the DM group (Fig. 6). It was found that in the non-DM group, the cerebellar tissue exhibited intact cerebellum with the linearly arranged Purkinje cells. Moreover, the molecular and granular layers were thicker. Otherwise, mice in the DM group had a reduction in the molecular and granular layers’ thickness with many areas exhibiting irregular thinning while other areas showed edema. The populations of Purkinje cells were also lesser with many cells exhibiting hydropic degeneration and lysed nuclei (pycnotic). Furthermore, those treated with V. gracilis decoction depicted thicker granular and molecular layers and denser Purkinje cell populations. In addition, the degenerated cells were also lesser in decoction-treated mice. The measurements of molecular layer thickness (Fig. 7A) revealed a significant decrement as compared with the non-DM group (p < 0.05). In contrast, mice treated with decoction had substantially thicker molecular layers than the DM group, particularly at the higher doses (50 and 100 g/l). Next, the measurements on the thickness of the granular layer (Fig. 7B) found that mice in the DM group also had significantly thinner granular layers as compared with the non-DM group (p < 0.05). Moreover, mice treated with higher doses of decoction (50 and 100 g/l) tended to have thicker granular layers as compared with the DM group, but it was apparently non-significant (p > 0.05). The counting on Purkinje cells (Fig. 7C) revealed that DM caused a substantial reduction in total Purkinje cells as compared with non-DM (p < 0.05) while those given with V. gracilis decoction at higher doses (50 and 100 g/l), but not at a lower dose (25 g/l), had significantly higher number of Purkinje cells in the cerebellum. Importantly, mice in the group treated with decoction at a dose of 100 g/l had a comparable Purkinje cell number as non-DM mice (p > 0.05). In addition, the DM also exhibited a marked increase of degenerated Purkinje cells (Fig. 7D) as compared with non-DM (p < 0.001). Otherwise, mice treated with decoction exhibited significantly lower degenerated Purkinje cell numbers than DM mice (p < 0.01) in a dose-dependent manner.
 | Figure 5. Effect of V. gracilis leaf decoction on oxidative stress in the brain of alloxan-induced diabetic mice. MDA (malondialdehyde). Different lower case letters above the bars represent statistical significance (p < 0.05). [Click here to view] |
 | Figure 6. Representative photomicrographs of mice cerebellum. (M) Molecular layer, (G) granular layer, black arrow in right panels indicated normal Purkinje cells, and yellow head arrows indicated degenerated Purkinje cells. Tissue was stained with HE. Scale bar in left panels = 200 µm, right panel = 100 µm. [Click here to view] |
The findings of this present study indicated that V. gracilis leaf decoction could effectively ameliorate sensory and motoric disorders while reducing MDA levels in brain tissue, particularly at higher doses, in alloxan-induced diabetic mice. Moreover, V. gracilis leaf decoction was capable of alleviating histopathological alterations in the cerebellum, a brain area involved in regulating motoric balance and sensory response. However, the decoction did not exert substantial beneficial effects against hyperglycemia and insulin intolerance, as well as body weight reduction. Moreover, the decoction did not affect food intake while failing to improve elevated water intake under diabetic conditions.
Oxidative stress and the accumulation of its by-products play a pivotal role in the pathological development of neurological disorders caused by hyperglycemia in diabetes [23,24]. Likewise, our present findings indicate that mice with diabetic conditions without any treatment exhibited a noticeable elevation of MDA in brain tissue and an apparent reduction in sensory response and motoric balance. Otherwise, daily administrations of V. gracilis leaf decoction appeared to be effective in sustaining high performance of sensory response and motoric balance alongside a marked depletion of MDA in brain tissue. This finding suggests that the V. gracilis decoction exerted a protective effect on neurological functions via an antioxidative mechanism. In previous studies, it was found that V. gracilis leaves contained some potent compounds that function as antioxidants [13,14,20]. Another report also suggests that V. gracilis leaf extract could suppress the MDA level while increasing superoxide dismutase (an endogenous antioxidant) in the blood plasma of rats [16]. Taken together, despite failing to manage blood glucose elevation, V. gracilis leaf decoction could effectively inhibit the elevation of oxidative stress, thereby alleviating the detrimental effect of hyperglycemia on neurological functions.
The cerebellum plays a crucial role in controlling motor function, and its impairment is significantly implicated in motoric function disorders [25]. Neuronal circuits involving the granular layer, molecular layer, and Purkinje cell layer in the cerebellum are essential for motor learning activities [26]. Furthermore, the cerebellum has been found to have functional connectivity with somatosensory areas, thereby participating in sensory response mechanisms [27]. As demonstrated in a previous report, chronic exposure of cerebellar tissue to hyperglycemia promotes the degeneration of Purkinje cells and granular cells in the cerebellum [28,29]. Similarly, in our current study, the hyperglycemic state in alloxan-induced diabetic mice resulted in a substantial reduction in Purkinje cells, the molecular layer, and the granular layer in the cerebellum. Interestingly, despite the sustained hyperglycemic condition, those treated with V. gracilis decoction exhibited cerebellar tissue features comparable to those of non-diabetic (healthy) individuals. This finding indicates that V. gracilis decoction could effectively protect the cerebellum against hyperglycemia-induced degeneration. It is speculated that the bioactive compounds in the decoction might play a role in inhibiting oxidative stress, thereby preventing subsequent damage to brain tissue. In addition, the decoction could also function to enhance the activity of endogenous antioxidants in the brain, including catalase, superoxide dismutase, and glutathione. However, our current study did not measure the levels of endogenous antioxidants. Therefore, further research is needed to clarify the speculations.
 | Figure 7. Effect of V. gracilis leaf decoction on histopathological alterations of the cerebellum in alloxan-induced diabetic mice. (A) Thickness of molecular layer, (B) thickness of granular layer, (C) total Purkinje cells, and (D) percentage of degenerated Purkinje cells. Different lower case letters above the bars represent statistical significance (p < 0.05). [Click here to view] |
While our present study did not investigate the inflammatory responses, their involvement in the development of central and peripheral neurological disturbances due to diabetes is highly likely [30]. Inhibiting proinflammatory cytokines is suggested as a key strategy for managing neurodegeneration caused by hyperglycemia [31]. In a prior study using a micro-colloidal preparation, we found that administering V. gracilis leaf extract significantly decreased the proinflammatory cytokine Tumor Necrosis Factor-alpha (TNF-α) while enhancing the anti-inflammatory cytokine Interleukin-10 in the circulatory system of rats [17]. This anti-inflammatory effect of V. gracilis may be attributed to certain compounds that could regulate systemic inflammatory responses. However, whether the decoction of V. gracilis leaf can also regulate inflammation in brain tissue to mitigate the pathological consequences of hyperglycemia remains to be elucidated.
The health benefits of V. gracilis decoction in managing the detrimental outcomes of diabetes on the brain, as indicated in this study, may be attributed to the various bioactive compounds found in the leaves of the plant. General phytochemical screening conducted by previous studies revealed that V. gracilis leaves contained flavonoids, alkaloids, glycosides, tannins, saponins, and terpenoids [14,15]. Furthermore, our previous study, employing a high-performance liquid chromatography-mass spectrometry technique [20], identified 26 phytochemical compounds in the leaf decoction of V. gracilis. Among these compounds, some potential substances include carthamoleusteron, methylbutanoate, valporic acid, curcubiten glucoside, lauric acid, dipropyleneglycol methyl ether acetate, arbutin, tetrahydroxypentanoic acid, phenyl salicylate, ribonic acids, ethylparaben, phenyl 2-hydroxybenzoate, arbutin, and norethindrone acetate. The common bioactivity of these compounds is as antioxidants, specifically acting as lipid peroxidase inhibitors, free radical scavengers, and NADPH peroxidase inhibitors. Alternatively, some compounds, such as phenyl 2-hydroxybenzoate, arbutin, lauric acid, and norethindrone acetate, may act as anti-inflammatory agents. Therefore, it is reasonable to suggest that the leaf decoction of V. gracilis exerts antioxidant and anti-inflammatory effects to protect the brain from damage caused by diabetes mellitus. Further studies emphasizing this mechanistic aspect are required to clarify these speculations.
Hyperglycemia is a pathological cause of various deleterious outcomes related to diabetes mellitus [4,5]. In this study, daily oral administrations of V. gracilis leaf decoction failed to manage hyperglycemia as indicated by the markedly higher blood glucose levels (> 400 mg/dl) until the end of treatment. Moreover, the exogenous administration of insulin was not effective in bringing blood glucose down to the normoglycemic level, indicating an insulin intolerance. Although we did not examine the histopathological alterations in the pancreatic tissue, we suggest that V. gracilis decoction might not be effective in alleviating the degeneration of pancreatic β cells caused by alloxan induction. As a consequence, the blood glucose homeostasis was not properly managed due to insulin insufficiency. Moreover, the fact that insulin intolerance remained observed could indicate the impairment of insulin receptors in the insulin-targeted cells (including myocytes, adipocytes, hepatocytes, and other cells). To our knowledge, there was no previous study investigating the effect of V. gracilis leaves on blood glucose profile under diabetes conditions. However, a study of another species in the same genus namely Vitis vinifera (red grape) demonstrated its ability to suppress blood glucose levels in individuals with diabetes mellitus [32]. Moreover, V. vinifera was also reported to exert an ameliorative effect on insulin sensitivity in human subjects with metabolic syndrome [33]. Therefore, besides species difference (V. gracilis vs. V. vinfera), it is assumed that this discrepancy may be also due to several plausible reasons. First, in our present study, the sample was prepared as a decoction, adopting a common traditional practice of the Karo tribe (in North Sumatra) in consuming V. gracilis leaves as a medicinal herb. In contrast, a previous study in V. vinifera employed extraction procedures. This might be implicated in the phytochemical constituents of the sample thereby affecting their effectivity in counteracting hyperglycemia and insulin intolerance. Second, the experimental treatment in our present study was conducted in a relatively short period (30 days). It is speculated that decoction may require a longer period of treatment (more than 30 days) to effectively exert alleviative effects against hyperglycemia and insulin intolerance under diabetic conditions. Future investigations with a longer period of administration and deploying different extraction techniques followed by comprehensive phytochemical screenings are needed to justify our assumptions.
Despite having comparable food intakes to those of non-treated groups, our current study also found that diabetic mice treated with V. gracilis decoction did not show a substantial body weight gain during the course of treatment. Under normal physiological circumstances, the energy from the food will be manifested in the body weight gain [34]. However, the hyperglycemia state in diabetes impairs the energy metabolic homeostasis [35]. Accordingly, instead of depositing as tissue biomass, including muscle and adipose tissue mass, energy from the food in diabetic individuals will be largely compensated to overcome physiological dysregulations promoted by hyperglycemia-associated disturbances [36]. Hence, a lesser body weight gain observed in diabetic mice treated with V. gracilis decoction could be a result of the inability of decoction treatment to manage hyperglycemia. In addition, the water intake was also remained higher in those treated with V. gracilis leaf decoction, suggesting the consequence of unmanageable hyperglycemia. As commonly observed in diabetic individuals, excessive water intake is associated with the physiological mechanism to compensate for elevated urine output due to glycosuria [36]. Therefore, as long as blood glucose levels remain in the state of hyperglycemia, the water intake also would be sustained at higher levels.
Although our current findings suggest the promising potential of V. gracilis leaf decoction in preventing neurological issues related to diabetes, it is important to acknowledge a notable limitation, namely the absence of a standard drug as a control group. The inclusion of such a standard drug group would have offered a benchmark for comparison, enabling a more comprehensive assessment of the efficacy of V. gracilis decoction in managing diabetes-related complications, including neurological problems. Future studies that incorporate a standard drug would contribute to a more robust evaluation of the potential therapeutic effects of V. gracilis decoction in comparison to established medical interventions for diabetes and associated outcomes.
CONCLUSION
In summary, this study investigated the effects of V. gracilis leaf decoction on various parameters in alloxan-induced diabetic mice. The findings indicated that the decoction demonstrated its efficacy in ameliorating sensory and motoric disorders as well as reducing oxidative stress in the brain tissue. In addition, an examination of the cerebellum histopathology revealed that V. gracilis decoction safeguarded against hyperglycemia-induced degeneration, preserving the integrity of cerebellar tissue. However, the decoction did not significantly affect blood glucose levels, insulin tolerance, or body weight, suggesting a lack of substantial anti-hyperglycemic effects. Therefore, despite its limited impact on hyperglycemia, V. gracilis leaf decoction demonstrated potential neuroprotective properties, indicating a potential role in alleviating neurological complications associated with diabetes. Subsequent research could delve into the underlying mechanisms, including potential anti-inflammatory effects, and investigate the long-term effects of decoction administration on hyperglycemia and its associated complications.
AUTHOR CONTRIBUTION
PS and RM designed the study and prepared the manuscript; PS, RM, and AY performed animal experiments; AT and AY conducted histopathological analysis; PS and DFB analyzed the data.
FINANCIAL SUPPORT
This study was funded by Research for Reputable Publication Grant of Andalas University 2023 (RPT, Contract No. T/71/UN16.19/PT.01.03/KO-RPT/2023).
CONFLICTS OF INTEREST
The authors report no financial or any other conflicts of interest in this work.
ETHICAL APPROVALS
The study protocol was approved by the Research Ethics Committee of the Faculty of Medicine, Andalas University, Padang, Indonesia with approval number 5210/2022.
DATA AVAILABILITY
All the data is available with the authors and shall be provided upon request.
PUBLISHER’S NOTE
All claims expressed in this article are solely those of the authors and do not necessarily represent those of the publisher, the editors and the reviewers. This journal remains neutral with regard to jurisdictional claims in published institutional affiliation.
USE OF ARTIFICIAL INTELLIGENCE (AI)-ASSISTED TECHNOLOGY
The authors declares that they have not used artificial intelligence (AI)-tools for writing and editing of the manuscript, and no images were manipulated using AI.
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