Effect of costus pictus D don methanolic leaf extract on induced prediabetic behavioral change in albino wistar rats

Jeneth Berlin Raj; S Parthasarathy; Manimekalai Kumarappan; AR Srinivasan

doi:10.4103/ajprhc.ajprhc_80_22

ORIGINAL ARTICLE

Year : 2022 | Volume : 14 | Issue : 4 | Page : 224-230

Effect of costus pictus D don methanolic leaf extract on induced prediabetic behavioral change in albino wistar rats

Jeneth Berlin Raj¹, S Parthasarathy², Manimekalai Kumarappan³, AR Srinivasan⁴
¹ Department of Physiology, Mahatma Gandhi Medical College and Research Institute, Sri Balaji Vidyapeeth, Puducherry, India
² Department of Anesthesiology, Mahatma Gandhi Medical College and Research Institute, Sri Balaji Vidyapeeth, Puducherry, India
³ Department of Pharmacology, Mahatma Gandhi Medical College and Research Institute, Sri Balaji Vidyapeeth, Puducherry, India
⁴ Department of Biochemistry, Mahatma Gandhi Medical College and Research Institute, Sri Balaji Vidyapeeth, Puducherry, India

Date of Submission	24-Sep-2022
Date of Decision	18-Oct-2022
Date of Acceptance	02-Nov-2022
Date of Web Publication	16-Dec-2022

Correspondence Address:
S Parthasarathy
Department of Anesthesiology, Mahatma Gandhi Medical College and Research Institute, Sri Balaji Vidyapeeth, Puducherry
India

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ajprhc.ajprhc_80_22

Abstract

Background: Prediabetes is a hyperglycemic intermediate state with glycemic parameters above normal values but below the threshold of diagnosis of diabetes. While the diagnostic criteria for prediabetes differ between international professional organizations, it is still a high-risk state for getting diabetes, with an annual conversion rate of 5%–10%. Plants and plant-derived products have been the first treatment choice in ancient India. Of late, they have regained a preference for allopathic medicine. Prediabetes, if identified early and treated, the intermittent hyperglycemia, as well as its complications, can be prevented or reverted. Costus pictus D Don would serve this purpose. Materials and Methods: The present study was aimed to study the effect of methanolic leaf extract of Costus pictus D Don on glycemic status and cognitive behavior in induced prediabetic status. Prediabetic status was induced by 35% sucrose solution. Glycemic status was assessed by the Oral glucose tolerance test, homeostatic model assessment for insulin resistance, and QUantitative Insulin sensitivity Check Index. Cognitive and anxiety-like behavior was assessed by elevated T maze and open arena maze. After the termination of the study, all the animals were sacrificed and their brain was sectioned and stained with H and E strain to check for any associated change in the hippocampal region to correlate with the observed behavioral change. Results: Methanolic leaf extract of Costus pictus D Don had a significant effect on normalizing glycemic status in prediabetic rats. Meanwhile, they did not cause hypoglycemia when given to normal rats. Methanolic leaf extract of Costus pictus D Don reduces blood glucose level and insulin level in both doses of 100 mg and 200 mg/kg BW. The extract exhibited an anxiolytic effect and improved cognition in prediabetic rats. When compared to control rats, the prediabetic rats exhibited slow learning. This was evident with an increase in transfer latency. Delta percentage was minimum in prediabetic animals indicating poor learning ability associated with prediabetes. The extract at the dose of 200 mg/Kg BW was found to revert this adverse effect back to near normal. Conclusion: Leaves of Costus pictus D Don can be a better choice for normalizing the hyperglycemic status and improving prediabetic induced mental disturbances such as anxiety and cognitive dysfunction.

Keywords: Cognition, Costus pictus D Don, glycemic status, prediabetes

How to cite this article:
Raj JB, Parthasarathy S, Kumarappan M, Srinivasan A R. Effect of costus pictus D don methanolic leaf extract on induced prediabetic behavioral change in albino wistar rats. Asian J Pharm Res Health Care 2022;14:224-30

How to cite this URL:
Raj JB, Parthasarathy S, Kumarappan M, Srinivasan A R. Effect of costus pictus D don methanolic leaf extract on induced prediabetic behavioral change in albino wistar rats. Asian J Pharm Res Health Care [serial online] 2022 [cited 2023 Jun 8];14:224-30. Available from: http://www.ajprhc.com/text.asp?2022/14/4/224/363946

Introduction

Prediabetes, a state of intermittent hyperglycemia, is prevalent globally and is predicted to affect more than 470 million people by 2030, affecting more people in South-East Asia and the Western Pacific Region. It is twice more common as diabetes mellitus and has been recognized as a risk factor for developing type 2 diabetes and diabetic-related microvascular and macrovascular complications.^[1] Prediabetics have increased glycemic variables compared to normal individuals but less than diabetic patients. Most of them are asymptomatic with only increased blood glycemic variables-impaired fasting glucose level and/or impaired glucose tolerance higher than normal but lower than diabetes thresholds. This disturbance in glucose homeostasis in the chronic stage may affect normal body metabolisms leading to organ dysfunction. It is observed that these metabolic disturbances are related to decreased insulin sensitivity and reduced pancreatic beta-cell function.^[2] Studies have reported an increased prevalence of neuropathies–autonomic, sensorimotor, and idiopathic polyneuropathy associated with prediabetes.^[3],[4],[5] Insulin resistance can cause mitochondrial dysfunction in brain cells leading to cognitive impairment.^[6] Behavioural changes like memory and learning disability, hyper-anxiety was reported in pre-diabetic rats.^[7] A healthy diet and increased physical activity can help revert the impaired glycemic status of prediabetes. Drugs such as metformin and acarbose are also being prescribed to treat uncontrolled glycemic status in prediabetes.^[8] Recently, more preference has been shown by the public toward medicinal plants and plant-derived products. Costus pictus D Don, commonly referred to as insulin plant/spiral ginger, has miraculous medicinal values in treating various diseases owing to its antidiabetic effect,^[9] hepatoprotective effect,^[10] and antioxidant effect,^[11] and anthelminthic property.^[12] The present study was aimed to study the effect of methanolic leaf extract of Costus pictus D Don on glycemic status and cognitive behavior in induced prediabetic status.

Materials and Methods

Plant material

Leaves from 1-year-old Costus pictus D. Don plant were collected from a local garden in Pondicherry. Authentication of the plant material was done by the Department of Botany, Annamalai University, Chidambaram (No. 326). The leaves were shade dried for 7 days and powdered. The extract was prepared by soxhalation with methanol.

Experimental animals

Forty-two male Albino Wistar rats aged 16 weeks were procured from Kings Institute, Chennai, after obtaining institute ethical clearance (01/IAEC/MG/2015). The animals were housed at the institute animal laboratory under standard conditions and diet. After a week of accommodation, the animals were randomly divided into seven groups [Table 1].

Table 1: Grouping of animals to study the effect of Costus Pictus D Don methanolic leaf extract on pre-diabetic status

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Induction of prediabetes

Prediabetes was induced by giving 35% Sucrose solution in drinking water for 9 weeks.^[13],[14] Prediabetic status was confirmed using an oral glucose tolerance test by giving 2 g/kg BW of glucose solution per oral in rats kept for 12 h fasting. Blood samples were collected from the tail vein at 0, 60 min, and 120 min. Fasting blood glucose level was determined by one touch select simple glucometer (Johnson and Johnson)^[15],[16] and serum insulin level by ELISA kit method (Crystal Chem Inc.-Downer Grove, USA) to confirm the impaired glycemic status. Insulin resistance was assessed noninvasively by HOmeostatic Model Assessment for Insulin Resistance (HOMA-IR) and insulin sensitivity by index QUantitative Insulin sensitivity Check Index (QUICKI). HOMA-IR was calculated using the formulae, HOMA-IR = ([Fasting Glucose level in mg/dl × Fasting Insulin level in mg/dl]/405) and QUICKI index = 1/(log [insulin] + log [glucose]). HOMA-IR more than 2 indicate Insulin resistance and QUICKI index between 0.30 and 0.45 indicate impaired insulin sensitivity. QUICKI index <0.30 is confirmation of diabetic status.

Delivery of test dose

After confirmation of prediabetic status, the methanolic leaf extract of Costus pictus D Don was given at a dose of 100 mg/kg BW to animals in Groups 3 and 6 and 200 mg/kg BW for animals in Groups 4 and 7. The extract was administered per oral using a standard rat intragastric lavage tube. The extract was given daily for 28 days during a fixed time of a day.

Determination of behavioral change

All animals in the study and control groups were assessed for cognitive and behavioral change with elevated plus maze and open field arena maze. Elevated plus maze was used to assess cognitive and behavioral parameters described by Rajashree et al. To assess learning and memory, a line of demarcation was made in the closed arm of the elevated T maze dividing it into two halves; the animals were then placed in the open arm and observed for 90 s to check its entry into the closed arm with all its four legs crossing the line of demarcation. This time taken to cross the line was noted as transfer latency (TL). If the animals failed to cross, a gentle push across the line of demarcation was done on day 1, and TL was noted as 90 s. The procedure was repeated on day 2. The values obtained on days 1 and 2 was used to calculate inflexion ratio (IR) using the formula (TL₁–TL₂)/TL_1. TL₁ represents TL on day 1, and TL₂ TL was recorded on the 2^nd day. Improvement or decline in learning ability will be reflecting as an increase or decrease in IR, respectively.^[17] Both elevated T maze and open file arena maze were used to assess anxiety-like behavioral change. With the elevated T maze, the rats were placed in the center facing the closed arm. Its movement and duration into the open and closed arm were noted along with other parameters such as rearing, grooming, and number of fecal droppings passed. With an open arena maze, the number of squares crossed by the animal for 5 min along with rearing, grooming, and the number of fecal droppings passed was noted.^[18]

Estimation of blood parameters

The prediabetic-induced animals in the study group, along with the control animals in Groups 3 and 4 were treated with 100 and 200 mg/Kg BW of Costus pictus D Don methanolic leaf extract for 28 days as mentioned earlier. At the end of the study period, blood samples were collected from tail vein to estimate blood glucose and insulin level. HOMA-IR and QUICKI were calculated from the estimated fasting blood glucose and insulin levels.

Tissue for histopathological study

After the termination of the study, all the animals were sacrificed and their brain was sectioned and stained with H and E strain to check for any associated change in the hippocampal region to correlate with the observed behavioral change.

Statistical analyses

All the data were collected in an excel sheet and transferred to IBM SPSS Statistics for Windows, Version 20.0. (Armonk, NY: IBM Corp) and unpaired Student's t-test, ANOVA followed by Tukey test were done according to the need and relevance of the tests. A P < 0.05 was considered statistically significant.

Results

Induction of prediabetic status

The induction rate of prediabetes in animals ingested with 35% sucrose solution in drinking water was 100%. Both impaired fasting glucose levels and impaired glucose tolerance were observed in prediabetic-induced animals. Fasting insulin level was also found to be significantly increased in prediabetic animals. Increase in insulin resistance checked by HOMA-IR and decrease in insulin sensitivity assessed by QUICKI was observed in prediabetic rats when compared to normal rats [Table 2]. Statistical analysis was done by unpaired t-test.

Table 2: Changes in blood glucose, insulin, and HOmeostatic Model Assessment for Insulin Resistance in the control group and study group animals fed with 35% sucrose solution confirming 100% induction of pre-diabetes in study group animals

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Effect on glucose, insulin and homeostatic model assessment for insulin resistance

Methanolic leaf extract of Costus pictus D Don reduces blood glucose level and insulin level in both doses of 100 mg and 200 mg/kg BW. This was evident when the extract was given to the animals in control group and prediabetic group. At the strength of 200 mg/kg BW, the extract was very efficient in bringing back the elevated blood glucose and insulin level in prediabetic animals. Although the reduction in these parameters was observed in the control group of animals treated with the extract, the hypoglycemic level was not observed [Table 3]. Elevated level of HOMA-IR of more than 2 and QUICKI index between 0.3 and 0.45 in prediabetic animal indicates early onset of insulin resistance and decrease in insulin sensitivity prediabetic state. These changes were seen reversed after treating with the extract. Significance between the groups was analyzed by One-Way ANOVA followed by Tukey t-test.

Table 3: Effect of methanolic leaf extract of Costus pictus D Don on fasting glucose, insulin and HOmeostatic Model Assessment for Insulin Resistance level in the study group compared to the control group

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Effect on cognition

When compared to control rats, the prediabetic rats exhibited slow learning. This was evident with an increase in TL. Delta percentage was minimum in prediabetic animals indicating poor learning ability associated with prediabetes. The extract at the dose of 200 mg/Kg BW was found to revert this adverse effect back to near normal. The same was evident with a decrease in IR observed in prediabetic rats and the ratio improved in prediabetic rats treated with both doses of the extract [Table 4].

Table 4: Effects of induced prediabetes on learning and memory in control and study group animals by an elevated plus maze

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Effect on behavior change

Decreased entry and decreased time spent in the open arm are indicative of anxiety-like behavior. Similar behavior changes were recorded in prediabetic-induced animals (Group 5). These anxiety-like behavior was reduced in rats treated with the plant extract (Groups 6 and 7), which was evident by the increase in the time spent by the animal in the open arm and the free movement in both open and closed arms. A similar result was observed when tested with an open arena maze where the animals induced with prediabetes showed restricted movement and spent less time in the central maze. The prediabetic rats in Groups 6 and 7 treated with the extract (100–200 mg/kg BW respectively) moved all around the arena, including the center maze. The animals in control groups (Group 1–4) were mostly seen staying in the closed arm and also seem to avoid entering open arm [Table 5] and [Table 6].

Table 5: Effect of Costus pictus D Don methanolic leaf extract on behaviour changes in control and study group animals by an elevated plus maze

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Table 6: Effect of Costus pictus D Don methanolic leaf extract on behaviour changes in control and study group animals by open arena maze

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Histological changes

In alignment with the results of our cognitive function assessment, inflammatory changes such as disorganized glial cells and increased vacuolation in Cornu ammonis and pyramidal cells in hippocampus was evident. These changes were not evident in the rats treated with the plant extract [Figure 1].

Figure 1: Histological changes in the hippocampus of prediabetic rats when compared to normal animals and prediabetic animals treated with methanolic leaf extract of Costus pictus D Don. CA1, CA3: Cornu ammonis 1 and 3, HL: Hippocampal pyramidal cells, DG: Dentate gyrus

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Discussion

Table sugar/sucrose is being commonly consumed by everyone. In higher concentration, it alters the body's glucose metabolism. Sucrose is easily digested and absorbed in the blood. In our study, we used 35% sucrose solution in drinking water to induce sustained elevated blood glucose level. The animals, when treated for 9 weeks with 35% sucrose, led to impaired fasting glucose and impaired glucose tolerance confirming prediabetic status. This result was in alignment with the previous studies by Alves et al. and Alves et al.^[13] Prediabetes, the stage before frank diabetes is associated with either impaired fasting glucose or/and impaired glucose tolerance resulting in elevated blood glucose levels. It is documented that, rats fed with a high concentration of sucrose, insulin level rapidly raises and stays elevated. This was in agreement with our research finding, where both serum glucose and insulin levels were raised in prediabetic animals. Our extract had a definite role in reducing glucose and serum insulin level in prediabetic rats. The reduction in blood glucose level in normal rats treated with both doses of the extract was not significant, proving the safeness of the extract by not causing hypoglycemia. Altered serum glucose level is due to abnormal insulin response and is considered the earliest detectable biochemical change in diabetes. In addition, β-cell response is diminished, leading to altered glucose-stimulated insulin secretion This later results in insulin resistance, increased triglycerides, and lipid deposition.^[19],[20]

Insulin resistance is associated with impaired insulin sensitivity. Insulin resistance can be determined by HOMA-IR, which is calculated by using the formulae HOMA-IR = (Glucose [mg/dl] × Insulin [mg/dl]/405. The accuracy of HOMA-IR to determine insulin resistance in rats is as accurate as Insulin Tolerance Test.^[21] Elevated values of HOMA-IR in prediabetic animals of our study result point to the fact insulin resistance are observed as early as in prediabetic status and decreased level of HOMA-IR observed in animals after treatment with Costus pictus D Don methanolic leaf extract indicates its ameliorating effect on insulin resistance in a rat model. Whereas, QUICKI index measure insulin sensitivity. These parameters will give a clue if the animal is in a diabetic state (Index value <0.3) or prediabetic state (index value between 0.30 and 0.45).^[22]

Freezing, tonic immobility, increased ambulation, aggression, increased vigilance/attention, irritability, restlessness, panting, urination, defecation, and sleep disturbances are behavioral signs of anxiety. These behavioral dysfunctions can compromise normal physiology. Anxiety develops in response to potential danger. Anxiety through amygdala and hypothalamic nuclei stimulates the sympathetic nervous system and hypothalamic–pituitary–adrenal axis to combat acute stress. Parallelly, the hippocampus and cerebral cortex are also activated.^[23] The hippocampus plays a major role in staying focused (spatial navigation), learning, and retaining memory. Rats fed with high-calorie diet for about 12 weeks were found to have decreased peripheral and neuronal (hippocampal) insulin receptor sensitivity.^[24] Insulin through its receptors in the hippocampal neurons, facilitates alpha-amino-3 hydroxy-5-methylisoxazole-4-propionic acid resulting in long-term depression, thus modulating activities of synaptic plasticity. The cholinergic and serotonergic neurons in the dorsal hippocampus balance between the anxiolytic and anxiogenic behavior respectively.^[25] Decreased neuronal insulin receptor sensitivity as observed in chronic hyperglycemic status affects these behaviors and is also associated with decline in cognition. It was demonstrated by our study that prediabetes cause anxiety-like behavior which is overcome by the methanolic leaf extract of Costus pictus D Don in both doses of 100 mg and 200 mg/kg BW proving its anxiolytic effect. The extract has proved its effectiveness in regaining cognitive function which is affected in prediabetic state. This was also evident in the histological changes observed in our study [Figure 1]. Similar changes were reported in Alzheimer rats treated with ethanolic leaf extract of Costus pictus.^[26] An earlier study suggests that Costus pictus Extract's potential anti-diabetic properties are linked to increased Glucagon-Like Peptide-1 (GLP-1) secretion and cell protection, which could be useful in the management of type 2 diabetes mellitus (T2DM)^[27] Even though we have deciphered the mechanism, we have found the same effect as their studies. It has been discovered that administering an aqueous extract of Costus Pictus to diabetic rats causes a significant decrease in blood glucose and an increase in plasma insulin levels.^[28] Costus speciosus rhizome possessed central nervous system depressant, anxiolytic and antidepressant-like activities.^[29] These actions were done with different species but we have shown better neurocognitive function. Yet another study^[30] suggests that costus pictus extract (CPE's) potential anti-diabetic properties are linked to increased GLP-1 secretion and cell protection, which could be beneficial in the management of T2DM.

Conclusion

With the increasing trend in preferring naturopathy, plants and plant-derived products are preferred over modern medicine owing to their complication(s). Many plants have proven to improve the elevated glycemic status in the diabetic state. Prediabetes when identified and treated early, can revert the glycemic status to normal. If untreated, it may lead to an increased probability of many avoidable complications. The fear of hypoglycemia maybe a cause of avoiding treatment in prediabetic status. Our extract has proved to be safe in normoglycemia and has also proved its efficacy in maintaining glucose levels in prediabetic status. The unseen tissue damage, decline in cognition, and development of anxiety-like behavior in prediabetes can also be handled effectively by the neuropharmacological effect of this miraculous plant Costus pictus D Don.

Authors contribution

JB – concept, design, write up, KM- data collection, ARS – data collection SPS – manuscript and overall supervision.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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