Investigating the effects of <i>Orthosiphon stamineus</i> on blood glucose regulation via pancreatic endocrine and exocrine functions in Type 3c Diabetes Mellitus Wistar Bonn/Kobori (WBN/Kob) pancreatitis rat model.

Ezarul Faradianna Lokman; Puteri Sofia Nadira Megat Kamaruddin; Hussin Muhammad; Azlina Zulkapli; Siti Zubaidah Che Lem

doi:10.12688/f1000research.163467.1

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Study Protocol

Investigating the effects of Orthosiphon stamineus on blood glucose regulation via pancreatic endocrine and exocrine functions in Type 3c Diabetes Mellitus Wistar Bonn/Kobori (WBN/Kob) pancreatitis rat model.

[version 1; peer review: 1 approved with reservations]

Ezarul Faradianna Lokman ¹, Puteri Sofia Nadira Megat Kamaruddin¹, Hussin Muhammad², Azlina Zulkapli³, Siti Zubaidah Che Lem³

Ezarul Faradianna Lokman ¹, Puteri Sofia Nadira Megat Kamaruddin¹, [...] Hussin Muhammad², Azlina Zulkapli³, Siti Zubaidah Che Lem³

PUBLISHED 24 Apr 2025

Author details Author details

¹ Endocrine and Metabolic Unit, NMCRC, Institute for Medical Research, National Institutes of Health Malaysia, Shah Alam, Selangor, 40170, Malaysia
² Toxicology and Pharmacology Unit, HMRC, Institute for Medical Research, National Institutes of Health Malaysia, Shah Alam, Selangor, 40170, Malaysia
³ Laboratory Animal Resources Unit, SRC, Institute for Medical Research, National Institutes of Health Malaysia, Jalan Pahang, Kuala Lumpur, 50588, Malaysia

Ezarul Faradianna Lokman
Roles: Conceptualization, Data Curation, Formal Analysis, Funding Acquisition, Investigation, Methodology, Project Administration, Resources, Software, Supervision, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing

Puteri Sofia Nadira Megat Kamaruddin
Roles: Formal Analysis, Funding Acquisition, Investigation, Methodology, Project Administration, Resources, Software, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing

Hussin Muhammad
Roles: Investigation, Methodology, Project Administration, Resources, Software, Supervision, Validation, Visualization, Writing – Review & Editing

Azlina Zulkapli
Roles: Investigation, Methodology, Project Administration, Resources, Validation, Visualization, Writing – Review & Editing

Siti Zubaidah Che Lem
Roles: Investigation, Methodology, Project Administration, Resources, Supervision, Validation, Visualization, Writing – Review & Editing

OPEN PEER REVIEW

REVIEWER STATUS

Abstract

Background

Type 3c diabetes mellitus (T3cDM) is a subtype that develops following pancreatic diseases, such as pancreatitis, impairing both exocrine and endocrine functions. Its management is challenging owing to exocrine enzyme deficiencies. Although plants have long been used for diabetes treatment, research on their role in T3cDM, particularly in exocrine enzymes, remains limited. The exact mechanisms by which enzymes and hormones interact remain unclear. Thus, this study aims to explore the role of Orthosiphon stamineus (OS) in glucose regulation by exploring both pancreatic endocrine and exocrine functions in the T3cDM Wistar Bonn/Kobori (WBN/Kob) pancreatitis rat model.

Methods

This protocol describes a planned, single-blind, randomized, controlled study conducted on animals to determine the efficacy of OS in regulating exocrine and endocrine pancreatic functions in a pancreatitis-induced WBN/Kob rat model. The groups will consist of control and different treatment groups. The duration of treatment is one month. A glucose tolerance test will be conducted orally before and after OS treatment. Blood glucose and insulin levels will be collected at different time points. Exocrine enzymes (amylase, lipase, and trypsinogen) and endocrine hormones (insulin, glucagon, ghrelin, pancreatic polypeptide, and somatostatin) will be measured in the serum. Hemoglobin A1c and lipid-soluble vitamins will also be evaluated.

Results

This pre-clinical study aims to explore pancreatic endocrine hormones and exocrine functions in response to OS treatment in rats with T3cDM pancreatitis. Body weight, diarrhea, and any adverse effects will be assessed as the secondary outcomes. Statistical analysis will be conducted to determine the significance of differences between the two groups.

Conclusion

This study protocol examines the efficacy of OS in addressing both exocrine and endocrine pancreatic dysfunction, ultimately aiding in blood glucose regulation. The findings of this study may help improve the health strategies of T3cDM patients and enhance patient outcomes.

Keywords

type 3c diabetes mellitus, T3cDM, Orthosiphon stamineus; pancreas; insulin; endocrine; exocrine; pancreatitis

Corresponding author: Ezarul Faradianna Lokman

Competing interests: No competing interests were disclosed.

Grant information: We received a Research Grant (NMRR ID-24-02224-O6O) from the Ministry of Health of Malaysia to perform the study.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2025 Lokman EF et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

How to cite: Lokman EF, Megat Kamaruddin PSN, Muhammad H et al. Investigating the effects of Orthosiphon stamineus on blood glucose regulation via pancreatic endocrine and exocrine functions in Type 3c Diabetes Mellitus Wistar Bonn/Kobori (WBN/Kob) pancreatitis rat model. [version 1; peer review: 1 approved with reservations]. F1000Research 2025, 14:461 (https://doi.org/10.12688/f1000research.163467.1) First published: 24 Apr 2025, 14:461 (https://doi.org/10.12688/f1000research.163467.1) Latest published: 24 Apr 2025, 14:461 (https://doi.org/10.12688/f1000research.163467.1)

Introduction

Type 3c Diabetes Mellitus (T3cDM), also referred to as pancreatogenic diabetes, develops as a result of diseases affecting the exocrine pancreatic islets. Nearly 80% of T3cDM cases and chronic pancreatitis are the underlying diseases, followed by pancreatic cancer (8.1%), hereditary hemochromatosis (7.0%), cystic fibrosis (4.1%), and post pancreatic resection for different reasons (2.3%).¹ This pancreatic damage leads to hyperglycemia caused by endocrine pancreatic dysfunction and is complicated by additional defects in pancreatic digestive enzyme production involving nutrient malabsorption and maldigestion.

Compared to the well-established prevalence of type 2 Diabetes Mellitus (T2DM) and type 1 diabetes (T1D), limited data are available on type 3c Diabetes Mellitus (T3cDM). This is due to a lack of awareness among clinicians of T3cDM existence, unavailability of tests, and the fact that most tests are not routinely performed in all clinical settings.² T3cDM is frequently misdiagnosed as T1D or T2D. The overlapping features of T3cDM with other types of diabetes lead to frequent misclassification.³ There are several proposed diagnostic criteria for T3cDM, but no universally accepted standard exists, and differing opinions on the condition remain.^2–6 An incorrect diagnosis of T3cDM can result in inappropriate medical treatment, making it essential to address this issue properly, as managing pancreatic exocrine dysfunction is necessary in this condition.

The precise mechanisms underlying the onset of T3cDM remain unclear; however, both the diagnosis and treatment of T3cDM should focus on exocrine pancreatic enzymes and endocrine pancreatic functions. Among the therapeutic approaches for managing T3cDM, plant-based treatments have gained considerable attention owing to their natural antidiabetic and anti-inflammatory properties. Over the years, there have been persistent efforts to identify plant sources that can improve insulin release and sensitivity to achieve glucose homeostasis. However, studies on its effects on exocrine pancreatic function are limited. The right treatment is meaningful because a special therapeutic option specifically for T3cDM is needed to appropriately manage the disease.

Our earlier research demonstrated that the medicinal plant Orthosiphon stamineus (OS), commonly known as ‘Misai kucing’ in Malaysia, induced insulin secretion in isolated Sprague Dawley rat islets when exposed to high glucose levels. Treatment with OS has also been found to lower blood glucose levels and improved insulin release in Gestational Diabetes Mellitus (GDM) streptozotocin-induced rats.⁷ The efficacy of OS in treating T2DM, specifically by lowering glucose and improving insulin secretion, has been shown in many studies.^8–10 However, the mechanisms underlying the treatment of exocrine pancreatic diseases associated with T3cDM remain unclear.

OS, a member of the Lamiaceae family, is extensively cultivated in various countries, especially Southeast Asia, including Malaysia. It has attracted significant attention for its therapeutic and pharmacological properties in the management of diabetes. OS helps to manage diabetes and its related problems through several key mechanisms. These include blocking certain enzymes (α-amylase and α-glucosidase), acting as antioxidants and reducing inflammation, controlling fat metabolism, encouraging insulin production, improving the body's response to insulin, increasing glucose absorption, supporting the breakdown of glucose, reducing glucose production, increasing glucagon-like peptide-1 (GLP-1) secretion, and preventing the modification of proteins by glucose. The hypoglycemic effects of OS are thought to stem from its key active compounds, including phenolic acids, flavonoids, and triterpenoids.⁸ The observed biological activity of OS can be attributed to its diverse array of bioactive components, including rosmarinic acid, phenolics, flavonoids, amino acids, coumarin derivatives⁹ and sineotine, eupatorine, rosmarinic acid, cichoric acid, and caffeic acid.⁸

The purpose of this study is to examine the effects of OS on both hormone production and digestive enzyme production by the pancreas in the context of T3cDM using the Wistar Bonn Kobori (WBN/Kob) rat model, which closely mirrors human pancreatitis. This study is driven by the possible significance of OS in managing diabetes.

Aim

We investigate the effects of Orthosiphon stamineus (OS) on glucose regulation by exploring both pancreatic endocrine and exocrine functions in a Wistar Bonn Kobori (WBN/Kob) pancreatitis rat model.

Objectives

Main objective

This study will evaluate the impact of Orthosiphon stamineus (OS) treatment on both endocrine and exocrine functions in rats with pancreatitis, specifically focusing on glucose regulation. The analysis will include data on blood glucose, hormone, and enzyme levels in the serum samples.

1) To assess the effect on blood glucose and hemoglobin A1c (HbA1c) levels in the treatment groups compared to the control group.
2) To investigate its impact on the endocrine function of the pancreas, focusing on the level of insulin and the levels of hemoglobin A1c (HbA1c) in the treatment groups with those in the control group.
3) To assess exocrine enzyme levels (amylase, lipase, trypsinogen) and lipid-soluble vitamins (A, D, E, and K) in the treatment groups compared to those in the control group.

Hypothesis

We hypothesize that Orthosiphon stamineus supplementation would improve blood glucose regulation in T3cDM rats through its effects on pancreatic endocrine function (insulin secretion) and exocrine function (digestive enzyme activity).

Methods

Study design: This is a prospective, single-blind, randomized, controlled animal study aimed at evaluating the safety and efficacy of OS in regulating blood glucose levels. In this study, we investigate the functions of the pancreas, both in hormone production (endocrine) and enzyme secretion (exocrine), using the T3cDM Wistar Bonn/Kobori (WBN/Kob) rat model of pancreatitis.

Study setting: The study will be conducted at the Non-Clinical Research Facility (NCRF), Laboratory Animal Resource Center, Institute for Medical Research Kuala Lumpur, Malaysia.

Study duration: One year.

Study population: This study will utilise Wistar rats (control, non-diabetic) and Wistar Bonn/Kobori (WBN/Kob) rats (diseased). Male WBN/Kob rats serve as a distinctive model for chronic pancreatitis, characterized by considerable fibrosis and parenchymal degeneration due to lymphocyte infiltration. WBN/Kob rats offer a unique model for studying autoimmune pancreatitis and autoimmune exocrinopathy, which affect organs beyond the pancreas.¹¹ Upon arrival is estimated to be between Upon arrival, the rats will undergo a 2-week quarantine followed by a 5-day acclimatization period to help them adapt to their new environment and achieve physiological stability.

WBN/Kob rats display distinct characteristics: (i) pancreatitis only affects male rats; (ii) they serve as a unique diabetic model, where the islets experience atrophy as a result of ongoing inflammatory fibrosis, which sets them apart from other diabetic models;^12,13 (iii) most infiltrating cells are lymphocytes, as opposed to plasma cells or eosinophils;¹⁴ and (iv) immunosuppressive therapies such as tacrolimus or corticosteroids may aid in preventing the onset of pancreatitis.¹⁵ These features are similar to those observed in human pancreatitis.

Sampling size

The sample size that calculates the suggested number of rats used in each group is based on a previously described formula.¹⁶ In a one-way ANOVA, the degrees of freedom (DF) that represent variability within groups were determined using the formula DF = N – k, which can also be written as DF = k (n – 1). Where N represents the total sample size, k is the number of groups being compared, and n is the number of subjects in each group.

We can rearrange this formula to solve for n = DF/k + 1:

To determine the smallest and largest acceptable sample sizes per group, we plugged in the minimum (10) and maximum (20) acceptable values for DF into the formula:

Minimum sample size per group : n = 10 / k + 1

Maximum sample size per group : n = 20 / k + 1

This calculation allows researchers to determine the appropriate range for the number of subjects needed in each group based on the desired degrees of freedom for ANOVA.

The required number of animals ranges from a minimum to a maximum total of:

Minimum N = Minimum n x k

Maximum N = Maximum n x k

There will be 5 groups, so, k = 5

Minimum n = 10 / 5 + 1 = 3

Maximum n = 20 / 5 + 1 = 5

Minimum N = 3 x 5

Maximum N = 5 x 5

5 rats per group x 5 group = 25 rats

Total sample size = 25 rats

Randomisation and blinding

Random numbers generated by a computer will be utilized to guarantee an impartial and completely random assignment of rats to experimental groups. Specifically, the rats will be randomly divided into a control group and one or more treatment groups using a randomization process. To maintain the integrity of the study and minimize the risk of bias, the researchers directly involved in the study did not have access to the randomization code.

The trial will remain blinded throughout its duration, meaning that neither the researchers nor the individuals responsible for administering the treatments will know which group each rat belongs to. This blinding approach will help eliminate potential biases in treatment administration and data interpretation.

Furthermore, all data collection will be conducted by qualified Medical Laboratory Technologists (MLTs) and trained Research Assistants who are not responsible for the formulation or assignment of the study drugs. This division of responsibilities guarantees that data collection remains impartial and is not affected by prior knowledge of treatment assignments. These thorough procedures aimed to improve the accuracy and dependability of the study results.

Preparation of Orthosiphon stamineus extract

The OS will be obtained from a commercial supplier or harvested from local sources and prepared as previously described.⁷ The extract will be standardized to ensure a consistent concentration of active compounds and other bioactive molecules known for their antidiabetic effects.

Intervention

The rats will be 12 weeks old at the beginning of the experiment. A total of 25 rats will be randomly allocated into five groups, each housed in separate cages (n=5/each cage);

Group 1= Control: Non-Diabetic Wistar Rat + Vehicle

Group 2= Control: WBN/Kob Rat + Vehicle

Group 3= Treatment: WBN/Kob Rat + OS

Group 4= Positive Control: WBN/Kob Rat + Insulin

Group 5= Treatment: WBN/Kob Rat + (Insulin+OS)

OS will be administered orally to the treatment group at a specific dose, based on our previous study,⁷ whereas the diabetic control and positive control groups will receive appropriate treatment according to their respective protocols. The treatment lasts for four weeks, with blood glucose and other relevant parameters monitored regularly throughout the study period.

This protocol evaluates the effects of OS treatment on glucose regulation by performing an Oral Glucose Tolerance Test and measuring exocrine and endocrine hormones.

i) Oral Glucose Tolerance Test (OGTT)
The OGTT will be performed at the end of treatment to evaluate how effectively insulin responds to a glucose challenge. Prior to the test, the rats underwent a 12-hour overnight fast. After an overnight fast the next day, baseline glucose will be measured before administering a glucose solution (0.2g/100 g body weight), with subsequent glucose monitoring performed on tail prick blood samples using a glucometer (Accu-Check Aviva Plus, Roche, USA). Blood glucose levels will be monitored at 0, 30, 60, 90, and 120 min. Blood samples will be collected at 0, 30, and 120 min to measure insulin levels, which will be analyzed using an Enzyme-Linked Immunosorbent Assay (ELISA) kit.
ii) Pancreatic exocrine enzymes and endocrine hormones
Following experimental treatment, the rats will be euthanized via cardiac puncture under Isoflurane (5%, Piramal Healthcare Limited, India) anesthesia. Blood samples will be then collected into EDTA-containing tubes, mixed thoroughly using ten gentle inversions, and subsequently centrifuged at 2000 × g for 10 min. The resulting plasma will be carefully separated and stored in 1.5 ml Eppendorf tubes at -20°C for analysis of endocrine and exocrine functions.
The collected blood will be analyzed for pancreatic exocrine enzymes (amylase, lipase, and trypsinogen) and endocrine markers (insulin, glucagon, pancreatic polypeptide, ghrelin, and somatostatin). Hormone levels will be assessed using either ELISA or the PrimePlex Rat Assay, following the manufacturer's protocol.
Pancreatic tissue from the treatment and control groups will be harvested and stored in 10% formalin for further histopathological and immunohistochemical analyses.
iii) Pancreatic endocrine hormones measurement
Endocrine function will be assessed by measuring endocrine markers (insulin, glucagon, pancreatic polypeptide, ghrelin, and somatostatin) using an enzyme-linked immunosorbent assay (ELISA) or PrimePlex Rat Assay, following the manufacturer's protocol.
Pancreatic tissue will be collected for histopathological analysis to assess the structural integrity of the pancreatic islets. Immunohistochemical staining for insulin-producing beta cells will be performed based on the manufacturer's protocol to evaluate the preservation of pancreatic islet function.
iv) Pancreatic exocrine function measurement
The exocrine function of the pancreas will be evaluated by measuring digestive enzymes (amylase, lipase, and trypsinogen) and conducting histological analysis of pancreatic acinar cells. Morphological changes in the exocrine pancreas will be assessed using hematoxylin and eosin (H&E) staining, according to the manufacturer's protocol.

Ethical considerations

This study protocol received approval from the Animal Care and Use Committee (ACUC) under the Ministry of Health, Malaysia, on [3 March 2025], as documented in the Ethics Committee approval letter (reference number: ACUC/KKM/02(1/2025⁹). No human samples will be utilized in this study.

Animal Welfare: All procedures involving animals will strictly adhere to the guidelines outlined in the Guide for the Care and Use of Laboratory Animals by the National Institutes of Health (NIH) (https://grants.nih.gov/grants/olaw/guide-for-the-care-and-use-of-laboratory-animals.pdf) and the Guidelines for the Handling of Laboratory Animals established by the Ministry of Health, Malaysia (MOH 2023). Every possible measure will be taken to reduce animal suffering and distress throughout the study. These measures include:

1. Administering appropriate anaesthesia (Isoflurane) for all surgical procedures, with continuous monitoring of anaesthetic depth.
2. Conducting regular assessments of animals for distress, with the application of humane endpoints when necessary.
3. Housing animals in a temperature-regulated environment, maintaining a 12-hour light/dark cycle, and providing unrestricted access to food and water.
4. Ensuring gentle handling: All procedures, including injections and oral gavage were carried out by trained personnel to minimize stress and discomfort.
5. Performing euthanasia in accordance with the American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals (https://www.avma.org/sites/default/files/2020-02/Guidelines-on-Euthanasia-2020.pdf).

The number of animals used in this study has been carefully calculated to achieve statistical significance while using the minimum number of animals possible. All personnel involved in animal handling and procedures have received appropriate training in laboratory animal science and welfare.

Outcome measures

Data collection

Data will be recorded by medical laboratory technologists and research assistants using standardized data collection forms to maintain consistency throughout data collection within the study period.

Statistical analysis

Statistical analysis will involve comparing the results of the control and treatment groups. The data will be summarized using descriptive statistics such as means, standard deviations, and percentages. To evaluate the significance of differences between the groups, inferential statistics, such as regression analysis, chi-square tests, and t-tests, will be applied.

Dissemination

The results of this study will be submitted for publication to well-regarded medical journals focused on diabetes and pancreatic cells.

Status of this study

We received a Research Grant (NMRR ID-24-02224-O6O) from the Ministry of Health of Malaysia to perform the study. The rats will be obtained from Japan. Computer-generated randomization will be used in the selection procedure to guarantee methodical and objective assignment of rats to the appropriate groups.

Scope

This study will focus on the potential therapeutic benefits of OS on T3cDM, which is associated with pancreatitis and often involves both endocrine and exocrine pancreatic dysfunction, using the WBN/Kob rat model as a representation of this condition. This study investigates the impact of OS on endocrine function of the pancreas, specifically its effect on insulin secretion, β-cell function, and overall glucose homeostasis.

Limitations

The most common causes of T3cDM are chronic pancreatitis, pancreatic cancer, hereditary hemochromatosis, cystic fibrosis, and post pancreatic resection. While the study is based on animal models, there is limited evidence or clinical data on the effects of OS in humans, particularly for blood glucose regulation and pancreatic function. Thus, the findings in rats may not directly translate to human physiology or therapeutic applications. This study will use the WBN/Kob rat model, which may not fully replicate humans with T3cDM or pancreatitis. Differences in species-specific responses to OS or variations in pancreatic function between rats and humans could limit the generalizability of these findings to human patients. To gain a more comprehensive understanding of pancreatic function impairment, future studies should include additional animal models. This will aid in further exploration of exocrine and endocrine functions of the pancreas and their clinical relevance. Expanding the scope of research beyond a single model may provide deeper insights into the disease mechanisms, variability in physiological responses, and potential therapeutic interventions.

Dissemination

The study findings and associated data will be disseminated through peer-reviewed publications, conference presentations, research databases, and open access repositories. Additionally, the results will be shared with policymakers, healthcare professionals, and the public through workshops, seminars, and digital platforms to maximize impact and accessibility.

Discussion

The efficacy of OS known as ‘Misai kucing’ in treating specifically T2DM by lowering glucose and improving insulin secretion has been shown previously in many studies.^8–10 However, the potential roles in treating particularly, the treatment of exocrine pancreatic disease associated with T3cDM is not elucidated. In our previous study, OS reduced blood glucose levels and stimulated insulin release in (GDM) streptozotocin-induced diabetic rats.⁷

In this study, we aim to explore the effects of OS on both exocrine and pancreatic functions in a Bonn WBN/Kob pancreatitis rat model to evaluate its clinical significance. An oral glucose tolerance test will be conducted to evaluate the effects of insulin and glucose utilization following OS treatment in comparison with the control group.¹⁷ HbA1c levels will be measured and compared between the treatment and control groups. Pancreatic exocrine enzymes (amylase, lipase, and trypsinogen) and endocrine hormones (insulin, glucagon, ghrelin, pancreatic polypeptide, and somatostatin) will be compared between the treatment and control groups. Given that T3cDM is complicated by other comorbidities, including maldigestion and concurrent qualitative malnutrition, this study also assess the levels of lipid-soluble vitamins (A, D, E, and K).

The pancreatic islets consist of endocrine cells, including alpha, beta, gamma, delta, and epsilon cells, which secrete hormones, such as glucagon, insulin, somatostatin, pancreatic polypeptide, and ghrelin. Additionally, exocrine cells produce digestive enzymes such as amylase, lipase, protease, and trypsinogen.¹⁸ Damage to the pancreas in T3cDM involves both exocrine and endocrine function. This affects the pancreas’s ability to produce insulin and leads to a lack of pancreatic enzyme production, which is important for digestion. Chronic pancreatitis is the underlying cause in almost 80% of T3cDM cases.¹

Recognizing the impact of pancreatic damage on the development of T3cDM is essential to formulate an effective management approach. In T3cDM, pancreatic beta cells are impaired, resulting in reduced insulin production. In addition, gamma cells producing pancreatic polypeptide, which upregulate insulin receptors in hepatic tissue, are reduced, causing hepatic insulin resistance. Furthermore, the glucagon levels produced by alpha cells diminish, causing severe hypoglycemia resulting from malabsorption and maldigestion.³ T3cDM is intricate and more challenging due to the presence of comorbidities such as maldigestion and concurrent qualitative malnutrition.⁴

Owing to the limitations of existing treatments, especially T3cDM, there is an increasing demand for studies on natural products with antidiabetic properties. Plants are considered safer alternatives because they tend to have fewer side effects and lower toxicity than synthetic therapies. So far, extensive research and literature have concentrated on identifying new antidiabetic medications derived from traditional plants and investigating their glucose-lowering effects through enhanced insulin secretion or increased tissue sensitivity. Although T3cDM exhibits traits common to both T1D and T2DM, they are distinct in terms of clinical and metabolic features. It requires individualized treatment owing to its unique characteristics and specific management needs.

Improving exocrine enzymes and fat-soluble vitamin deficiencies in T3cDM are required as additional therapeutic options for T3cDM. Therefore, this study aims to identify whether OS treatment may have therapeutic properties in managing T3cDM, thus preventing further complications.

Data availability

No data are associated with this article.

Reporting guidelines

OSF Repository: ARRIVE checklist for “Investigating the effects of orthosiphon stamineus on blood glucose regulation via pancreatic endocrine and exocrine functions in a type 3c diabetes mellitus wistar bonn/kobori (wbn/kob) pancreatitis rat model”. https://doi.org/10.17605/OSF.IO/P8QDN¹⁹

Data are available under the terms of the Creative Commons Attribution-ShareAlike 4.0 International Public License (CC BY-SA 4.0).

Acknowledgements

The authors express their gratitude to the Director General of Health, Malaysia, the Director of the Institute for Medical Research, and the Ministry of Health Malaysia for providing the necessary authorization to publish this study.

References

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19. Lokman EF, Kamaruddin PSNM, Muhammad H, et al.: Investigating the effects of orthosiphon stamineus on blood glucose regulation via pancreatic endocrine and exocrine functions in a type 3c diabetes mellitus wistar bonn/kobori (wbn/kob) pancreatitis rat model. OSF. 2025.

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Author details Author details

¹ Endocrine and Metabolic Unit, NMCRC, Institute for Medical Research, National Institutes of Health Malaysia, Shah Alam, Selangor, 40170, Malaysia
² Toxicology and Pharmacology Unit, HMRC, Institute for Medical Research, National Institutes of Health Malaysia, Shah Alam, Selangor, 40170, Malaysia
³ Laboratory Animal Resources Unit, SRC, Institute for Medical Research, National Institutes of Health Malaysia, Jalan Pahang, Kuala Lumpur, 50588, Malaysia

Ezarul Faradianna Lokman
Roles: Conceptualization, Data Curation, Formal Analysis, Funding Acquisition, Investigation, Methodology, Project Administration, Resources, Software, Supervision, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing

Puteri Sofia Nadira Megat Kamaruddin
Roles: Formal Analysis, Funding Acquisition, Investigation, Methodology, Project Administration, Resources, Software, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing

Hussin Muhammad
Roles: Investigation, Methodology, Project Administration, Resources, Software, Supervision, Validation, Visualization, Writing – Review & Editing

Azlina Zulkapli
Roles: Investigation, Methodology, Project Administration, Resources, Validation, Visualization, Writing – Review & Editing

Siti Zubaidah Che Lem
Roles: Investigation, Methodology, Project Administration, Resources, Supervision, Validation, Visualization, Writing – Review & Editing

Competing interests

No competing interests were disclosed.

Grant information

We received a Research Grant (NMRR ID-24-02224-O6O) from the Ministry of Health of Malaysia to perform the study.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Article Versions (1)

version 1

Published: 24 Apr 2025, 14:461

https://doi.org/10.12688/f1000research.163467.1

Copyright

© 2025 Lokman EF et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Lokman EF, Megat Kamaruddin PSN, Muhammad H et al. Investigating the effects of Orthosiphon stamineus on blood glucose regulation via pancreatic endocrine and exocrine functions in Type 3c Diabetes Mellitus Wistar Bonn/Kobori (WBN/Kob) pancreatitis rat model. [version 1; peer review: 1 approved with reservations]. F1000Research 2025, 14:461 (https://doi.org/10.12688/f1000research.163467.1)

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Open Peer Review

Version 1

VERSION 1

PUBLISHED 24 Apr 2025

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7

Reviewer Report 25 Jul 2025

Majed Ahmed Al-Mansoub, Universiti Sains Malaysia, Minden Heights, Pulau Pinang, Malaysia

Approved with Reservations

https://doi.org/10.5256/f1000research.179823.r392016

Thank you very much for choosing me to review the manuscript entitled ‘’Investigating the effects of Orthosiphon stamineus on blood glucose regulation via pancreatic endocrine and exocrine functions in Type 3c Diabetes Mellitus Wistar Bonn/Kobori (WBN/Kob) pancreatitis rat model’’.
... Continue reading

Thank you very much for choosing me to review the manuscript entitled ‘’Investigating the effects of Orthosiphon stamineus on blood glucose regulation via pancreatic endocrine and exocrine functions in Type 3c Diabetes Mellitus Wistar Bonn/Kobori (WBN/Kob) pancreatitis rat model’’.

This manuscript investigates the potential regulatory effects of Orthosiphon stamineus (OS) on glucose metabolism in a Type 3c diabetes model, which is focused on both endocrine and exocrine pancreatic functions using the WBN/Kob rat model. The study protocol addresses a less-explored area of secondary diabetes (Type 3c) and integrates phytotherapeutic intervention with disease-specific pathology. The authors should ensure a consistent scientific tone and structure aligned with preclinical protocol reporting standards (e.g., ARRIVE guidelines). However, this protocol requires revisions in methodological transparency, data interpretation, and clarity in the mechanistic hypothesis to be suitable for indexing. A revised version including these adjustments should be submitted for further consideration.

Some specific comments on the text include:

In the Abstract:

The authors should be more specific… Blood glucose and insulin levels will be collected at different time points.

Provide brief details about grouping. It is also advisable to mention any exclusion criteria in this protocol before defining the groups, including the control and different treatment groups.

They should clarify that results are expected, as no data have been collected yet.

Avoid over-promising; use cautious terms like “may provide insights” or “may support”.

In the Introduction:

Clearly define the hypothesis at the end of the introduction, such as: "We hypothesise that OS improves glycaemic control in T3cDM by modulating both endocrine and exocrine pancreatic functions."

Use a transitional sentence (e.g., “Given this background…”) to link the bioactivity of OS with the justification for testing it in a pancreatitis-induced diabetic model.

Multiple studies about OS effects in T2DM are cited, but their relevance to T3cDM or pancreatic exocrine dysfunction is not fully established. This could mislead readers to assume similar mechanisms without supporting evidence. Therefore, I recommend acknowledging that T2DM and T3cDM differ pathophysiologically and that direct evidence of OS on exocrine pancreatic function is lacking, which is the novelty of your study.

While the model is mentioned, no references or justification are provided for why WBN/Kob rats are suitable for mimicking both endocrine and exocrine dysfunction seen in human T3cDM. Briefly explain why this model is appropriate and cite relevant literature validating its use for pancreatitis-induced diabetes.

There is some redundancy in explaining the pharmacological properties of OS and its bioactive components (e.g., antioxidant and anti-inflammatory are mentioned more than once).

Aim, objectives, and hypothesis are OK.

In the Methods:

Please define clearly the extraction method and include standardisation data (e.g., HPLC profile or marker compound concentration). Reference the dose used and its justification from prior efficacy or toxicity studies.

The authors should note that the sample size calculation is vague and only refers to formulae based on degrees of freedom, not statistical power. This approach is insufficient for determining appropriate power for detecting significant differences in biochemical outcomes (e.g., glucose, enzymes). The sample size critically affects the hypothesis and the study design; thus, I suggest recalculating the sample size using a power analysis based on anticipated effect size, alpha (e.g., 0.05), and power (e.g., 0.80). Include the parameters and assumptions used in the calculation.

The study measures multiple parameters (glucose, insulin, enzymes, hormones, histology), but there is no distinction between primary and secondary outcomes. Please add it.

Consolidate the endocrine and exocrine assessment methods under unified subheadings. Please avoid repeating protocols already described earlier.

The text mentions H&E and immunohistochemistry but does not describe the scoring system, staining targets, or how the data will be quantified. Specify the histological grading system (e.g., fibrosis, islet area, inflammation score), etc.
The protocol mentions single-blind procedures, yet states that "neither the researchers nor the individuals administering the treatments will know the group assignments." This describes a double-blind design, not a single-blind. Clarify whether the study is single or double-blind (e.g., animal handlers blinded). If double-blind, explain how treatment coding and data analysis will remain blinded throughout.

Justify why these lipid-soluble vitamins (A, D, E, K) are being measured.

In the next sections:

The statistical methods (t-test, regression, chi-square) are listed without clarification of which test is used for which data type or variable. Please indicate normality testing and corrections for multiple comparisons.

Fat-soluble vitamins are mentioned again, but no mechanistic rationale or reference links them directly to OS or pancreatitis-induced T3cDM. Briefly explain why these vitamins are relevant biomarkers in the context of exocrine dysfunction or OS therapy.

Strengthen discussion by explaining how OS might mechanistically improve both beta-cell function and enzyme production in the WBN/Kob model (e.g., through anti-inflammatory or anti-fibrotic effects).

In the limitations section, please add a sentence acknowledging that the extract’s multi-compound nature could limit the identification of specific active constituents responsible for the effects.

For References:

The authors should standardise all references using the short or full name of journals. References such as 3,13, 14, 15, 16, and 18 are not consistent with others. Make sure all links provided are accessible.

Overall, the study is conceptually strong; however, revisions are required prior to resubmission to address the lack of clear outcome classification, vague statistical analysis, insufficient rationale for vitamin assessment, and an underdeveloped discussion.

Is the rationale for, and objectives of, the study clearly described?

Yes
Is the study design appropriate for the research question?

Yes
Are sufficient details of the methods provided to allow replication by others?

Yes
Are the datasets clearly presented in a useable and accessible format?

Yes

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Drug discovery in preclinical and clinical studies.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

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Majed Ahmed Al-Mansoub, Universiti Sains Malaysia, Minden Heights, Malaysia

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Reviewer Report

7 Views

25 Jul 2025 | for Version 1

Majed Ahmed Al-Mansoub, Universiti Sains Malaysia, Minden Heights, Pulau Pinang, Malaysia

7 Views Cite this report Responses(0)

Approved With Reservations

Thank you very much for choosing me to review the manuscript entitled ‘’Investigating the effects of Orthosiphon stamineus on blood glucose regulation via pancreatic endocrine and exocrine functions in Type 3c Diabetes Mellitus Wistar Bonn/Kobori (WBN/Kob) pancreatitis rat model’’.

This manuscript investigates the potential regulatory effects of Orthosiphon stamineus (OS) on glucose metabolism in a Type 3c diabetes model, which is focused on both endocrine and exocrine pancreatic functions using the WBN/Kob rat model. The study protocol addresses a less-explored area of secondary diabetes (Type 3c) and integrates phytotherapeutic intervention with disease-specific pathology. The authors should ensure a consistent scientific tone and structure aligned with preclinical protocol reporting standards (e.g., ARRIVE guidelines). However, this protocol requires revisions in methodological transparency, data interpretation, and clarity in the mechanistic hypothesis to be suitable for indexing. A revised version including these adjustments should be submitted for further consideration.

Some specific comments on the text include:

In the Abstract:

The authors should be more specific… Blood glucose and insulin levels will be collected at different time points.

Provide brief details about grouping. It is also advisable to mention any exclusion criteria in this protocol before defining the groups, including the control and different treatment groups.

They should clarify that results are expected, as no data have been collected yet.

Avoid over-promising; use cautious terms like “may provide insights” or “may support”.

In the Introduction:

Clearly define the hypothesis at the end of the introduction, such as: "We hypothesise that OS improves glycaemic control in T3cDM by modulating both endocrine and exocrine pancreatic functions."

Use a transitional sentence (e.g., “Given this background…”) to link the bioactivity of OS with the justification for testing it in a pancreatitis-induced diabetic model.

Multiple studies about OS effects in T2DM are cited, but their relevance to T3cDM or pancreatic exocrine dysfunction is not fully established. This could mislead readers to assume similar mechanisms without supporting evidence. Therefore, I recommend acknowledging that T2DM and T3cDM differ pathophysiologically and that direct evidence of OS on exocrine pancreatic function is lacking, which is the novelty of your study.

While the model is mentioned, no references or justification are provided for why WBN/Kob rats are suitable for mimicking both endocrine and exocrine dysfunction seen in human T3cDM. Briefly explain why this model is appropriate and cite relevant literature validating its use for pancreatitis-induced diabetes.

There is some redundancy in explaining the pharmacological properties of OS and its bioactive components (e.g., antioxidant and anti-inflammatory are mentioned more than once).

Aim, objectives, and hypothesis are OK.

In the Methods:

Please define clearly the extraction method and include standardisation data (e.g., HPLC profile or marker compound concentration). Reference the dose used and its justification from prior efficacy or toxicity studies.

The authors should note that the sample size calculation is vague and only refers to formulae based on degrees of freedom, not statistical power. This approach is insufficient for determining appropriate power for detecting significant differences in biochemical outcomes (e.g., glucose, enzymes). The sample size critically affects the hypothesis and the study design; thus, I suggest recalculating the sample size using a power analysis based on anticipated effect size, alpha (e.g., 0.05), and power (e.g., 0.80). Include the parameters and assumptions used in the calculation.

The study measures multiple parameters (glucose, insulin, enzymes, hormones, histology), but there is no distinction between primary and secondary outcomes. Please add it.

Consolidate the endocrine and exocrine assessment methods under unified subheadings. Please avoid repeating protocols already described earlier.

The text mentions H&E and immunohistochemistry but does not describe the scoring system, staining targets, or how the data will be quantified. Specify the histological grading system (e.g., fibrosis, islet area, inflammation score), etc.
The protocol mentions single-blind procedures, yet states that "neither the researchers nor the individuals administering the treatments will know the group assignments." This describes a double-blind design, not a single-blind. Clarify whether the study is single or double-blind (e.g., animal handlers blinded). If double-blind, explain how treatment coding and data analysis will remain blinded throughout.

Justify why these lipid-soluble vitamins (A, D, E, K) are being measured.

In the next sections:

The statistical methods (t-test, regression, chi-square) are listed without clarification of which test is used for which data type or variable. Please indicate normality testing and corrections for multiple comparisons.

Fat-soluble vitamins are mentioned again, but no mechanistic rationale or reference links them directly to OS or pancreatitis-induced T3cDM. Briefly explain why these vitamins are relevant biomarkers in the context of exocrine dysfunction or OS therapy.

Strengthen discussion by explaining how OS might mechanistically improve both beta-cell function and enzyme production in the WBN/Kob model (e.g., through anti-inflammatory or anti-fibrotic effects).

In the limitations section, please add a sentence acknowledging that the extract’s multi-compound nature could limit the identification of specific active constituents responsible for the effects.

For References:

The authors should standardise all references using the short or full name of journals. References such as 3,13, 14, 15, 16, and 18 are not consistent with others. Make sure all links provided are accessible.

Overall, the study is conceptually strong; however, revisions are required prior to resubmission to address the lack of clear outcome classification, vague statistical analysis, insufficient rationale for vitamin assessment, and an underdeveloped discussion.

Is the rationale for, and objectives of, the study clearly described?

Yes
Is the study design appropriate for the research question?

Yes
Are sufficient details of the methods provided to allow replication by others?

Yes
Are the datasets clearly presented in a useable and accessible format?

Yes

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Drug discovery in preclinical and clinical studies.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

Respond to this report

Responses (0)

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[2] 2. Vujasinovic M, Zaletel J, Tepes B, et al.: Low prevalence of exocrine pancreatic insufficiency in patients with diabetes mellitus. Pancreatology. 2013; 13(4): 343–346. Publisher Full Text

[3] 3. Duggan SN, Conlon KC: Pancreatogenic Type 3c Diabetes:Underestimated, Underappreciatedand Poorly Managed. Pract. Gastroenterol. 2017; 163: 14–23.

[4] 4. Ewald N, Hardt PD: Diagnosis and treatment of diabetes mellitus in chronic pancreatitis. World J. Gastroenterol. 2013 Nov 14; 19(42): 7276–7281. PubMed Abstract | Publisher Full Text | Free Full Text

[5] 5. Hart PA, Bellin MD, Andersen DK, et al.: Type 3c (pancreatogenic) diabetes mellitus secondary to chronic pancreatitis and pancreatic cancer. Lancet Gastroenterol. Hepatol. 2016 Nov; 1(3): 226–237. PubMed Abstract | Publisher Full Text | Free Full Text

[6] 6. Petrov MS, Basina M: Diagnosis of Endocrine Disease: Diagnosing and classifying diabetes in diseases of the exocrine pancreas. Eur. J. Endocrinol. 2021 Apr; 184(4): R151–R163. PubMed Abstract | Publisher Full Text

[7] 7. Lokman EF, Saparuddin F, Muhammad H, et al.: Orthosiphon stamineus as a potential antidiabetic drug in maternal hyperglycemia in streptozotocin-induced diabetic rats. Integrative Medicine Research. 2019 Sep 1; 8(3): 173–179. PubMed Abstract | Publisher Full Text | Free Full Text

[8] 8. Wang Q, Wang J, Li N, et al.: A Systematic Review of Orthosiphon stamineus Benth. in the Treatment of Diabetes and Its Complications. Molecules. 2022 Jan [cited 2024 Jun 11]; 27(2). Publisher Full Text Reference Source

[9] 9. Olah NK, Radu L, Mogoşan C, et al.: Phytochemical and pharmacological studies on Orthosiphon stamineus Benth. (Lamiaceae) hydroalcoholic extracts. J. Pharm. Biomed. Anal. 2003; 33(1): 117–123. PubMed Abstract | Publisher Full Text

[10] 10. Abdullah NR, Ismail Z, Ismail Z: Acute toxicity of Orthosiphon stamineus Benth standardized extract in sprague dawley rats. Phytomedicine. 2009; 16(2–3): 222–226. PubMed Abstract | Publisher Full Text

[11] 11. Sakaguchi Y, Inaba M, Tsuda M, et al.: The Wistar Bonn Kobori rat, a unique animal model for autoimmune pancreatitis with extrapancreatic exocrinopathy. Clin. Exp. Immunol. 2008 Apr; 152(1): 1–12. PubMed Abstract | Publisher Full Text | Free Full Text

[12] 12. Piccirillo CA, Tritt M, Sgouroudis E, et al.: Control of type 1 autoimmune diabetes by naturally occurring CD4+CD25+ regulatory T lymphocytes in neonatal NOD mice. Ann. N. Y. Acad. Sci. 2005 Jun; 1051: 72–87. PubMed Abstract | Publisher Full Text

[13] 13. Zipris D: Evidence that Th1 Lymphocytes Predominate in Islet Inflammation and Thyroiditis in the BioBreeding (BB) Rat. J. Autoimmun. 1996 Jun 1; 9(3): 315–319. PubMed Abstract | Publisher Full Text

[14] 14. Mori Y, Yokoyama J, Nishimura M, et al.: Diabetic Strain (WBN/Kob) of Rat Characterized by Endocrine-Exocrine Pancreatic Impairment Due to Distinct Fibrosis. Pancreas. 1990 Jul; 5(4): 452–459. PubMed Abstract | Publisher Full Text

[15] 15. Hashimoto T, Yamada T, Yokoi T, et al.: Apoptosis of Acinar Cells Is Involved in WBN/Kob Rats: Role of Glucocorticoids.[cited 2025 Jan 27]. Reference Source

[16] 16. Arifin W: Sample Size Calculation in Animal Studies Using Resource Equation Approach. The Malaysian Journal of Medical Sciences. 2017; 24(5): 101–105. PubMed Abstract | Publisher Full Text

[17] 17. Lokman EF: Guidance protocols to oral glucose tolerance test, pancreatic islets isolation and glucose stimulated insulin secretion in rats. International Medical Research Journal. 10(1): 39–51.

[18] 18. Wei Q, Qi L, Lin H, et al.: Pathological Mechanisms in Diabetes of the Exocrine Pancreas: What’s Known and What’s to Know. Front. Physiol. 2020 Oct 28 [cited 2024 Apr 16]; 11. PubMed Abstract | Publisher Full Text | Free Full Text

[19] 19. Lokman EF, Kamaruddin PSNM, Muhammad H, et al.: Investigating the effects of orthosiphon stamineus on blood glucose regulation via pancreatic endocrine and exocrine functions in a type 3c diabetes mellitus wistar bonn/kobori (wbn/kob) pancreatitis rat model. OSF. 2025.

Investigating the effects of Orthosiphon stamineus on blood glucose regulation via pancreatic endocrine and exocrine functions in Type 3c Diabetes Mellitus Wistar Bonn/Kobori (WBN/Kob) pancreatitis rat model.

Abstract

Background

Methods

Results

Conclusion

Keywords

Introduction

Aim

Objectives

Main objective

Hypothesis

Methods

Sampling size

Randomisation and blinding

Preparation of Orthosiphon stamineus extract

Intervention

Ethical considerations

Outcome measures

Data collection

Statistical analysis

Dissemination

Status of this study

Scope

Limitations

Dissemination

Discussion

Data availability

Reporting guidelines

Acknowledgements

References

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