[version 1; peer review: 2 approved with reservations]
No competing interests were disclosed.
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
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.
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.
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.
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%).
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.
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
OS, a member of the
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.
We investigate the effects of
This study will evaluate the impact of
To assess the effect on blood glucose and hemoglobin A1c (HbA1c) levels in the treatment groups compared to the control group. 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. 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.
We hypothesize that
The sample size that calculates the suggested number of rats used in each group is based on a previously described formula.
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:
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:
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.
The OS will be obtained from a commercial supplier or harvested from local sources and prepared as previously described.
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,
This protocol evaluates the effects of OS treatment on glucose regulation by performing an Oral Glucose Tolerance Test and measuring exocrine and endocrine hormones.
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. 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. 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. 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.
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(1/2025
Animal Welfare: All procedures involving animals will strictly adhere to the guidelines outlined in the
Administering appropriate anaesthesia (Isoflurane) for all surgical procedures, with continuous monitoring of anaesthetic depth. Conducting regular assessments of animals for distress, with the application of humane endpoints when necessary. Housing animals in a temperature-regulated environment, maintaining a 12-hour light/dark cycle, and providing unrestricted access to food and water. Ensuring gentle handling: All procedures, including injections and oral gavage were carried out by trained personnel to minimize stress and discomfort. Performing euthanasia in accordance with the American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals (
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.
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 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.
The results of this study will be submitted for publication to well-regarded medical journals focused on diabetes and pancreatic cells.
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.
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.
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.
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.
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.
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.
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.
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.
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.
No data are associated with this article.
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”.
Data are available under the terms of the Creative Commons Attribution-ShareAlike 4.0 International Public License (CC BY-SA 4.0).
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.
1. The manuscript adequately summarizes the antidiabetic properties of Orthosiphon stamineus in T2DM models; however, the novelty regarding T3cDM remains insufficiently articulated.
The authors should explicitly clarify:
a) why T3cDM requires distinct therapeutic approaches compared to T1DM and T2DM.
b) how endocrine-exocrine pancreatic interactions differ in T3cDM.
c) why OS may uniquely benefit pancreatitis-associated diabetes.The current introduction occasionally implies that mechanisms established in T2DM are directly applicable to T3cDM, which may oversimplify the pathophysiology.
2. This is a major methodological concern because phytochemical variability significantly affects reproducibility and interpretation.
The authors are strongly encouraged to include:
a) HPLC or LC-MS profiling,
b) concentrations of key compounds such as rosmarinic acid, sinensetin, eupatorin, or caffeic acid
c) justification for dose selection based on prior efficacy and toxicity studies.
3. The manuscript describes the study as “single-blind,” yet later states that neither researchers nor treatment administrators will know group assignments, which actually describes a double-blind design.
The authors should clarify:
a) who specifically remains blinded,
b) how treatment coding will be maintained,
c) whether histopathological assessments will also be blinded.
4. The manuscript repeatedly discusses endocrine and exocrine analyses across several subsections, resulting in redundancy.
The Methods section would benefit from restructuring into endocrine assessment, exocrine assessment, histopathological assessment and metabolic assessment.
6. The protocol mentions H&E staining and immunohistochemistry but lacks information regarding:
a. scoring systems,
b. fibrosis grading,
c. inflammatory infiltration scoring,
d. β-cell quantification,
e. islet size analysis.
7. The Discussion would benefit from a more clinically oriented perspective.
The authors should discuss:
a. human dose translation,
b. bioavailability,
c. long-term safety concerns,
d. potential herb-drug interactions,
e. comparison with current T3cDM therapies such as insulin or pancreatic enzyme replacement.
8. The abstract should clearly indicate that this is a study protocol and that no experimental data have yet been collected.
9. The phrase “results” in the abstract should be revised to indicate anticipated outcomes rather than completed findings.
10. ARRIVE guideline compliance should be emphasized more explicitly.
11. Reference formatting is inconsistent, particularly references 3, 13, 14, 15, 16, and 18.
12. Ethical approval formatting requires correction “ACUC/KKM/02(1/20259” appears incomplete (maybe, Please check)
13. The manuscript would benefit from a graphical workflow or experimental timeline diagram.
This protocol addresses an important and underexplored area of pancreatogenic diabetes research and demonstrates potential translational relevance. However, revisions are required to improve methodological transparency, mechanistic depth, statistical rigor, and clarity regarding endocrine-exocrine pancreatic assessments before the study can be considered sufficiently robust and reproducible for indexing and future clinical translation.
Is the study design appropriate for the research question?
Yes
Is the rationale for, and objectives of, the study clearly described?
Partly
Are sufficient details of the methods provided to allow replication by others?
Yes
Are the datasets clearly presented in a useable and accessible format?
Partly
Reviewer Expertise:
Neuroscience/Neurodegenerative disease/Microscopic study/Molecular study/Natural Products
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.
Thank you very much for choosing me to review the manuscript entitled ‘’
This manuscript investigates the potential regulatory effects of
The authors should be more specific…
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”.
Clearly define the hypothesis at the end of the introduction, such as: "
Use a transitional sentence (e.g., “
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).
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 "
Justify why these lipid-soluble vitamins (A, D, E, K) are being measured.
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.
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 study design appropriate for the research question?
Yes
Is the rationale for, and objectives of, the study clearly described?
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
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.