INTRODUCTION:

International Diabetes Federation (IDF) Atlas guideline report mentioned that more than half a billion people are living with diabetes worldwide. In year, 2021, it is estimated that 537 million people are with diabetes and this number predicted to be 643 million in year 2030 and 783 million in year 2045. It is said that worldwide, in the age group of 20 to 79 years, more than 1 in 10 adults is affected with diabetes. In some countries, the prevalence of diabetes is 1 in 5 or even more¹. Diabetes affects the functional capacities of individuals and their quality of life resulting in increase in morbidity and premature mortality².

The oral antidiabetic drugs are reported to have certain disadvantages such as drug resistance, adverse effects, and toxicity. For example, sulfonylureas, showed reduced efficacy after 6 years of treatment in almost 44% of the patients. Glucose lowering drugs are not effective in limiting the hyperlipidemia to certain extent³.

Apart from synthetic modern medicines, many diabetic patients prefer to use plant-based medicines for the treatment of diabetes as complementary and alternative medicine (CAM)⁴. Approximately 1200 plants are reported to have antidiabetic activity⁵. Around 400 plants and over 700 recipes and bio-functional molecules are evaluated for the treatment of type-2 diabetes⁶. It is reported that up to 72.8% of diabetic people use herbal medicine, dietary supplements, and CAM for various chronic metabolic disorders⁷. However, actual number of people taking herbal or CAM therapy could be still higher as many patients did not reveal that they are taking herbal medicines and physicians also do not ask about herbal medicines while prescribing medicines⁸. Such concomitant usage of herbal medicines and modern medicines may lead to herb-drug interactions. Herbal medicines upon co-administration with modern synthetic medicines, may increase or decrease the pharmacological or toxicological effect of any medicine leading to challenging clinical condition⁹. Popular Nigerian herbal medicines (Annona senegalensis, Bridellia ferruginea, Cassytha filiformis, Daniellia ogea, Khaya ivorensis, Syzygium guineense, Terminalia avicennioides, and X. americana.) used for diabetic patients are shown to potentially affect glibenclamide absorption at concentrations in the intestinal tract¹⁰. Gymnema sylvestre used as an antidiabetic herb showed improved glycemic control in presence of glibenclamide without affecting pharmacokinetics of glibenclamide¹¹. Curcumin and glibenclamide co-administration in STZ-induced diabetic rats showed longer elimination half-life and long residence of glibenclamide compared to glibenclamide alone treated rats¹². Many herbal medicines which are concomitantly taken along with modern synthetic medicines are reported to have serious herb-drug interactions leading to loss of efficacy or unwanted adverse clinical signs. Therefore, physicians need to know if patients are on any herbal medicines^{13, 14}.

Momordica charantia (MC), commonly known as bitter melon; bitter gourd or Karela, is one of the medicinal plants used not only as medicine but also as a vegetable for cooking in many countries. MC has many biological activities such as antidiabetic, hypolipidemic, anti-inflammatory, antioxidant, anti-obesity, antitumor, antimicrobial and anthelmintic^{15,16,17,18,19,20}. Many patients consume MC in the form of vegetable in daily diet or take fruit juice or extract for the treatment of chronic metabolic disorders. MC fruit contains various bioactive components which may interact with drug metabolizing enzymes and or drug transporters leading to modulation of drug disposition or drug targets or signaling pathways²¹. To best of our knowledge, there is limited information or data available in the public domain regarding the effect of MCE coadministration on pharmacokinetics and pharmacodynamic parameters of commonly prescribed antidiabetic drugs,glibenclamide in diabetic condition. Hence, current study was planned with an objective to evaluate herb-drug interaction potential of MCE in STZ induced diabetic rats with respect to pharmacokinetics and pharmacodynamic end points together in the same study. The outcome of current study will help physicians to titrate the dose of glibenclamide when patients are taking MC in the form of vegetable or fruit juice in daily life to avoid unwanted side effects like hypoglycemia or lack of efficacy.

Materials and methods:

Materials:

Glibenclamide, Streptozotocin were purchased from Bio Organics, Bengaluru, India. Methyl cellulose (400 cP), Verapamil, Tween 80^®and formic acid (MS grade) were purchased from Sigma-Aldrich Chemicals Limited, Bangalore, India. HPLC-grade acetonitrile, methanol, isopropyl alcohol, dimethyl sulphoxide were obtained from JT Baker (California, USA). Isoflurane was purchased from Piramal Life Sciences, Mumbai. Reagents for clinical chemistry were purchased from Siemens, India.

Momordica Charantia extract:

Momordica charantia extract was kindly provided by Natural Remedies, Bangalore as gift sample for research purpose. As per the certificate of analysis, Momordica charantia fruits were used for preparing methanolic extract. The appearance of the extract was dark brown powder. The phytochemical analysis of the extract revealed extract contains around 8.01% (w/w) of the total bitter contents.

Animals:

Male Sprague Dawley rats (275±25g body weight) were obtained from authorized and approved animal vendor. Upon receipt at facility, rats were quarantined for one week for health monitoring. The rats were acclimatized in the laboratory for one week prior to experiments and were maintained under standard environmental conditions with 12h light/dark cycle with free access to rodent chow and filtered water. Prior to start of experiments, animals were kept for 10-12h fasting and water was supplied ad libitum. Feed was provided 4h post-dose. All animal experiments were approved by the Institutional Animal Ethics Committee (SYNGENE/IAEC/1183-08-2020) and were in accordance with the Committee for Control and Supervision of Experiments on Animals (CCSEA), Ministry of Fisheries, Animal Husbandry and Dairying, Department of Animal Husbandry and Dairying, Government of India.

Induction of Diabetes:

Streptozotocin (STZ) is widely used chemical for induction of diabetes in rodents. Therefore, in the current study, diabetes mellitus was induced by intravenous administration of a single dose (50mg/kg) of STZ²². The dosing solution was prepared in 0.1 M citrate buffer (pH 4.5) at 25mg/mL concentration and administered at the dose volume of 2mL/kg. The blood glucose levels were checked at periodic intervals post 5^th Day of STZ administration. The rats showing consistent fasting blood glucose ≥ 200mg/dL were considered as diabetic and selected for the study.

Study design:

The study consists of total four groups. Each group consist of four SD rats. Non-diabetic (NC) and diabetic (DC) groups were maintained as vehicle control groups (Group-1 and group-2 respectively). Glibenclamide (GLB) was administered without or with (-/+) Momordica charantia extract (MCE; Group-3 and group-4 respectively). During the study period all animal received food and water ad libitum. The treatment group animals received GLB at 10mg/kg/day and MCE at 200 mg/kg/day, once daily for 28 consecutive days by oral gavage administration. The MCE dose was selected based on the results obtained in oral glucose tolerance test (OGTT) in rats. In OGTT, MCE at 200mg/kg dose showed significant reduction in blood glucose levels, hence 200mg/kg dose was selected for further studies (data not shown here). The dose volume for single drug treatment administration was 10mL/kg. Combination group received 5mL/kg dose volume for each formulation (total dose volume: 10mL/kg). The formulation vehicle comprised of 1% Tween 80^® and 0.5% (w/v) methyl cellulose in water q.s.

Pharmacokinetic Herb-Drug Interaction by oral administration in male SD rats:

The pharmacokinetic Herb-Drug-Interaction potential of GLB with MCE was evaluated on Day-1 and Day-28. Blood samples (~150 µL) were collected at predetermined time points post dose administration from each rat. The blood samples were collected from an alternate eye by retro-orbital plexus bleeding under light isoflurane anesthesia. Blood samples were centrifuged and the obtained plasma samples were transferred in labelled eppendorff tubes stored frozen below -70ºC until bioanalysis. All the plasma samples were analyzed by fit-for purpose liquid chromatography-tandem mass spectroscopy (LC-MS/MS) bioanalytical method with slight modification as reported in the literature²³for estimation of GLB in the plasma samples. The samples (CC and QCs) along with study samples were extracted with 15-fold volumes of ice-cold acetonitrile containing 50ng/mL of the internal standards (verapamil). All the samples were vortex mixed; centrifuged and 2µL of supernatant was injected into LC-MS/MS for analysis. The lower limit of quantification of GLB was 2ng/mL. Pharmacokinetic parameters (Cmax, tmax, AUC_0-t,AUC_0-inf, Vd/f, Cl/f and elimination half-life (t_1/2) of GLB were calculated by non-compartmental analysis module of Phoenix^®WinNonlin^® 8.3 version (Certara Inc, USA) for individual animals and were reported as the mean± SD. Maximum plasma concentration (C_max) and time to achieve maximum concentration (T_max) were observed values. The area under the plasma concentration versus time curve (AUC_0-tand AUC_0-inf) were calculated using a linear-log trapezoidal method. The Vd/f and Cl/f are the volume of distribution and plasma clearance obtained after oral administration. The elimination half-life (T_1/2) was calculated by selecting at least three sampling points after Cmax during elimination phase (r²>0.80).

Pharmacodynamic Herb-Drug Interaction by oral administration in male SD rats:

Pharmacodynamic interaction of GLB with MCE was evaluated the effect on various parameters like oral glucose tolerance (OGTT), body weight, feed consumption, water intake, urine output and biochemical parameters (blood glucose, alanine aminotransferase (ALT), aspartate transaminase (AST), blood urea nitrogen (BUN), total cholesterol (TC), triglycerides (TG)) were determined.

a) Oral glucose tolerance test:

The oral glucose tolerance test was performed in overnight fasted animals on study Day-1 and Day-28 as per the method described in literature^24,25,26. All animals received the drug treatment as mentioned in animal study section on the respective study days. Glucose (2 g/kg; 40% w/v solution; 5mL/kg dose volume) was administered 1 hour post drug treatment. Blood samples (~10-20µL) were collected from lateral tail vein at pre-dose, 0.25, 0.50-, 1.0-, 1.5- and 2-hour post glucose administration. Blood glucose level was immediately measured using glucometer (Accu Chek, Roche, USA).

b) Determination of biochemical parameters:

Effect of MCE co-administration with GLB on biochemical parameters like blood glucose, AST, ALT, BUN, total cholesterol, triglycerides were determined by collecting a terminal blood sample on study Day-29. An aliquot of whole blood was collected in Eppendorf tubes containing sodium fluoride for blood glucose estimation and another aliquot was collected in clot activator tubes for generating the serum. Biochemical parameters like AST, ALT, BUN, total cholesterol, and triglycerides were evaluated in serum. All these parameters were estimated by using fully automated Dimension Xpandplus analyzer, (Siemens, India).

c) Histopathological examination:

After euthanizing animals, pancrease were isolated, rinsed with phosphate buffer saline and fixed in 10% neutral buffered formalin (NBF). The fixed tissues were dehydrated in an ethyl alcohol gradient (70, 80, 95, 100 %). The dehydrated tissues were cleared using two changes of xylene then embedded in paraffin blocks. The paraffin embedded pancreases tissues were sliced in 4-5 µm thickness and were stained with haematoxylin and eosin (H & E)²⁷. The slides were examined under light microscope equipped with digital camera for determination of histopathological changes.

Statistical analysis:

Statistical analysis was performed using GraphPad Prism 9 for Windows (GraphPad Software, Inc. San Diego, CA, USA). Plasma concentrations time profile, pharmacokinetic parameters, Glucose AUC obtained during OGTT test, physiological and biochemical parameters obtained following GLB with and without MCE coadministration were analyzed by using Two-way ANOVA with Tukey's multiple comparisons test. A p value (p<0.05) was considered statistically significant.

Results:

Pharmacokinetic Herb-Drug Interaction by oral administration in male SD rats:

The plasma concentration time profile following single and repeat oral gavage administration of GLB (10 mg/kg/day) with or without MCE (200mg/kg/day) on Day-1 and Day- 28 days in STZ induced diabetic rats is shown in Figure 1. Mean Pharmacokinetic parameters of GLB is presented in Table 1.

Figure 1: Plasma concentration-time profile of GLB with and without MCE repeat dose administration in STZ induced diabetic rats: A) GLB (+/- MCE) administration on Day-1 and Day-28

Two-way ANOVA followed by Tukey multiple comparison test was applied. **P<0.001 compared to Day-1 treatment of same group (glibenclamide +MCE) whereas ^$$P<0.001 compared to respective antidiabetic drug control group on Day-28.

Pharmacokinetic profile of GLB:

Following single and repeat oral dose administration of GLB in male STZ induced diabetic rats, mean plasma C_maxof GLB was 597±77.1, and 698±66ng/mL, respectively, on study Day-1 and study Day-28 and the corresponding mean plasma AUC_last,AUC_infvalues of GLB were 7420±165, 9730 h*ng/mL and 8440±1010, 9640 h*ng/mL, respectively. The median time to reach peak plasma concentrations (T_max) was 6 hours post GLB administration on both the study days. The volume of distribution, clearance, and elimination half-life values of GLB were 15.1 L/kg, 17.1mL/min/kg and 10.2 h on study Day-1 whereas 15.2 L/kg, 17.4mL/min/Kg, 10.1h on study Day-28 respectively. There was no significant difference in PK parameters of GLB observed with single and once daily repeat dose administration for 28 consecutive days.

Following single and repeat oral dose administration of GLB with MCE in male STZ induced diabetic rats, mean plasma C_max of GLB was 660±89.3, and 1730± 490ng/mL, respectively, on study Day-1 and study Day-28 and the corresponding mean plasma AUC_last, AUC_inf values of GLB were 7320±391, 8560±824h*ng/mL and 13600±2630, 14100±2630 h*ng/mL, respectively. The median time to reach peak plasma concentrations (T_max) was 4 hours post GLB administration on both the study days. The volume of distribution, clearance, and elimination half-life values of GLB were 13.4±0.9L/kg, 19.7±2.0mL/min/kg and 7.9±1.0h on study Day-1 whereas 5.13±1.3 L/kg, 12.1±2.4mL/min/Kg, 4.85±0.42 h on study Day-28 respectively. The plasma Cmax and AUClast values of GLB, following GLB and MCE co-administration, on Day-28 are approximately 2.6-folds and 1.85-folds higher compared to Day-1 Cmax and AUClast values of GLB following GLB and MCE co-administration (P<0.001). Due to delayed GLB absorption, sufficient time points (excluding Tmax point), were not available in the elimination phase of GLB for characterizing the proper elimination rate constant. Hence, PK parameters (AUCinf, Vd/f, Cl/f and T1/2) associated with elimination rate constant could not be calculated for few animals receiving GLB alone or GLB and MCE coadministration.

Pharmacodynamic Herb-Drug Interaction by oral administration in male SD rats:

a) Oral glucose tolerance test:

The Area under the blood glucose concentration-time curve (AUC) is presented in Table-2. At the initiation of the study on Day-1, the basal mean blood glucose concentration in non-diabetic normal rats (NC) was 91 mg/dL whereas in STZ induced diabetic rats it ranges between 330 to 360mg/dL. Following oral glucose (2 g/kg) administration on Day-1, the mean blood glucose AUC values were 328, 1010, 890, and 818h*mg/dL in NC, DC, GLB, and GLB + MCE groups, respectively. The blood glucose AUC value in DC group was approximately 3-fold higher compared to NC group (P<0.001). Diabetic animals treated with GLB alone and in combination with MCE showed significant reduction in mean blood glucose AUC values compared to DC group (P<0.001). On study Day-28, mean blood glucose AUC values were 330, 1040, 825, and 646h*mg/dL in NC, DC, GLB, and GLB +MCE groups, respectively. Both the treatment groups showed significant reduction in mean blood glucose AUC values compared to DC group (P<0.001). MCE co-administered with GLB showed significant reduction in mean blood glucose values compared to GLB alone treated groups (P<0.001).

b) Determination of biochemical parameters:

The effect of MCE co-administration with GLB on various biochemical parameters like blood glucose, triglycerides, total cholesterol, AST, ALT, and BUN is presented in Figure-2. Diabetic animals from DC group showed increased levels of blood glucose, total cholesterol, triglycerides, AST, ALT, and BUN compared to animals from NC group (P<0.001).

Table 2: Summary of blood glucose area under the curve in oral glucose tolerance test following oral administration of GLB with and without MCE coadministration in STZ induced diabetic rats.

Group	*AUC_last (hmg/dL)**
Group	Day-1	Day-28
Normal control (NC)	328 ± 8.38	330 ± 12.2
Diabetic control (DC)	1010 ± 57.2^**	1040 ± 32.4**
GLB (10 mg/kg, PO)	890 ± 56.8^**$	825 ± 20.9**^$$
GLB (10 mg/kg, PO) + MCE (200 mg/kg, PO)	818 ± 26.6^**$$	646 ± 27.4^**$$##

Two-way ANOVA followed by Tukey multiple comparison test was applied. *P<0.05, **P<0.001 compared to normal control group whereas ^$P<0.05, ^$$P<0.001 compared to diabetic control group, ^#P<0.05, ^##P<0.001 compared to glibenclamide control group

GLB alone and in combination with MCE significantly reduced the blood glucose level in animals compared to diabetic control animals in DC group (P<0.001). The blood glucose level in animals receiving MCE co-administration with GLB showed significantly lower blood glucose levels compared to the animals who received GLB alone (P<0.05). The total cholesterol and triglycerides in GLB alone treated group were comparable with DC group animals. MCE co-administered with GLB showed significant reduction in total cholesterol level compared to GLB alone treated group animals (P<0.05), however triglycerides levels were comparable with that of GLB alone treated group animals(P>0.05) but lower than DC group animals (P<0.001). Both the treatment group animals showed reduction in AST, ALT, and BUN levels compared to DC group animals. MCE co-administered with GLB showed comparable AST, ALT, and BUN levels compared to GLB alone treated group animals.

c) Histopathological examination:

The pancreatic tissue of animals from the NC group after staining with H&E stain showed normal architecture (Grade 0). Diabetic animals from DC group showed severe atrophy of the islet of Langerhans, macrophage infiltration leading to decrease in size and number of islets (Grade++++). GLB treated diabetic rats showed moderate atrophy of the islet of Langerhans and moderate reduction in size and number of islet cells (Grade+++). Animals treated with MCE in combination with GLB showed mild degree of atrophy and histological changes in islets of Langerhans (Grade++). The representative photographs of histopathological examinations are presented in Figure-3.

Figure 3: Histopathological images of pancreas

A) Vehicle Control stained with H&E revealed normal architecture.

B) Diabetic control; yellow arrow indicates marked atrophy of islets.

C) GLB alone; yellow arrow indicating moderate atrophy of islets.

D) GLB + MCE; yellow arrow indicates mild atrophy of islets. .

Discussion:

Diabetes is known metabolic disorder associated with increased blood glucose levels and other associated complications affecting large population world-wide. Various modern synthetic medicines are being prescribed for the treatment of diabetes. Majority of diabetic population also takes herbal medicines in the form of dietary supplements, juice, extracts as supplementary treatment for the treatment of chronic metabolic disorders. In current study, we investigated the herb-drug interaction (HDI) potential of Momordica charantia extract (MCE) following co-administration with widely prescribed antidiabetic agents GLB in STZ induced diabetic rats for 28 consecutive days. The study evaluated the PK and PD based HDI in the same animal. We believe this might be the first study of its kind evaluating the potential of PK and PD HDI of MCE with antidiabetic agents in same diabetic animals, mimicking the clinical scenario. Bitter gourd or MC is commonly used as vegetable in Indian diet and many diabetic patients also consumes bitter gourd or MC in the form of fruit juice, extract, or tablets along with prescribed antidiabetic agents for lowering the hyperglycemia. Therefore, it is essential to evaluate the HDI potential of MCE with antidiabetic drugs in diabetic rats mimicking the clinical scenario which can help or guide physicians to titrate the doses of antidiabetic agents to avoid potential side effects or lack of efficacy.

The dose of MCE (200 mg/kg) was selected based on the outcome of oral glucose tolerance test conducted in STZ induced diabetic rats (data not shown here). The dose of GLB was selected from the previously reported literature^28,29. In this study, for GLB, median time (Tmax) to reach peak plasma concentration (C_max) was around 6 h post dose administration suggesting delayed rate of oral absorption. The plasma exposure (AUC_last) of GLB was comparable between study Day-1 and Day-28 following GLB alone administration suggesting there was no accumulation of GLB following once daily repeat dose administration for 28-consecutive days. Following once daily repeat dose co-administration of MCE and GLB for 28-consecutive days, the plasma AUC_lastand C_maxof GLB were approximately 2.6-folds and 1.85-folds higher respectively, on Day-28 compared to Day-1. The increase in plasma exposure could be associated with inhibition of CYP2C9 drug metabolizing enzyme because of MCE. GLB is metabolized by CYP2C9 enzyme^28,30whereas MCE is an inhibitor of CYP2C9 and CYP3A4³¹. MCE might have inhibited the CYP2C9 enzyme therefore, clearance of GLB was inhibited leading to increased plasma concentrations on Day-28 of the study. The pharmacodynamic HDI was assessed by evaluating the reduction in blood glucose AUC values in OGTT, effect on biochemical parameters along with histopathological evaluation. The reduction in blood glucose AUC values with MCE and GLB co-administration are significantly lower compared to GLB alone treated groups. MCE co-administration with GLB showed significant reduction in blood glucose, total cholesterol levels, and improved protection of beta cells of Langerhans (histopathological observation) compared to GLB alone treated groups suggesting synergistic or additive effect of MCE. However, MCE and GLB co-administration did not show significant difference in triglycerides, AST, ALT as compared to GLB alone treated groups. Aqueous extract (0.02% w/v) of sun-dried pulps of M. Charantia prevented pancreatic beta cell death and showed recovery of partially destroyed pancreatic beta cells in alloxan and STZ induced diabetic rats³². The protective effect of M. Charantia on pancreatic beta cells is in line with our observation that MC co-administration with GLB has prevented or facilitated recovery of partially destroyed pancreatic beta cells in STZ induced diabetic rats. As MCE coadministration with GLB resulted in increased plasma exposures of GLB, physicians need to be more cautious while prescribing GLB to patients if they are taking MCE as supplement. Alternatively, physician should advise patients not to take MCE while on GLB treatment or reduce GLB dose to avoid excessive hypoglycemia.

Conclusions:

MCE co-administration with GLB showed positive pharmacodynamic herb-drug interaction by reducing the blood glucose, and improved glucose tolerance with protective effect on pancreatic beta cells. The plasma exposure of GLB was increased with MCE co-administration. All these findings suggests that MCE co-administration with GLB produced desired antidiabetic effects. The doses of glibenclamide can be lowered when patients are on MCE coadministration to avoid excessive hypoglycemia.

Conflict of interest:

The authors report no conflict of interest.

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Received on 01.11.2023 Modified on 15.01.2024

Research J. Pharm. and Tech 2024; 17(8):3757-3763.

DOI: 10.52711/0974-360X.2024.00584

Group	Day	T_max (h)	C_max (ng/mL)	*AUC_last (hng/mL)**	*AUC_INF (hng/mL)**	Vd/f (L/kg)	Cl/f (mL/min/kg)	T_1/2 (h)
GLB (PO: 10 mg/kg)	1	6.0 (4-8)	597±77.1	7420±165	9730^a	15.1^a	17.1^a	10.2^a
GLB (PO: 10 mg/kg)	28	6.0(4-8)	698±66^NS	8440±1010^NS	9640^b	15.2^b	17.4^b	10.1^b
GLB (PO: 10 mg/kg) + MCE (PO: 200 mg/kg)	1	4.0 (4-8)	660±89.3^NS	7320±391^NS	8560 ±824	13.4 ±0.9	19.7±2.0	7.9±1.0
GLB (PO: 10 mg/kg) + MCE (PO: 200 mg/kg)	28	4.0(4-4)	1730±490^**##	13600±2630^**##	14100±2630	5.13 ±1.3	12.1±2.4	4.85±0.42