INTRODUCTION:

The antidiabetic effect of plant extracts represents a growing field of research, emphasizing the potential of diverse bioactive compounds from plants in the effective management of diabetes¹. Phytochemicals, bioactive compounds present in plant extracts, are essential for modulating glucose metabolism, improving sensitivity to insulin, and aiding pancreatic β-cell function².

α-glucosidase inhibitors, including those sourced from mulberry, are significant for their capacity to decelerate carbohydrate digestion and lower postprandial glucose levels, positioning them as potential options for managing type-2 diabetes³. Medicinal plants serve as abundant sources of various bioactive compounds, such as alkaloids, flavonoids, and terpenoids, which demonstrate notable antidiabetic properties⁴. Polyphenols, a class of bioactive compounds, have been studied for their mechanisms of action, including the enhancement of insulin sensitivity and the reduction of oxidative stress, a significant factor in the progression of diabetes⁵. The antioxidant characteristics of these herbal extracts may reduce oxidative stress, thereby improving their potential for use in diabetes management⁶. In vitro and in vivo models are employed to evaluate the efficacy and safety of these plant extracts, facilitating the identification of effective diabetes treatments⁷. Clinical trials are crucial for evaluating the security and effectiveness of these extracts in human beings, highlighting their promise as supplement therapies in diabetes management⁸. Comprehending the processes of action of these extracts is essential for their advancement as therapeutic agents, as it elucidates the pathways through which they produce their beneficial effects. The amalgamation of age-old wisdom with trendy pharmacological techniques via ethnopharmacology is facilitating the development of innovative antidiabetic therapies from plant sources.

Hibiscus sabdariffa, or roselle, has attracted considerable interest for its various pharmacological qualities, including its potential in the management of diabetes mellitus⁹. This plant, part of the Malvaceae family and native to India, Sudan, and Malaysia, is known by several names, including hibiscus, rosela, and azedinha¹⁰. Studies highlighting the health benefits of hibiscus, particularly its antioxidant properties and positive effects on hypertension, gastrointestinal disorders, liver issues, and cholesterol lipid levels, are driving up demand for it¹¹. Many researchers are looking into how Hibiscus sabdariffa can help people with diabetes because it has many different chemicals in it, such as anthocyanins, polyphenols, and other bioactive compounds that have been shown to help break down glucose and make cells more sensitive to insulin^12–14. Hibiscus sabdariffa extracts have been shown to lower blood sugar, lower cholesterol, and protect cells from damage¹⁵. This means they could be used to treat diabetes and other metabolic disorders. The current scientific literature offers compelling justification for exploring the antidiabetic properties of mucilage extracted from Hibiscus sabdariffa leaves.

Hibiscus sabdariffa leaves contain mucilage, which is a complex polysaccharide matrix that may have anti-diabetic effects that need to be carefully studied¹⁶. Mucilage, a soluble fibre, is recognized for its capacity to regulate the absorption of glucose in the intestine, which may enhance glycaemic management¹⁷. Previous studies have shown that plants have mucilage-containing cells in their leaves and external mucilage-filled holes in their leaf veins and stem cortex, which means that a lot of mucilage builds up in these areas¹⁸. These chemicals may function as effective chemo preventive agents and nutritious foods¹⁹. The investigation of mucilage derived from Hibiscus sabdariffa leaves may provide an innovative strategy for diabetes management, differing from the action mechanisms linked to the calyces. The study of Hibiscus sabdariffa mucilage's effectiveness in treating diabetes is an important part of a larger effort to find safer and more natural ways to treat diabetes. Therefore, there is an urgent warrant for innovative therapeutic approaches that tackle the complex aspects of diabetes while reducing side effects. This study looked at how H. sabdariffa leaf mucilage extracts affected fasting blood glucose (FBG) and biochemical parameters, such as serum total cholesterol (TC), LDL, HDL, creatinine, urea, and alkaline phosphatase, in rats that had been given STZ to make them diabetic for up to 21 days. Consequently, till now it may be a first research report conducted on the mucilage of H. sabdariffa in STZ-induced diabetic rats.

MATERIALS AND METHODS:

Collection, identification and authentication of plant material:

Hibiscus sabdariffa leaves were acquired from the local market located in Anjora, district Rajnandgaon, during the month of November in the year 2019. The authentication of plant specimens was conducted by Dr. P. K. Joshi, who serves as the Professor and Head of the Department of Dravya Guna at the Government Ayurvedic College located in Raipur, Chhattisgarh, India.

Preparation of Hibiscus sabdariffamucilage extracts:

The mucilage from the fresh leaves of Hibiscus sabdariffa was extracted utilizing the conventional method. In summary, the Hibiscus sabdariffa underwent a thorough washing process and was subsequently immersed in distilled water for a duration of 8 to 9 hours. Subsequently, the mixture was subjected to heating in a water bath using continuous agitation for a duration of 30 minutes at a temperature of 60°C, thereby promoting the complete release of the mucilage into the aqueous medium. The concentrated thick solution underwent filtration using a muslin cloth, resulting in the isolation of the residual leaves of Hibiscus sabdariffa for subsequent applications. The viscous solution that had been filtered was allowed to cool to room temperature²⁰. The filtrated mucilage was applied onto a non-adhesive substrate positioned on a tray. Subsequently, the mucilage solution underwent drying to achieve an even weight within a cabinet dryer that was maintained at 45°C for an estimated duration of 24hours. Subsequently, the desiccated mucilage underwent grinding into a fine powder utilizing a mortar and pestle apparatus. The pulverized mucilage (PM) was subsequently subjected to a 80 sieve size and subsequently stored in sealed containers for future utilization and analysis.

Calculation of percentage yield:

The percentage yield was determined by calculating the ratio of the weight of dried mucilage obtained to the weight of fresh material, as described in the following equation.

Weight of dried mucilage extracted

Percentage yiled (%) = ----------------------------------------------

Weight of fresh plant material used for extraction

Organoleptic properties of Hibiscus sabdariffa mucilage studies:

The organoleptic properties of Hibiscus sabdariffa mucilage were assessed to evaluate its colour, odour, shape, size, and apex as per the method Bahadur et al²¹

Solubility studies of Hibiscus sabdariffa mucilage:

The solubility of the extracted muco-adhesive material was evaluated through a solubility test in various solvents, in accordance with the specifications outlined by Bhadur et al²¹

Phytochemical screening of Hibiscus sabdariffa dried mucilage extract:

Preliminary experiments were conducted to verify the characteristics of the mucilage obtained²². The conducted chemical analyses include the assessment for carbohydrates utilizing Molisch’s test, the evaluation for tannins through the Ferric chloride test, the determination of proteins via the Ninhydrin test, the identification of alkaloids employing Wagner’s test, the examination for glycosides using the Keller-Killaini test, the analysis for mucilage with the Ruthenium red test, the detection of flavonoids through the Shinoda test, and the qualitative estimation of reducing sugars via Fehling’s test.

Total phenolic content (TPC):

The total phenolic content of mucilage obtained from Hibiscus sabdariffa was assessed utilizing the Folin–Ciocalteu method, as outlined by Chouhan et al²³. Approximately 1 ml of mucilage extracts was combined with 2ml of Folin–Ciocalteu reagent, which had been diluted to one-tenth of its original concentration. The solution was subsequently allowed to equilibrate at ambient temperature for a duration of 3 minutes. Subsequently, 10ml of a 20% sodium carbonate solution was introduced into the mixture and allowed to incubate at ambient temperature for a duration of one hour. The absorbance of the resulting mixture was quantified at a wavelength of 765nm using a UV spectrophotometer, with a blank solution serving as the reference. The blank solution comprised the complete reagent mixture, excluding both the extract and the standard sample. The quantification of total phenolic contents was achieved through the utilization of a gallic acid standard curve, with results articulated as milligrams of gallic acid equivalent (GAE) per gram of dried weight.

In vitro Antioxidant activity (DPPH assay):

The evaluation of free radical scavenging activity across different extracts was conducted utilizing a DPPH assay²⁴. The decrease in absorption of the DPPH solution upon the addition of an antioxidant was measured at 517nm. Ascorbic acid was employed as a standard at a concentration of 10mg/mL in DMSO. Percentage scavenging activity was calculated by using below equation.

Absorbance of control – Absorbance of Test sample

% DPPH scavenging activity = ----------------------------- × 100

Absorbance of control

In-vitro animal study:

Selection and procurement of experimentalanimal:

Male Wistar rats, with an average weight of 200 ± 10 g, were procured from the Animal House at the Columbia Institute of Pharmacy, located in Raipur, Chhattisgarh. The experimental protocol received approval from the Institutional Animal Ethical Committee at Columbia Institute of Pharmacy, Raipur (Reg. No. 1321/PO/ReBi/S/10/CPCSEA). The subjects were accommodated in ventilated enclosures and provided with a standard pellet diet along with unrestricted access to water. The subjects were kept under standard environmental conditions in accordance with CPCSEA guidelines.

Acute oral toxicity studies:

In order to facilitate acclimatization to the laboratory environment, rats were randomly selected, marked for individual identification, and housed in their cages for a minimum duration of 5 days prior to the administration of the dosing regimen. Preparation of the group the subjects were deprived of food for a duration of one night preceding the administration of the dosage, although they were permitted access to water²⁵. A limit test for acute toxicity was conducted using a single oral dose of 2000mg/kg. The dosage was administered to the subject in accordance with its body weight. After meticulously monitoring the subject for 30 minutes, a prolonged observation period of 4 hours followed. Food was administered following a duration of 1 to 2 hours post-dosing. Subsequent to the survival of the initially treated subject, an additional four subjects were administered the identical dosage.

A control group of rats (n = 5) received distilled water in an equivalent volume to that administered to the treated group. For a total of 14 days, the two groups underwent systematic monitoring at regular intervals after 6 hours of meticulous observation. Surviving specimens were monitored for additional toxicological effects. The mass of the subjects was systematically recorded from the onset of the investigation, and blood specimens were obtained via cardiac puncture while under halothane-induced anaesthesia, followed by serum separation for subsequent biochemical and haematological analyses.

Induction of Diabetes:

After not eating or drinking for one night, diabetes was caused by injecting STZ (Sigma, St. Louis, MO) into the abdomen. The drug was mixed in a 0.1 M cold sodium citrate buffer with a pH of 4.5 and given at a dose of 55 mg/kg of body weight. The control group of rats was administered the vehicle exclusively²⁶. The subjects were permitted to ingest a 5% glucose solution overnight to mitigate the effects of drug-induced hypoglycemia. Twenty-four hours post-STZ injection, the fasting blood glucose levels were assessed. Animals subjected to STZ treatment were classified as diabetic when their fasting blood glucose levels exceeded 250 mg/dl.

Experimental design:

The animal experiments were categorized into five distinct cohorts, each comprising six animals, arranged as follows: Group I consisted of normal controls that received 0.1 M citrate buffers at a pH of 4.5. Group II included diabetic controls that were administered STZ. Groups III and IV comprised diabetic rats treated with Hibiscus sabdariffa mucilage at dosages of 300 mg/kg and 600 mg/kg, respectively. Lastly, group V consisted of diabetic rats that were given Metformin at a dosage of 65 mg/kg. The mucilage of Hibiscus sabdariffa and metformin were dissolved in distilled water and administered orally to the animals on a daily basis for a duration of 21 days, with blood samples obtained through pricking the tail of the rats. The blood samples were obtained on the 0th, 3rd, 7th, 14th, and 21st days of the treatment. The doses were determined through an acute oral toxicity study and administered via the oral route.

Collection of blood samples:

Blood samples from animals, encompassing both treated and vehicle control groups, were collected in tubes containing EDTA for the purpose of conducting a hematological study²⁷. The collected blood samples were preserved at −30°C prior to the assessment of multiple hematological parameters, including complete blood count (CBC) metrics, hemoglobin (Hb) levels, total red blood cell (RBC) count, platelet count, white blood cell (WBC) count, as well as the counts of neutrophils (N), lymphocytes (L), monocytes (M), and eosinophils (E).

Biochemical analysis:

Various biochemical parameters were assessed utilizing the Humalyzer 3000, employing a commercial kit sourced from Randox Laboratories Limited (United Kingdom) within a biochemical analyser²⁸. The parameters examined included total protein (TP), total cholesterol (TC), high-density lipoprotein (HDL), and triglyceride (TG) levels. The levels of low-density lipoprotein (LDL) were determined through calculated through below equation.

Total cholesterol – (HDL + Triglyceride)

LDL = -------------------------------------------------------

Statistical analysis:

All experiments were carried out in triplicate, with values presented as mean ± standard deviation. Mean comparisons were conducted utilizing the student’s t-test. The threshold for statistical significance was established at p < 0.05.

RESULTS:

Morphological studies:

The organoleptic evaluation of the plant extract provides essential preliminary insights into its physical and sensory characteristics, which are crucial for quality assessment and standardization. The study concentrated on various parameters such as colour, odour, flavour, and texture, as these attributes may indicate the existence of specific phytochemicals and potential bioactive compounds. The organoleptic properties demonstrate variability that can be affected by multiple factors, such as plant species, geographical origin, extraction methods, and the polarity of solvents. Table 1 presents the organoleptic evaluation of the mucilage extract from Hibiscus sabdariffa leaves, focusing on attributes such as colour, odour, and taste. The mucilage exhibited an irregular morphology and possessed a mucilaginous flavour profile.

Table 1: Organoleptic properties of Hibiscus sabdariffa mucilage

S. No.	Parameters	Organoleptic properties of H. Sabdariffa
1	Colour	Creamy or pale white
2	Odour	Odourless
3	Taste	Mucilaginous

Solubility studies:

The ability to dissolve of plant extracts is a fundamental factor influencing their absorption and pharmacological efficacy. This investigation evaluated the solubility characteristics of the chosen extract across different solvents, taking into account its physical and chemical characteristics and polarity. The results elucidate the interactions between solvents and extracts, which are critical for enhancing formulation strategies in both pharmaceutical and nutraceutical domains. The dissolution of extracted mucilage from H. sabdariffa was assessed in various solvents. The findings are illustrated in Table 2.

Table 2: Solubility studies of Hibiscus sabdariffa mucilage.

Sr. No.	Solvent	Solubility
1	Cold Water (8⁰C -25⁰C)	Swell to form gel
2	Hot water (30⁰C -40⁰C)	Soluble
3	Ether	Insoluble
4	Acetone	insoluble
5	Chloroform	Insoluble
6	Methanol	Insoluble
7	Ethanol	Insoluble
8	Benzene	Insoluble

Preliminary Phytochemical analysis:

The preliminary phytochemical examination of the plant extract indicated the existence of multiple bioactive compounds, such as alkaloids, flavonoids, tannins, saponins, and phenolics. The pharmacological properties of each of these secondary metabolites are well-documented, underscoring their contribution to the therapeutic value of the extract. The qualitative assessment serves as a crucial basis for subsequent quantitative analyses and investigations into bioactivity. The phytochemical analysis of the test extract showed that the H. sabdariffa extract is made up of carbohydrates, polysaccharides, and mucilage. Table 3 presents the phytochemical findings and phenolic content derived from the analyses conducted on the extract.

The measurement of phenol content was conducted utilizing a calibration curve based on gallic acid. The mucilage from Hibiscus sabdariffa that was extracted had a total phenol content of 55.41 ± 0.6 mg Gallic acid equivalent (GAE) per gram of dried extract, as shown in Table 3. statistically significant difference (p < .05) was observed between the groups.

Antioxidant assay (DPPH assay scavenging activity):

The DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging assay, a recognized method for evaluating free radical inhibition, was employed to assess the antioxidant capacity of the isolated plant mucilage. The mucilage extract demonstrated concentration-dependent scavenging action, suggesting the presence of phytoconstituents that can donate hydrogen atoms to neutralize DPPH radicals. The DPPH assay of mucilage was conducted using the spectrophotometric method, with ascorbic acid serving as the reference. Aliquots ranging from 5 to 30 µg/ml were generated, and absorbance was recorded. Table 4 illustrates the DPPH scavenging activity of mucilage extract from Hibiscus sabdariffa leaves, juxtaposed with ascorbic acid as a reference across different concentrations.The half maximal inhibitory concentration (IC₅₀) of ascorbic acid was determined to be 13.60μg/ml. Conversely, the IC₅₀ value for free radicals of the isolated mucilage was determined to be 79.12g/ml, as presented in Table 3. In comparison to ascorbic acid, it is evident that the mucilage extracted from Hibiscus sabdariffa exhibits greater anti-radical activity than the mucilage itself.

Table 3: Preliminary identification tests for Hibiscussabdariffa mucilage

Preliminary Test	Observation	Inferences
Ruthenium test	Pink colour develops	Mucilage present
Molisch’s test	Violet ring is observed at the junction of liquids	Carbohydrate present
Iodine test	No colour develops in solution	Polysaccharides present (starch absent)
Enzyme test	No blue color is produced	Enzyme absent
Total phenolic content (TPC)	55.41± 0.6 mg/gm of dried extract

Toxicity study:

The oral administration of Hibiscus sabdariffa mucilage extract demonstrated safety at a dosage of up to 2000 mg/kg of body weight in rat subjects. No toxicological effects or mortality were observed. The doses of 300 and 600 mg/kg body weight were ultimately chosen for the study. The acute oral toxicity assessment of Hibiscus sabdariffa mucilage extract was conducted using Wistar rats as the test subjects. Throughout the 14-day observation period, there were no recorded fatalities or indications of toxicity. Both the vehicle-treated and test sample-treated groups demonstrated standard physiological and behavioural metrics, encompassing respiration, salivation, urination, and somatomotor activity, as well as the condition of fur, skin, and mucous membranes. Throughout the duration of the study, there were no notable adverse effects, including coma, convulsions, tremors, or itching (Table 5). The results indicate that the mucilage extract demonstrates a favourable tolerance profile at the administered dose and may be classified as non-toxic following acute oral exposure. This is consistent with earlier studies that endorse the safety of mucilage-derived plant extracts for applications in pharmaceuticals.

Table 5. Observational parameters for the assessment of acute oral toxicity of Hibiscus sabdariffa mucilage extract in rats over a duration of 14 days.

Parameters

30 min

4 hours

24 hours

48 hours

7 days

14 days

TST

Coma

Convulsant and tremors

Eyes

Faeces consistency

Fur and skin

Itching

Mortality

Mucus membrane

Respiration

Salivation

Sleep

Somatomotor activity and behaviour pattern

Urination (colour)

All parameters were observed at designated time intervals following administration. VT = vehicle that has undergone treatment; TST = test sample that has been treated; A = absence; and N = normal conditions.

Table 6: Body weight of rats recorded during the acute toxicity study of Test sample (TST)

Group	Dose	Body weight (mg/kg body weight)
		Day 0		Day 7		Day 14
		Male	Female	Male	Female	Male	Female
Control	1 ml	164 ± 8.3	158.8 ± 10.3	166.3 ± 2.8	160.2 ± 2.5	167.3 ± 7.8	163 ± 2.5
Treatment (TST)	2000 mg/kg body wt.	166.28 ± 2.6	160.3 ± 93	167.2 ± 5.6	161.4 ± 9.4	168.4 ± 7.6	164.2 ± 2.4

Values are presented as mean ± SEM; n=10. *p ˃0.05 when compared with the vehicle group (Warm distilled water)

The acute oral toxicity assessment of test sample TST) administered at a dosage of 2000mg/kg body weight demonstrated no notable changes in the body weight of male rats throughout the 14-day observation period (Table 6). The body weights observed in all groups, including the control, exhibited a typical physiological increase, with no indications of weight loss or anomalies associated with toxicity. The absence of statistically significant differences (p> 0.05) between the control and treated groups indicates that test sample (TST) is well-tolerated and exhibits non-toxic properties at the administered dose. The results obtained substantiate the safety profile of TST, warranting additional pharmacological assessment.

Antidiabetic activity:

Effects of mucilage on body weight of diabetic rats:

Diabetes mellitus represents a persistent medical condition characterized by increased blood glucose concentrations, frequently resulting in complications, including unintentional weight loss. Plant extracts, characterized by their abundance of bioactive molecules, have been extensively investigated for their potential in combating diabetes. Mucilage, a thick polysaccharide found in various plant species, is recognized for its capacity to modulate glucose absorption and enhance metabolic health. The evaluation of the effect of Hibiscus sabdariffa mucilage extract on weight loss in diabetic rats was conducted, with results presented in Figure 1. The administration of the extract to diabetic rats resulted in a notable alteration in body weight when contrasted with the control group. The treated rats demonstrated stability in body weight, in contrast to the untreated diabetic controls, which exhibited a progressive decline. This indicates the use of mucilage extract could potentially alleviate weight loss associated with diabetes, likely through mechanisms that enhance nutrient absorption or improve insulin sensitivity. Previous research demonstrates that extracts of Hibiscus sabdariffa possess the capability to lower blood glucose levels and enhance lipid metabolism, thereby corroborating these findings. These results necessitate additional exploration into the fundamental mechanisms and the potential for a dose-dependent relationship. The findings highlight the potential of the extract as a supplementary therapeutic agent in the management of diabetes, while also indicating the necessity for further research to confirm these preliminary observations.

Figure 1. Effect of Hibiscus sabdariffamucilage extract body weight in diabetic rats

All data are expressed as mean±SE. Means±SE within the column bearing different superscriptsis significantly different (p < .05).

Impact of Hibiscus sabdariffa mucilage extract on the blood glucose levels in streptozotocin-induced diabetic rats.

The therapy of diabetes mellitus, which is defined by elevated blood glucose levels, progressively integrates plant-derived extracts, including Hibiscus sabdariffa mucilage. As shown in Figure 2, this study looked at how Hibiscus sabdariffa mucilage extract affected the blood sugar levels of rats that had been given streptozotocin to make them diabetic. The treated group demonstrated a notable decrease in blood glucose levels, declining from an initial mean of 350 mg/dL to 245 mg/dL, representing a 30% reduction throughout the experimental duration. Conversely, the control group exhibited stable glucose levels, remaining at about 340 mg/dL, with no significant variation observed. The low blood sugar effect seen suggests that the bioactive compounds in the mucilage may help make insulin work better or stop glucose absorption, which is in line with what other research has found about Hibiscus sabdariffapotential to help people with diabetes. Nevertheless, the precise mechanisms and enduring consequences necessitate additional exploration.

Figure 2. Effect of Hibiscus sabdariffamucilage extract on blood glucose level of streptozotocin-induced diabetic rats

All data are expressed as mean±SE. Means±SE within the column bearing different superscriptsis significantly different (p < 05)

Impact of Hibiscus sabdariffa mucilage extract on lipid profile of diabetic rats:

Dyslipidemia is a common problem in people with diabetes. Higher levels of total cholesterol, triglycerides, and low-density lipoprotein (LDL) and lower levels of high-density lipoprotein (HDL) are the defining characteristics. This makes cardiovascular risk factors worse. Current investigation indicates that plant extracts, including Hibiscus sabdariffa mucilage, may possess the capability to enhance lipid profiles. This study investigated the effects of Hibiscus sabdariffa mucilage extract on lipid levels in diabetic rats. The findings are presented in Table 7. The extract demonstrated a significant reduction in total cholesterol, triglycerides, and LDL levels, while raising HDL in a dose-dependent manner. At a dosage of 600mg/kg, there was a notable reduction in total cholesterol by 18.9%, triglycerides by 27.1%, and LDL by 24.5%, while HDL exhibited an increase of 19.9% in comparison to the diabetic control group. The results indicate that the mucilage extract of Hibiscus sabdariffa could play a role in the management of diabetic dyslipidemia, thereby necessitating additional investigation into its underlying mechanisms and potential clinical applications.

Table 7 Effect of Hibiscus sabdariffamucilage extract on Lipid profile in diabetic rats

Groups	Total cholesterol (mg/dl)	Triglyceride (mg/dl)	LDL (mg/dl)	HDL (mg/dl)
Normal	77.5 ± 2.5	54.2 ± 1.6	44.2 ± 1.8	46.5 ± 2.9
Diabetic control	136 ± 3.17	88.5 ± 2.7	78.5 ± 2.3	31.2 ± 1.2
Test-I (300mg/kg)	125.2 ± 1.1	80.2 ± 1.3	68.2 ± 2.6	34.1 ± 1.3
Test-II (600mg/kg)	110.3 ± 2.6	64.5 ± 2.5	59.3 ± 2.8	37.4 ± 1.81
Standard	82.5 ± 4.15	64.5 ± 4.31	51.2 ± 3.23	43.45 ± 2.4

All data are expressed as mean ± SE. Means ± SE within the column bearing different superscripts is significantly different (p < .05).

Effect of Hibiscus sabdariffa mucilage extract on various biochemical parameters in mice:

As part of a study on acute oral toxicity, the safety profile of Hibiscus sabdariffa mucilage extract was looked into by looking at how it affected rat haematological parameters. The extract was administered at a dosage of 2000mg/kg body weight to both male and female rats, while a control group was provided with distilled water. The findings indicated that there were no statistically significant differences in the haematological parameters assessed among the therapy and control groups, as presented in Table 8. The parameters assessed, such as white blood cell count, red blood cell count, haemoglobin levels, platelet count, and various blood cell indices, exhibited no significant alterations. This finding suggests that the extract does not induce acute haematological toxic effects at the specified dosage. In the study, male rats' haemoglobin levels dropped from 13.5±1.14g/dL in the control group to 12.8±1.56g/dL in the treatment group. This was a decrease of about 5.2% that was not statistically significant. The white blood cell counts in males were found to be consistent, recorded at 6.3±1.02 and 6.3± 1.21 x 10³/µL, indicating no variation. The red blood cell counts in males exhibited a minor increase from 6.8 ±1.07 to 6.9±1.84 x 10⁶/µL, representing a 1.5% change; however, this variation did not reach statistical significance. The observed minimal variations, all remaining within established normal ranges, support the conclusion that there is no acute haematological toxicity. This finding is essential for evaluating the extract's safety in potential applications related to diabetes. The results demonstrate that the mucilage extract of Hibiscus sabdariffa administered at a dosage of 2000 mg/kg is safe in terms of haematological parameters in healthy rats, with no negative effects noted.

DISCUSSION:

Plant-based natural alternatives to synthetic diabetes medicines are still a very interesting area of research, because they work well, have fewer side effects, and are cheaper than synthetic drugs²⁹. A wide range of culinary and medicinal plants have been found to contain bioactive compounds that could help fight type 2 diabetes and its complications³⁰. As a result, many species have been identified as complementary anti-hyperglycaemic agents. Several plant chemicals, such as alkaloids, terpenoids, steroids, saponins, anthranoids, flavonoids, and polyphenols, have been shown to help fight diabetes by working with different targets in different ways³¹. Nonetheless, various other macromolecules have been recognized as the primary anti-diabetic constituents of plants, encompassing proteins, glycans, and mucilage³².

This study examined the antidiabetic efficacy of mucilage derived from Hibiscus sabdariffa leaves in STZ-induced diabetic rats, revealing substantial decreases in fasting blood glucose, enhancements in lipid profiles, and antioxidant activity without toxicity, indicating its promise as a natural therapeutic agent for diabetes management.

The treatment of Hibiscus sabdariffa leaf mucilage at 600mg/kg led to a 30% decrease in fasting blood glucose levels in diabetic rats over 21 days, similar to the impact of metformin. This findingcorresponds with prior studies on Hibiscus sabdariffa, which have indicated its hypoglycaemic attributes, including a comprehensive review and meta-analysis that identified substantial decreases in fasting plasma glucose in clinical trials³³. Nevertheless, the majority of research concentrates on calyx or floral extracts, while our investigation exclusively analysed leaf mucilage. The observed antidiabetic effect may be ascribed to the elevated polysaccharide content in the mucilage, which functions as soluble fibres that impede glucose absorption in the colon, thereby diminishing postprandial glucose surge³⁴. Moreover, the presence of phenolic compounds, recognized for their antioxidant qualities, may improve insulin sensitivity and diminish oxidative stress, aiding glycaemic control³⁵.

Diabetes frequently coexists with dyslipidaemia, marked by increased total cholesterol, triglycerides, and LDL levels, and diminished HDL levels²⁸. Our research showed that treatment with Hibiscus sabdariffa leaf mucilage markedly enhanced the lipid profile in diabetic rats, resulting in decreases of total cholesterol (18.9%), triglycerides (27.1%), and LDL, alongside an elevation in HDL at the higher dosage. The findings align with those documented in prior research on Hibiscus sabdariffa, including a meta-analysis demonstrating substantial decreases in LDL and total cholesterol levels³⁶. The lipid-lowering effects may result from the mucilage's capacity to bind bile acids, thus enhancing their excretion and diminishing cholesterol levels, a mechanism akin to that of other soluble fibres³⁷. Furthermore, the antioxidant characteristics of the phenolic compounds in the mucilage may inhibit lipid peroxidation, hence enhancing lipid profiles³⁸.

In the DPPH test, the mucilage had only moderate antioxidant activity, with an IC₅₀value of 79.12μg/ml compared to 13.60μg/ml for ascorbic acid. Although less powerful than ascorbic acid, this antioxidant ability is significant, given that oxidative stress is pivotal in the etiology of diabetes and its consequences. The antioxidant action is probably attributable to the presence of phenolic chemicals, which can neutralize free radicals and safeguard cells from oxidative damage³⁹. Prior research has documented the antioxidant capabilities of Hibiscus sabdariffa extracts, especially from the calyx, which is abundant in anthocyanins and other polyphenols⁴⁰. Our findings indicate that the leaf mucilage contains substantial quantities of these beneficial chemicals.

The acute toxicity investigation demonstrated no fatalities or negative effects at levels up to 2000mg/kg, suggesting that the mucilage is safe for ingestion. Moreover, haematological indicators were within normal limits, indicating an absence of haematotoxic effects. The safety profile is essential for the prospective development of mucilage as a medicinal agent, as it guarantees that the advantageous benefits are not overshadowed by detrimental side effects.

The phytochemical analysis of the mucilage verified the existence of carbohydrates, polysaccharides, and phenolic compounds. The total phenolic content was 55.41±0.6mg GAE/g, indicating a significant contribution to the observed antioxidant and antidiabetic effects. Polysaccharides, especially mucilage, provide numerous health benefits, including immune-modulating, anti-inflammatory, and hypoglycaemic properties⁴¹. In the realm of diabetes, fibres that dissolve, such as mucilage, can enhance glycaemic regulation by prolonging stomach emptying and diminishing glucose absorption⁴².

Although studies explicitly examining Hibiscus sabdariffa leaf mucilage are scarce, investigation into mucilage from other botanical sources corroborates our conclusions. Mucilage derived from fenugreek seeds has been shown to possess substantial antidiabetic properties in both animal studies and human trials⁴³. Psyllium husk, an important source of soluble fibre that dissolves, is commonly utilized to regulate blood sugar levels in diabetic individuals⁴⁴. The similarities indicate that the mucilage derived from Hibiscus sabdariffa leaves may function via analogous processes.

Research on Hibiscus sabdariffa calyx extracts indicates its potential in combating diabetes through mechanisms such as the inhibition of carbohydrate-degrading enzymes (α-glucosidase and α-amylase)⁴⁵. The leaf mucilage may potentially exert its effects via enzyme inhibition; however, this was not specifically examined in our work⁴⁶.

Notwithstanding the encouraging outcomes, our study possesses certain limitations. The study was first performed on an animal model, and the results may not be immediately applicable to people owing to variations in metabolic processes and physiology. Consequently, clinical trials are essential to validate the efficacy and safety of Hibiscus sabdariffa leaf mucilage in individuals with diabetes. Secondly, the precise mechanisms behind the antidiabetic benefits were not clarified. Subsequent research ought to concentrate on examining the molecular processes implicated, including the effects on insulin signalling, glucose transporters, or gut microbiota. Furthermore, identifying and characterizing the distinct bioactive chemicals within the mucilage may elucidate their unique contributions to the reported benefits.

CONCLUSION:

Diabetes is a chronic metabolic disorder characterized by high blood glucose levels. Hyperglycaemia, or diabetes mellitus, results from an inherent or acquired shortfall in insulin production by the pancreas or from the inefficiency of the insulin produced. Chronic hyperglycaemia in diabetes results in secondary problems impacting the eyes, kidneys, nerves, and arteries. Diabetes is linked to microvascular and macrovascular diseases, which are primary contributors to morbidity and mortality in diabetes mellitus. Diabetes can be controlled by exercise, nutrition, and medication. The pursuit of improved and safer hypoglycaemic drugs is a significant topic of research focus. Our work demonstrates that the mucilage of Hibiscus sabdariffa reduces blood glucose levels in streptozotocin-induced diabetic rats. The mechanism by which mucilage extract of H. sabdariffa mitigates diabetes remains unidentified. Research indicates that traditional medications employed in diabetes exhibit considerable antioxidant activity. The mucilage may potentially diminish the release of free radicals during diabetes, which could contribute to tissue damage and insulin resistance. Treatment with antioxidants and reactive oxygen species scavengers slows the progression of problems and tissue damage in people with diabetes and animals used in experiments. This study examined the mucilage of Hibiscus Sabdariffa for its antioxidant and anti-diabetic properties. It was determined that it possesses antioxidant and anti-diabetic properties, making it a significant source of antioxidants for nutraceutical formulation. The sample presents significant potential for evaluating its antioxidant and free radical scavenging properties in vivo. In conclusion, the findings of this study indicate that the mucilage of Hibiscus sabdariffa restores normal blood glucose levels in diabetes-induced rats, demonstrating hypoglycaemic activity.

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Received on 05.01.2025 Revised on 12.03.2025

Accepted on 19.05.2025 Published on 12.06.2025

Available online from June 14, 2025

Research J. Pharmacy and Technology. 2025;18(6):2686-2696.

DOI: 10.52711/0974-360X.2025.00386

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

Concentration µg/ml	% DPPH radical scavenging
Concentration µg/ml	% inhibition of mucilage	IC₅₀	% inhibition of ascorbic acid	IC₅₀
5	0.18 ± 0.2 %	79.12 ± 0.14	9.01 ± 0.15%	13.76 ± 0.28
10	0.92 ± 0.15%		21.25 ± 0.14%
15	5.12 ± 0.34 %		44.22 ± 0.2%
20	9.94 ± 0.12%		64.17 ± 0.24%
25	13.4 ± 0.12%		71.28 ± 0.21%
30	19.96 ± 0.12%		83.32 ± 0.27%

Biochemical parameters	Control (1 ml in warm distilled water)		Treatment (2000mg/kg body weight)
Biochemical parameters	Male	Female	Male	Female
WBC (X10³/µL)	6.3 ± 1.02	5.7 ± 1.1	6.3 ± 1.21	5.8 ± 1.04
RBC (X10⁶/µL)	6.8 ± 1.07	5.7 ± 0.97	6.9 ± 1.84	5.6 ± 1.03
Hg (g/dL)	13.5 ± 1.14	13.02 ± 1.24	12.8 ± 1.56	12.6 ± 1.06
Neutrophils (%)	54.5 ± 2.1	56.3 ± 2.4	55.01 ± 2.7	54 ± 3.01
Lymphocytes (%)	34 ± 2.8	34.3 ± 2.5	35.2 ± 3.1	35.4 ± 3.5
Eosinophils (%)	4.2 ± 1.2	3.9 ± 1.04	4.1 ± 1.3	3.8 ± 1.6
Monocytes (%)	4.7 ± 2.01	4.3 ± 2.07	4.6 ± 2.1	4.3 ± 2.06
Basophils (%)	0.6 ± 0.6	0.57 ± 0.41	0.52 ± 0.53	0.34 ± 0.62
Platelet count (X10³/µL)	2.8 ± 0.42	2.9 ± 0.41	2.7 ± 0.43	2.7 ± 0.41
MPV (fl)	9.6 ± 0.7	9.6 ± 0.6	9.5 ± 0.6	9.3 ± 0.5
PCV (%)	40.2 ± 2.1	39.8 ± 3.2	39.7 ± 3.4	39.8 ± 3.2
MCV (fl)	86.4 ± 8.2	86.5 ± 9.03	85.6 ± 9.6	85.4 ± 9.7
MCH (pg)	28.74 ± 4.3	27.08 ± 4.02	27.2 ± 4.5	28.03 ± 4.6
MCHC (g/dl)	34.2 ± 1.5	34.03 ± 1.7	34.5 ± 1.04	34.14 ± 1.8