Effects of cassiavera extract-cocoa liquor mixture on blood glucose, inflammation, immune response, and histopathological of pancreas in alloxan-treated Wistar rats

2.1 Preparation of cassiavera extract and CCM

Cassiavera samples were collected from Sumatera Tropical Spices, West Sumatra, Indonesia. After collection of samples, cassiavera samples (200 g) were ground (Wilman, Indonesia) to obtain a cassiavera powder and then sieved through a 40-mesh sieve. The cassiavera powder was macerated with 96% food-grade ethanol (Novalindo, Indonesia) at a powder-to-solvent ratio of 1:5 for 24 h with occasional stirring at room temperature. The residue was filtered through No. 1 Whatman filtration paper. The resulting filtrate was then concentrated under reduced pressure at 40°C using rotary evaporator (Buchi, Switzerland) (Sari et al., 2021). The procedure continued until all of the ethanol had evaporated (with 0% residual solvent found) yielding 45.71 g of thick extract (22.85%), which was later studied in an experimental animal test. Cocoa liquor samples were obtained from Chokato, West Sumatra, Indonesia. The crude extracts of cassiavera were mixed with cocoa liquor at a ratio of 1:1 (w/w). We prepared a primary stock solution (200 mg/mL) by dissolving 1 g of the mixture in 5 mL of sterile distilled water, and then diluted it to a working concentration of 21.6 mg/mL. The final administration doses were calculated based on individual rats body weight, with 1 mL/200 g body weight (equivalent to 108 mg/kg) delivered via oral gavage. Given that this study was an initial exploration of the therapeutic potential of the CCM, the dose of 108 mg/kg was chosen to ensure the appearance of a real biological response.

2.2 Antioxidant activity (IC50)

DPPH was used as the radical indicator in the spectrophotometric method to assess the antioxidant activity to scavenge free radicals. A range of CCM concentrations were tested (2, 4, 6, 8, and 10 ppm). 2 mL of DPPH solution (200 μM) and 1 mL of sample were added to each test tube. For half an hour, the mixture was allowed to sit at room temperature in the dark. The absorbance values at 517 nm were used to calculate the percentage of DPPH radical scavenging activity (Nayak et al., 2024).

2.3 Animals’s acquisition, housing environment, and handling procedures

Therefore, 20 normoglycemic male adults Wistar rats (Rattus norvegicus) weighing 150-200 g aged 2-3 months were obtained from the Central Animal House of Universitas Andalas, Indonesia and acclimated for the period of 7 days before treatment. Rats were housed in polypropylene plastic cages, with rice husk bedding as the floor material, which was changed twice every week in controlled environment (25± 2°C, 12 h light/dark) to maintain cleanliness and keep them away from illness. Standard diet (Citrafeed, RatBio) and fresh water were given to rats ad libitum. This study received approval from the Research Ethics Committee of the Medical Faculty at Universitas Andalas (No:882/UN.16.2/KEP-FK/2023).

2.4 Experimental procedure

The rats were separated randomly into four experimental groups, i.e., K-: negative control; K+: positive control administered a single dose of alloxan monohydrate (Aldrich, Germany) 100 mg/kg body weight, M: diabetic groups that received a single dose of alloxan monohydrate and given metformin dose 45 mg/kg body weight, and CC: diabetic group treatment given CCM at 108 mg/kg body weight for 7 weeks. The metformin dose (45 mg/kg body weight) was chosen based on well-known ways to convert doses for humans to rats and its known high bioavailability in rodent models (Reagan-Shaw et al., 2008). In contrast, the higher CCM dose (108 mg/kg) was required due to the formulation’s naturally derived composition (1:1 cassiavera extract-cocoa liquor), which generally exhibits lower bioavailability compared to synthetic compounds. This dose was established through preliminary in vitro antioxidant assays, which demonstrated that CCM required concentrations approximately 2.4-fold higher than those of metformin to achieved comparable bioactivity.

After seven days of environmental adaptation, the rats were fasted overnight (12–16 hours) before receiving alloxan. Rats were given an intraperitoneal injection of alloxan monohydrate 100 mg/kg body weight which was dissolved in aquabidest as a single dose. Rats were given free access to food and drink after the injection. Thirty-six hours after the alloxan injection, a fasting blood sample was collected from the tip of the rats tail and glycemia was determined (Allmedicus, South Korea). The rats that had a postprandial blood glucose level greater than 200 mg/dL were chosen for treatment and divided into groups randomly. The experiment lasted 7 weeks following the induction of diabetes. The treatment was administered three times a week orally. Animal’s body weight was evaluated on the initial and last days of the experiment.

2.5 Measurement of fasting blood glucose levels and serum preparation

The rats experienced a two-hour fasting period before blood collection. After the rats were fasted, they were subsequently anesthetized by inhaling diethyl ether (Merck, Germany) vapor delivered by ether-saturated cotton swabs until they lost consciousness, and blood was taken out from the orbital sinus vessels using micro haematocrit capillary tubes (Nris, Denmark). Blood droplets were applied to glucose strips to assess blood glucose levels utilizing a glucometer (Allmedicus, South Korea). Approximately ±2 mL of rat blood was collected using a vacuum tube and gel activator. The blood was then centrifuged (Hettich-Centrifuge Benchtop EBA 20, Germany) at 4000 rpm for 10 mins, resulting in blood serum and plasma. Blood serum was pipetted into a microtube and stored at -80 °C (Parasuraman et al., 2010).

2.6 Pro-inflammatory cytokines analysis

Pro-inflammatory cytokines were determined using an enzyme-linked immunosorbent assay (ELISA) using the BT Lab ELISA kit. The standard well was filled with 50 μL of standard solution, and the sample wells were filled with 40 μL of blood serum, followed by anti-IL-6 (10 μL) or anti-TNF-ɑ (μL) antibodies. Streptavidin-HRP (50 μL) was added to both standard and samples wells, mixed and incubated at 37°C for 60 mins (Biosan PST-100HL, Germany). Wash buffer (2-Morpholinoethanesulfonic acid, monohydrate buffer) was used to wash the plate five times. Substrate solutions A and B (50 μL) were added to each well, and the plate was incubated at 37 °C for 10 mins. 50 μL stop solution was added to each well, and the optical density (OD) value was measured at 450 nm absorbance using a microplate reader (XmarkTM BIORAD, California) (Lely et al., 2023).

2.7 Macrophage’s activity and capacity

On the final treatment day, a 1×10⁸ CFU/mL Staphylococcus aureus suspension (1 mL) was infected to the rats and they were left for 60 minutes before being euthanized and dissected. A smear preparation was made on an object glass using a 1 mL sample of peritoneal fluid. The fluid was fixated with methanol, colored with Giemsa stain (Segara Husada, Indonesia), washed with flowing water, and allowed to dry. Giemsa-stained intraperitoneal fluid smears were used to determine phagocytic activity (percentage of macrophages engulfing bacteria) and capacity (mean bacteria per active phagocyte) examined using a 1000x microscope (Olympus CX33, Japan). All procedures followed Azima et al., (2023) modified protocols.

2.8 Histopathological examination

Histopathological examination following modified methods by Ferreira et al., 2010. On the termination day, rats were placed supine on a surgical board, with their limbs pinned for abdominal access. After equipment sterilization with 70% ethanol, a longitudinal skin incision revealed the muscle layer, which was precisely separated to enter the peritoneal cavity. The pancreas was recognized by its anatomical markers (the stomach/spleen/duodenum junction), taken out using micro-dissection equipment to preserve histoarchitecture, washed with physiological solution (NaCl, Merck) to eliminate debris, and right away fixed in 10% buffered formalin (ParaFORM, Indonesia) for structural preservation. Specimens of pancreas were paraffin-embedded, and 3 μm thick sections were stained for regular histopathological evaluation with hematoxylin-eosin (HE). Each sample was examined under a light microscope in 5 fields of view at 10x magnification (Olympus CX33, Japan). The pathologist scored the degree of injury visible under light microscopy in a single-blind style for the animal study group. Endocrine pancreatic damage was evaluated by examining changes in the Langerhans islets, including shape (architecture), inflammatory infiltrate, fibrosis, vacuolization, and intra-islet congestion. Every component received a semiquantitative rating on a score of 0 (< 25% cell damage, normal), 1 (25-50%, mild damage), 2 (50-75%, moderate damage), and 3 (>75%, severe and extensive damage).

2.9 Statistics

The data was analyzed statistically using One-way Variation Analysis (ANOVA) and Duncan’s test at a 5% significance level using SPPS 16.0 version.

3.1 Antioxidant activity (IC50)

The CCM exhibited radical scavenging activity in the DPPH assay (IC50 = 18.85 ± 6.60 ppm, n=3 replicates), which indicates a very strong antioxidant. The IC50 value consists of a scale of very strong (<50 ppm), strong (50-100 ppm), medium (101-150 ppm), and weak (250-500 ppm). Cassiavera has high antioxidant activity as it is rich in bioactive compounds (Menggala & Damme, 2018). The radical scavenging activity of cassiavera extract has an IC50 value of around 31.5-33.3 ppm. It is related to various phenolic compounds, such as catechin, EC, quercitrin, and protocatechuic acid in the cassiavera extract (Muhammad et al., 2021). On the other side, the analysis of cassiavera oil revealed that its main constituents are cinnamaldehyde (68.3%–82%), cinnamyl acetate (2.5%–16%), cinnamyl alcohol (2.25%–4.6%), and cinnamic acid (3%–8%) which acts as an antioxidant (Fajar et al., 2019).

Meanwhile, the IC50 value of cocoa liquor ranged from 33.04-139.01 ppm, indicating that antioxidant capacity varies across cocoa types. Cocoa contains several important components, including procyanidins, EC, and catechin, which are potent antioxidants (Nazario et al., 2014). Many polyphenolic compounds can be found in cocoa, but flavonoids—more specifically, flavanols abundant in the cocoa. Cocoa also contains methylxanthine compounds-theobromine, ranging from 2%-3% by weight, in addition to polyphenols (Katz et al., 2011). The formulation of cassiavera and cocoa significantly enhances antioxidant activity beyond the individual effects of each ingredient.

3.2 Blood glucose

The longitudinal evaluation of glycemic control depicted in Fig. 1 showed significant therapeutic effects of CCM treatment in alloxan-treated diabetic rats. Initial alloxan injection (week 0) resulted in chronic hyperglycemia in untreated diabetic controls (K+ group; p < 0.05 compared to normoglycemic K- controls), establishing the disease model. The CCM-treated group showed rapid therapeutic results, with significant blood glucose reduction occurring during the first week of treatment, an effect that was both sustained throughout the research duration and more noticeable than metformin (M group) during early interventions. Notably, CCM treatment restored and maintained euglycemic levels comparable to healthy controls, but untreated diabetic rats (K+) exhibited chronic hyperglycemia. These findings underline CCM’s advantages in terms of more rapid therapeutic onset and sustained efficacy when compared to standard antidiabetic therapy.

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Fig. 1.

Blood glucose of alloxan-treated rats during treatment.

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In hyperglycemic mice, cinnamon extract significantly reduces blood glucose levels. It may improve insulin action, produce more insulin, or release bound insulin (Begum et al., 2014). Cinnamon’s antioxidants, such as cinnamaldehyde and MHCP (methylhydroxychalcone polymer), contribute to its strong effect on lowering blood glucose (Asmira et al., 2024; Azima et al., 2025; Azzahra et al., 2025). Cao et al., 2010 found that purified cinnamon polyphenols specifically increased insulin receptor β (IRβ) levels, which enhanced insulin sensitivity and elevated glucose transporter 4 (GLUT4), improving glucose uptake. Cinnamon extract markedly lowered blood glucose levels in diabetic rats by modulating essential insulin signaling genes with tissue-specific effects; it affected protein tyrosine phosphatase 1B (PTP-1B), insulin receptor substrate 1 (IRS-1), protein kinase B (PKB), phosphoinositide-dependent kinase (PDK1), and phosphoinositide 3-kinase (PI3K) in muscle, and predominantly PTP-1B, protein kinase c theta (PKCθ), and IRS-1 in adipose tissue (Eijaz et al., 2014).

Cocoa and its major flavanols may help prevent or delay T2DM by regulating insulin release, improving lipid metabolism, and increasing glucose uptake. It also has protective effects against oxidative and inflammatory damages linked to the illness and helps enhance glycemic control and insulin response (Martin et al., 2016). Cocoa liquor procyanidins decrease glucose levels through their ability to increase the secretion of glucagon-like peptide-1 (GLP-1) and activate AMP-activated protein kinase (AMPK), which promotes GLUT4 translocation in skeletal muscle and prevents postprandial hyperglycemia (Yamashita et al., 2019). Cocoa polyphenol extract and the essential cocoa flavanol, (-)- EC, improved insulin sensitivity of liver cells (HepG2), by suppressing the levels of relieving and tyrosine-phosphorylated insulin receptor (IR), IRS-1, and IRS-2 owing to high glucose levels (Cordero-Herrera et al., 2013). Bowser et al., 2017 also reported that cocoa procyanidin extracts had an insulinomimetic effect in human main skeletal muscle cells. The synergistic effect of this plant combination is a result of the complementary actions of bioactive compounds in both ingredients, which collectively improve insulin sensitivity and alleviate DM-related metabolic dysregulation. Notably, the selected CCM dose demonstrated an excellent safety profile in the 7-week treatment period, with no observed behavioral or toxicity effects. Further studies are urgently needed to evaluate the minimum effective dose of CCM and compare it with metformin as a positive control.

3.3 Body weight

Analysis of body weight patterns revealed significant metabolic differences among treatment groups (Fig. 2). The untreated diabetic control group (K+) experienced significant weight loss compared to all other groups (p < 0.05), which reflects the catabolic state caused by persistent hyperglycemia. Rats treated with metformin (Group M) exhibited an intermediate weight loss, which was significantly greater than that of both the healthy control group (K-) and the CC intervention group (p < 0.05). Interestingly, the combination of cassiavera extract and cocoa liquor (CC) totally prevented diabetes-related weight loss. Final body weights were statistically indistinguishable from those of healthy controls (K-, p > 0.05). Given these results, tthe CC formulation is more effective at maintaining metabolic homeostasis under diabetic states.

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Fig. 2.

Body weight of alloxan-treated rats during treatment.

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In hyperglycemia, insulin deficiency or resistance impairs glucose uptake by cells. The body then uses alternative energy sources, breaking down fat (lipolysis) and muscle protein (proteolysis). This catabolic state causes muscle and fat loss, leading to weight loss (Saltiel & Kahn, 2001). Studies on diabetic rats highlight the beneficial impact of the cassiavera extract intake on body weight management. Cassiavera extract administration is associated with controlled body weight and reduced adiposity index, primarily through enhanced glucose utilization and reduced intestinal glucose absorption (Mohsin et al., 2023). Eijaz et al., (2014) found that cinnamon extract acted as an insulin sensitizer, increasing cellular glucose uptake via AMPK-mediated GLUT4 translocation in skeletal muscle and adipocytes, significantly increasing the body weights of diabetic rats. Increasing body weight means that as glucose uptake normalizes, the body begins to use glucose rather than fats.

Moreover, cocoa is known to normalize the body weight in a diabetic animal model. Alloxan-induced diabetic rats with a cocoa diet for 3 weeks helped to reduce weight loss. Cocoa, rich in polyphenols, helped stabilize body weight (Olasope et al., 2016). This observation is in line with Ruzaidi et al., (2008), who found that cocoa extract could normalize the rats’ weight loss caused by streptozotocin (STZ) within 4 weeks. Fidaleo et al., (2014) discovered that cocoa supplementation in high-fat diet (HFD)-fed animals normalized body weight to levels comparable to chow-fed controls after 4 weeks of treatment. This beneficial effect led to significant improvements in glucose homeostasis, implying that cocoa’s ability to enhance insulin-mediated glucose uptake may prevent the metabolic dysregulation commonly seen in diabetogenic conditions.

3.4 Pro-inflammatory cytokines

The analysis of pro-inflammatory markers showed different patterns in the groups that were studied (see Fig. 3). The untreated diabetic group (K+) demonstrated notable pro-inflammatory activation compared to the baseline, with a significant increase in both ΔTNF-α (89.12%) and ΔIL-6 (45.45%) (both p < 0.05). This finding aligns with the inflammation caused by hyperglycemia. The anti-inflammatory efficacy of both therapeutic interventions (M and CC) was demonstrated by their ability to significantly attenuate these responses (p < 0.05 compared to K+). The normal control group (K-) exhibited low ΔTNF-α and ΔIL-6 levels (-5.38% and -11.41%), which indicated physiological homeostasis.

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Fig. 3.

Inflammatory level of alloxan-treated rats (TNF-ɑ and IL-6).

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Increasing inflammation, associated with elevated TNF-α and IL-6 levels, is linked to obesity, hyperglycemia, and the development of T2DM. Newly diagnosed T2DM patients show significantly higher TNF-α levels, underscoring their role in the disease’s pathophysiology. Furthermore, the rise in IL-6 levels in the diabetic rat group suggests its influence on glucose metabolism and homeostasis across various tissues, indicating a comprehensive impact on T2DM pathology (Rehman et al., 2017) (Majeed et al., 2022). CCM administration significantly reduced inflammation throughout the body in alloxan-induced diabetic rats. Numerous studies reported the anti-inflammatory properties of cassiavera. Khatib et al., (2005) found that C. burmannii is the most potent anti-inflammatory agent among 20 Indonesian medicinal herbs. The ethyl acetate fraction yields 2-hydroxy-cinnamaldehyde, which inhibits lipoxygenase (IC50 = 60 μM). Cassiavera extract was shown to downregulate multiple pro-inflammatory cytokines, including TNF-α, IL-1β, COX-1, and IL-6, when given to rats orally. This was accomplished by decreasing the rate of production or gene expression of these cytokines in the body (HelmyAbdou et al., 2019). Cao et al. (2010) reported that aqueous cassiavera extract (100 μg/mL) treatment in mouse adipocytes increased tristetraprolin (TTP) mRNA expression six-fold, indicating anti-inflammatory potential through TTP-mediated degradation of pro-inflammatory cytokine mRNAs (e.g., TNF-α). Furthermore, cinnamaldehyde inhibits NF-κB by reducing IKappaB Kinase (IKK) phosphorylation, preventing NF-κB nuclear translocation, and subsequently decreasing TNF-α, IL-1β, and IL-6 levels (Almoiliqy et al., 2020).

Additionally, consuming cocoa and catechins is associated with a reduction in insulin resistance with a decrease in pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β) by modulating cytokine production through NF-κB and MAPK pathways (Ellinger & Stehle, 2016; Mao et al., 2002). Cocoa flavonoids neutralize reactive oxygen species (ROS), thereby reducing oxidative stress, which inhibits NF-κB activation and pro-inflammatory cytokine production (Ramiro-Puig & Castell, 2009). Cocoa polyphenols also reduced inflammation by inhibiting NACHT, LRR, and PYD domains-containing protein 3 (NLRP3) activation through opioid and γ-aminobutyric acid (GABA)ergic pathways. Furthermore, they regulated NO signaling through iNOS downregulation, thereby reducing pro-inflammatory cytokine production and oxidative stress (De Feo et al., 2020). CCM treatment effectively reduced inflammation markers in alloxan-treated rats, alleviating diabetes-related inflammatory responses.

3.5 Macrophage’s activity and capacity

Macrophage functional analysis revealed marked treatment effects throughout the experimental groups (Fig. 4). Notably, the CC treatment group exhibited significantly enhanced macrophage activity (82 ± 2.12%) and phagocytic capacity (235 ± 9.19 cells) (p < 0.05 compared to K+). Conversely, untreated diabetic rats (K+) exhibited considerably impaired macrophage function, demonstrating the lowest observed values that differed significantly from both healthy controls and treated groups (p < 0.05). The impaired macrophage activity observed in the K+ group possibly reflects the immunosuppressive effects of chronic hyperglycemia. Thus, the advantageous results in the CC group suggest the immunomodulatory benefits of the combined treatment. Phagocytosis was impaired due to exposure to a bacterial infection. High glucose levels increase the susceptibility of macrophages to cytokine stimulation, thereby reducing their ability to produce nitric oxide and phagocytose. This may be due to decreased glycolytic capacity (Sousa et al., 2023).

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Fig. 4.

Alloxan-treated rats’ activity and capacity of macrophage.

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Cassiavera enhances immune system stimulation by improving macrophage function and activity and boosting the immune response, especially macrophage activation, which results in high phagocytic efficiency for a robust immune response against various foreign agents (Kang et al., 2014). Kim et al. (2018) demonstrated that trans-cinnamaldehyde (TCA) promoted the polarization of macrophages from a pro-inflammatory (M1) state to an anti-inflammatory (M2) state. TCA inhibits M1 polarization in lipopolysaccharide (LPS)-stimulated macrophages by blocking NF-κB signaling. This reduces inflammatory mediators such as iNOS, TNF-α, IL-6, and IL-1β. TCA also promotes M2-associated markers (Arg1, CD206), improving tissue repair.

Low catechin intake improves the nonspecific immune response. Even a tiny amount of cocoa extract effectively increases both phagocytic capacity and macrophage activity (Sari et al., 2016). Cocoa’s flavonoids contribute to regulating immune cells (Ramiro-Puig & Castell, 2009). Cocoa polyphenols reduce pro-inflammatory M1 macrophage activity by reducing cytokines like TNF-α and IL-6 and increasing anti-inflammatory IL-10, promoting a shift toward the M2 phenotype. It also improves oxidative metabolism, increasing mitochondrial respiration and ATP production, which aids in anti-inflammatory and tissue repair functions (Dugo et al., 2017).

3.6 Histopathological examination

Quantitative assessment of Langerhans islet area and cellular damage among experimental groups has been presented in Table 1. Histopathological examination revealed that diabetic untreated rats (K+ group) resulted in significant islet damage (p < 0.05), including atrophy, reduced islet size, and cellular degeneration, as evidenced by shrunken cells and pyknotic nuclei (Fig. 5). CCM-treated diabetic rats (CC) showed preserved islet morphology, including restored size and reduced necrosis, similar to metformin’s protective effects (M) (p > 0.05). The normal control (K- group) had normal islet architecture, confirming the baseline structural integrity.

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Fig. 5.

The histopathology of the pancreas of experimental animals shows the exocrine (E) and the endocrine cells of the islets of Langerhans (P). (Staining: Hematoxylin-eosin). Original objective magnification 40x. Scale 100 µm. The endocrine cells exhibited signs of degeneration and necrosis, characterized by cells with shrinking pyknotic nuclei (↓), as well as cells that lysis (▼).

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Oxidative stress has been linked to an increase in β-cell apoptosis, which contributes to the gradual loss of β-cell mass in the prediabetic state. Overproduction of ROS activates multiple signaling cascades involved in β-cell apoptosis (Drews et al., 2010). The improvement in pancreatic islet cells in the group of rats given a CCM is related to the active compounds in both plant components. Several studies have shown that cinnamaldehyde has protective effects against STZ-induced β-cell injury. Cinnamaldehyde has strong antioxidant properties that contribute to the neutralization of ROS and the protection of pancreatic β-cells from STZ-induced damage (Yuan et al., 2011). Flavanol compounds in cocoa have been shown to protect Langerhans islet cells; dietary cocoa prevents β-cell apoptosis by reducing oxidative stress, increases the size of small islets, and maintains the total islet mass, indicating β-cell proliferation (Basu et al., 2015). Fernández-Millán et al. (2015) also reported that a cocoa-rich diet in Zucker Diabetic Fatty (ZDF) rats preserved pancreatic islet architecture, significantly increased β-cell area and insulin content, and shifted islet size distribution toward smaller islets (<1000 μm²), indicating enhanced islet neogenesis and functional β-cell mass maintenance to counteract insulin resistance. This protective effect was linked to increased anti-apoptotic Bcl-xL protein levels, indicating cocoa’s role in preventing β-cell apoptosis, which is consistent with findings on flavanol EC in diabetic models. Cocoa-diet also increased pancreatic antioxidant defenses, particularly GPx, which reduced oxidative stress-mediated beta-cell damage and improved survival in diabetic condition.