INTRODUCTION:

DM is a degenerative disease caused by metabolic syndrome, characterized by elevated blood glucose levels. This condition arises due to insufficient insulin production, reduced responsiveness of body cells to existing insulin, or a combination of both factors. In Indonesia, DM ranks fourth in prevalence after the United States, China, and India¹.

In long-term cases, diabetes mellitus (DM) can lead to serious complications impacting multiple organs, such as the eyes (retinopathy), kidneys (nephropathy), nerves (neuropathy), and the cardiovascular system^2,3. Conventional management of DM involves adopting a healthy lifestyle and using oral hyperglycemic agents (OHA) and insulin injections. The prolonged use of OHA and insulin may potentially lead to side effects ^4,5,6. Certain medicinal plants can serve as alternative sources in the search for new treatments for DM.

Indonesia is one of the countries abundant in natural resources, estimated to have around 30.000 species of medicinal plants that have been utilized in traditional remedies, with 16,218 species successfully identified⁷. Several plants have demonstrated efficacy as blood glucose-lowering agents, supported by both empirical and scientific evidence, such as Mulberry (M. alba L.) and Cinnamon (C. burmannii Nees and T.Nees).

M. alba L., locally known as murbei and belonging to the Moraceae family, has been used both as an edible plant and for treating various diseases. Ethnopharmacological studies demonstrated that various parts of the Mulberry plant, including its fruits, leaves, branches, and roots, have been utilized by communities to address high blood glucose levels⁸. A study on Mulberry leaf powder and the isolated flavonol glycoside compound (quercetin 3-(6-malonyl glucoside)) exhibited their capability to reduce blood glucose levels. This was achieved through the activation of enzymes involved in glycolysis and the suppression of thiobarbituric acid-reactive substances in the liver of a diabetic mouse model^9,10. Ethanol extract of Mulberry leaves can ameliorate hyperglycemia, and insulin resistance, hyperlipidemia, and enhance the function of Langerhans beta cells. This is attributed to its capacity to induce autophagy in Langerhans beta cells in both DM rat models and in vitro studies¹¹. Asano (2001)¹² successfully isolated 18 types of polyhydroxylated alkaloid compounds that exhibit alpha-glucosidase enzyme inhibition activity, with the alkaloid 1-Deoxynojirimycin (DNJ) being the major compound with the highest activity. These active compounds, particularly the alpha-glucosidase inhibitor content, contribute to Mulberry's glucose-lowering property in both in vivo and clinical evidence.

Cinnamon, belonging to the Lauraceae family, has been used by people for health maintenance. Previous studies indicated that cinnamon was utilized for its hypoglycemic, anti-inflammatory, anti-lipidemic, and antioxidant properties¹³. The water extract of cinnamon decreased the expression of two key genes involved in liver gluconeogenesis, namely glucose-6-phosphatase and phosphoenolpyruvate carboxykinase, thus improving the condition of type 2 DM mice¹⁴. The primary compound in cinnamon, methylhydroxychalcone polymer, can modulate glucose levels on 3T3-L1 adipose cells. This compound interacts with insulin receptors and stimulate a series of processes that enhance glucose uptake in adipose cells. Moreover, it was reported that the combination of this polymer with insulin provided an additive effect¹⁵.

A polyherbal preparation is a combination of various plant-derived extracts used for treating various of disorders. These combinations are designed to achieve enhanced potency through synergistic interactions between the individual components, rather than relying on single herbal treatments. The benefit of these herbal mixtures is that they allow for lower doses of each individual herb, thereby reducing the risk of dose-related side effects while maintaining the same bio-effective dose as a single herb^16,17. This study was conducted to investigate the blood glucose-lowering activity of a combination of M. alba L. leaf and C. burmannii bark extract in the DM animal model. The effectiveness of the combined extract was conducted in vivo using DM animal models. The DM animal can be generated, one of which was by inducting using the combination of a high-fat diet and low-dose STZ. This high-fat-STZ-induced DM rat was characterized by hyperglycemia associated with hypertriglyceridemia and mimicked many other alterations resembling human DM type 2 symptoms¹⁸.

MATERIALS AND METHODS:

Sample preparation:

Mulberry leaves were collected from the BRIN plantation in Lampung, South Sumatera, Indonesia. The leaves were dried in an oven at 50⁰C and then powdered. One kg of the powdered leaves was extracted using 5 L of ethanol with agitation at room temperature for 16h, and then the filtrate was separated. Filtrate was evaporated using a vacuum evaporator until a semisolid mass was obtained. The dried bark cinnamon bought from the local market located in Serpong, South Tangerang, Banten Indonesia was powdered, and then 1 kg of the bark powder was extracted with 5 L of ethanol. The cinnamon filtrate was treated in the same way as the mulberry leaves extract. Formula extract was prepared with homogenous mixing in the proportionated combination of these two semisolid extracts. The formula extract was kept in the dark glass bottle for the next experiment.

Formula extract determination:

Extract characterization was conducted by phytochemical screening to identify the compound groups, namely, flavonoid, alkaloid, saponin, tannin, quinone, steroid, terpenoid, essential oil, and coumarin compounds, as described by a previous study¹⁹. Subsequently, the formula extract quality was also determined based on the previous source²⁰ by measuring some parameters, such as water and ethanol soluble content, drying loss, water content, total ash, lead and microbial contamination. The level of total phenolic compounds in the formula extract was also determined based on the previous study²¹.

Animal experiment:

Male rats of the Sprague Dawley strain utilized in this experiment were bought from BPOM (Indonesia FDA) with an age of 6-8 months. Animals were kept in the polycarbonate cage, 4 rats per cage, with normal food and tap water ad-libitum. The environment was under control with a temperature of 23±2⁰C, humidity 50-70%, and 12/12 cycle dark/light. After 7 d of acclimatization, the healthy animals were maintained on a high-fat diet (HFD, a combination of egg yolk, fructose, and lard) for 50 d, followed by STZ induction (30mg/kg bw i.p. twice) with a 7-d difference between treatments. STZ was prepared in a cold citric buffer at pH 4.5 before being injected. Seven days after the last STZ induction, the animal was fasted overnight but kept on watering, and blood was collected from the sinus orbitalis vein. Plasma EDTA was separated using cold centrifugation (4⁰C, 5000rpm, 10min). The glucose level was measured using the reagent kit GOD-PAP (Diasys^®). Animals with blood glucose levels greater than 200mg/dL were used for the next experiment and grouped randomly into 5 groups with 4 rats per group. Groups 1, 2, and 3 were administered formula extract at doses of 120, 250, and 500mg/kg bw. Group 4 was the control group treated with the oral diabetic standard drug, metformin, and Group 5, as the model group, was treated with the carrier (CMC 0.5% solution). Formula extract and metformin were suspended in a CMC 0.5% solution. All treatments were conducted for 21 days peroral, and the animals were fed with HFD as long as the period of experiment. The blood glucose level was then determined at days 4, 7, and 21 after treatment. The normal group was prepared by using normal rats without HFD treatment and induced citric buffer i.p. Animal experiments had been approved by the Ethics Committee of the Faculty of Medicine, University of Indonesia (Number: Ket-193/UN2.F1/ETIK/2017).

Data analysis:

Data were served as mean±SD. Statistical analysis was carried out with the ANOVA method and followed by LSD for determination of the specific differences between groups (for parametric data) and for non-parametric data, were with Kruskal Wallis followed by the Mann Whitney method. The statistically significant difference between groups was set at P < 0.05.

RESULTS:

Formula extract characterization:

The phytochemical screening indicated that the formula extract contained alkaloid, flavonoid, saponin, tannin, coumarin, and steroid compound groups. Subsequently, evaluation of formula extract quality was exhibited in Table 1. All the tested parameters demonstrated that the quality of formula extract fulfilled the guidance standard of the regulator (Indonesian Food and Drug Authority, BPOM). Evaluation of the loss on drying of the formula extract is one of the requirements that must be met in the quality test of medicinal plants. This parameter indicated the remaining water and volatile ingredients in the formula extract, which were 9.82%. The assessment of water content aimed to quantify the amount of water present in the formula extract, yielding a value of 2.31%. Maintaining an appropriate water content is crucial for preserving the quality of the extract and preventing microbial growth. A lower water content in the extract corresponds to a reduced risk of microbial growth, fungi, and damage inflicted by microorganisms. The determination of total ash and acid insoluble content was intended to provide the impurities of the internal and external minerals originated from the raw material and at the extraction process until the extract is produced. The total ash and acid insoluble content are 7.41 and 0.33%, respectively. The determination of the levels of heavy metal content (Pb and Cd) and microbial contaminants was to provide assurance that the extract was safe due to the heavy metals and microorganisms that were more than a certain level that were harmful (toxic) for health. Furthermore, quantitative analysis of the total phenolic content of the formula extract indicated that this value was 1.39±0.07%.

Table 1. The quality of the formula extract

No.	Parameter tested	Results	Requirement
1	Water soluble content	53.58%	-
2	Ethanol soluble content	37.85%	-
3	Drying loss	9.82%	-
4	Water content	2.31%	< 10.00%
5	Total ash content	7.41%	-
6	Insoluble acid ash content	0.33%	-
7	Solvent residue	0.02%	< 1.00%
8	Lead contamination - Pb - Cd	1.1 mg/kg 0.0028 mg/kg	< 10 mg/kg < 0.3 mg/kg
9	Microbial contamination - Total plate count. - Mold and yeast count.	1.72 x 10² < 10	< 10⁴colony/g < 10³colony/g

Noted: * based on (BPOM, 2014)

The total phenolic content of the formula extract was 1.39 ±0.07%.

Animal experiment:

The hypoglycemic activity of formula extract was evaluated on the combined HFD-STZ low dose-induced DM rat. The rat was stated as DM when the fasting blood glucose level was more than 200 mg/dL. Treatment HFD for 50 days increased animal’s body weight gain compared to the normal group (Figure 1).

Figure 1. Body weight change of all groups during the experiment. W-0: the early body weight of animals before HFD treatment. W0: body weight of animals before sample treatment. N = 4 rats.

Treatment formula extract on DM rats was carried out per orally for a period of experiment 21 d. Blood glucose level was determined periodically at the day of 4^th, 7^th, and 21^st after formula extract treatment. Generally, all dose of formula extract exhibited the glucose lowering activity, especially at the 4^th and 7^th measurements, significantly (P < 0.05) compared to the Model group. However, at day 21 after treatment, it was shown that animals that received the Dose2 of formula extract demonstrated rise of blood glucose levels in which it did no differ significantly (P > 0.05) compared to the Model group. It was exhibited that the glycose lowering activity of formula extract was not in dose dependent manner. Meanwhile, the metformin as a positive control showed the ability to lower blood glucose in the duration of experiment significantly compared to the model Group (P < 0.05) (Figure 2).

Figure 2. Fasting blood glucose level after sample treatment. Value: average±SD. n = 4 rats. * significantly difference from normal (P < 0.05). # significantly difference from model (P <0.05).

DISCUSSION:

The utilization of medicinal plant resources as agents for health has gained great attention, especially when challenged with the side effects of conventional medicine. The study revealed that the biological activities of plant-derived extracts were contributed by phytochemical secondary metabolites, including alkaloids, phenols, saponins, carbohydrates, terpenoids, steroids, flavonoids, and volatile oils. Interaction between compound groups presents synergistic, cumulative, and additive properties that bore out the beneficial biology effect and improved the pharmacological bioavailability. Due to the sometimes-unpredictable interactions among these compounds, investigating their effectiveness through in vitro, in vivo, and clinical investigations becomes a crucial prerequisite for establishing them as medicinal agents ^16,22. This research aimed to scientifically demonstrate the effects of a proportioned mixture of mulberry and cinnamon extract (formula extract) on reducing blood glucose levels in a DM animal model.

The phytochemical evaluation of these studies revealed that the formula extract contains groups of compounds such as alkaloids, flavonoids, saponins, tannins, coumarins, and steroid compounds, while coumarin and essential oil compound groups were not detected. Furthermore, the quantitative determination of the total phenolic compounds in the formula extract showed a value of 1.39±0.07%. These compounds were contributed to the biological activity²². Studies showed that the plant-derived polyphenol compounds exhibited pharmacological benefits in both controlling homeostatic disorder or stress response through some mechanisms, such as radical scavenging and anti-oxidant activities, modulating inflammation, and inhibiting human enzymes^22,23. The next quality evaluation of formula extract showed that it met the quality requirements of the standard extract according to BPOM guideline.

Pharmacodynamic activity as a lowering of blood glucose of the formula extract was conducted on diabetic rats induced with the combination of HFD and STZ. A study conducted by Barrière and team (2018)²⁴ demonstrated that the HF/STZ-induced diabetic rat produced DM animals with progression resembling various stages of human DM type 2 during a 56-week observation. In the initial prediabetic phase, it was marked by elevated insulin levels and slight dysglycemia. This is followed by an advanced T2DM phase featuring significant hyperglycemia, normalized insulin levels, severe dyslipidemia, hepatic fibrosis, and failure of pancreatic beta cells. Our experiment was in accordance with the previous study. Administering rats with a high-fat diet for a duration of 50 days and a low dose of STZ (30mg/kg body weight, twice i.p.) resulted in hyperglycemic rats with fasting blood glucose levels of more than 200mg/kg bw. This value was significantly different from the normal group (P<0.05) (Figure 2).

Afterward, the pharmacodynamic evaluation of formula extract on DM rats indicated that the extract administered at three different dosage levels was generally capable of reducing fasting blood glucose levels. The blood glucose levels measured on the 4th and 7th days exhibited a notable decrease compared to the Model group (P <0.05). However, at the 21-d post-treatment, group that received dose 2 demonstrated the increase of blood glucose that was significantly not difference from the model group (P > 0.05). This was probably due to be interactions among the chemical compounds present in the formula extract. It is mentioned that the formula extract was composed from a proportionated combination of plant-derived extract that was containing complex and intricate chemical substances.

Medicinal plant-derived extracts comprise numerous types of compounds, each present in different quantities. The pharmacological effectiveness of these extracts was a result of the interaction that arises between these compounds, displaying synergistic, additive, or antagonistic properties. In several cases, the activity of plant extracts sometimes delivered a better impact than that of an isolated compound at the equivalent level. Therefore, a thorough assessment of synergistic effects within medicinal plant extracts requires meticulous analytical methods and validation through scientific research ²². Our results demonstrated that the proper dosing of formula extract was needed to produce the proper hypoglycemic effect. This demonstrates that synergistic, antagonistic, or agonistic interactions between compounds is highly influenced by the concentration of the compound used in the experiment.

Previous studies have proven that mulberry exhibited hypoglycemic activity. The diabetic activity observed was attributed to the abundance of bioactive compounds, particularly flavonoids, alkaloids, polysaccharides, and polyphenols, which exhibit a strong correlation with diabetes management. The mechanisms underlying blood glucose reduction include inhibiting alpha-glucosidase activity in the intestine, regulating lipid metabolism in liver, protecting beta cells of pancreas, repairing insulin resistance, modulating glucose uptake by target tissues, and improving oxidative stress levels ^10,25.

Polyphenols present in mulberry leaves have been reported to lower blood glucose levels in high-fat diet-induced diabetic Wistar rats ²⁴. Following a 4-week treatment period, the polyphenol-rich extract was found to significantly reduce blood glucose levels, decrease serum triglyceride and total cholesterol levels, lower serum urea and creatinine levels, and increase serum HDL-cholesterol levels. In addition to the mulberry compound, the ethanolic crude extract of mulberry leaves also demonstrated hypoglycemic activity after being administered for 11 weeks to DM rats. This ethanolic extract improved insulin resistance and islet function, reduced islet damage, and managed dyslipidemia in the DM model²⁵. A clinical study revealed that a single oral intake of 0.8 and 1.2 g of DNJ-enriched leaf powder effectively reduced the increase in postprandial blood glucose levels and insulin secretion. DNJ, known as 1-deoxynojirimycin, was an alkaloid group and the major compound in mulberry²⁶. The study mentioned above indicates that mulberry leaves exhibit noteworthy effects in managing diabetes when utilized in their natural form, as extracted materials, and through their active compounds¹⁰. It had been proven that cinnamon exhibited an effective blood lowering agent. The source stated that the dried bark of cinnamon contains a significant number of polyphenols and has been employed to enhance health, including DM ^27.

It was reported that the major active components of C. zeylanicum (C. burmannii) are cinnamaldehyde, cinnamyl acetate, beta-caryophyllene, alpha-terpineol, eugenol, and proanthocyanidin cinnammtannin B1. The antidiabetic mechanism of cinnamon was, one of which, by inhibition of intestinal enzymes such as alpha-amylase and alpha-glucosidase activities, which hindered carbohydrate digestion and absorption²⁸. Administration of cinnamaldehyde, the major compound found in cinnamon, demonstrated the ability to reduce the activity of phosphoenolpyruvate carboxykinase (PEPCK) and normalize PEPCK messenger RNA (mRNA) levels in the liver and kidney. Additionally, this compound increases the activity of the glycolytic liver enzyme pyruvate kinase²⁹. A clinical study also claimed that cinnamon demonstrated a blood glucose lowering effect, especially in DM type 2 patients, with overwhelming scientific proof²⁷.

Based on the findings of this research, it can be concluded that the formula extract, consisting of a proportional combination of mulberry and cinnamon extracts, has been successfully developed. This formula extract exhibited hypoglycemic activity in the HFD-STZ-induced DM model animal. The hypoglycemic effect of the formula was observed in a non-dose dependent manner, with the lowest effective dose being 125mg/kg bw. This dose is lower than the doses needed for hypoglycemic effects in diabetic animal models from single extracts found in previous studies, which were 600 mg/kg for mulberry leaves ³⁰ and 200 mg/kg bw for cinnamon bark ethanolic extract³¹, respectively. It is likely that a synergistic effect exists between the compounds in the formula extract. However, further exploration is needed to confirm this results.

CONCLUSIONS:

From these researches, we obtained the proportionate combination of mulberry and cinnamon extract that was termed with formula extract. Phytochemical screening demonstrated that this extract contained alkaloid, flavonoid, saponin, tannin, coumarin, and steroid compound groups, and the quantitative analysis of the total phenolic level was 1.39±0.07%. The pharmacodynamics study on the FHD-STZ-induced diabetic rats demonstrated that Formula Extract exhibited the lowering blood glucose with non-dose dependent properties. It was found that the smallest effective dose of formula extract as hypoglycemic was 125mg/kg bw rat.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

This work was supported by a research collaboration between the National Research and Innovation Agency (BRIN) Indonesia and the Korea Research Institute of Bioscience and Biotechnology (KRIBB).

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Received on 27.05.2024 Revised on 24.10.2024

Accepted on 31.01.2025 Published on 01.07.2025

Available online from July 05, 2025

Research J. Pharmacy and Technology. 2025;18(7):3127-3132.

DOI: 10.52711/0974-360X.2025.00449

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