In Vitro, Molecular Docking, and Meta-analysis Studies of Screening Antidiabetic Bioactive Compounds
from Roselle (Hibiscus sabdariffa Linn.)
Ira Erdiandini1, Baso Manguntungi2*, Apon Zaenal Mustopa3, Lita Meilina3, Leggina Rezzy Vanggy4, Ahmad5, Handoko6, Kharmila Rahmadani7,
Fahri Sinulingga8, Haslinda DS9, La Ode Fitradiansyah10, Andi Masniawati11, and Heru Pitria Hastuti12
1Department of Agrotechnology, Universitas Tanjungpura, Pontianak 78124 Indonesia
2Department of Biology Education, Universitas Sulawesi Barat, Majene 91412 Indonesia
3Research Center for Biotechnology, National Research and Innovation Agency, Indonesia
4Department of Research, Innovation, and Development, Sumbawa Technopark, Sumbawa 84316 Indonesia
5Department of Food Science and Technology, IPB University, Bogor 16680 Indonesia
6Department of Physics, IPB University, Bogor 16680 Indonesia
7Department of Biotechnology, Sumbawa University of Technology, Sumbawa 84371 Indonesia
8Department of Aquatic Product Technology, IPB University, Bogor 16680 Indonesia
9Department of Anatomy, Physiology, and Pharmacology, IPB University, Bogor 16680 Indonesia
10Animal Bioscience Department, IPB University, Bogor 16680 Indonesia
11Department of Biology, Hasanuddin University, Makassar 90245 Indonesia
¹²National Research and Innovation Agency, Indonesia
Diabetes mellitus is a chronic metabolic disorder due to insulin function insufficiency. This study aimed to test the effectiveness of flower extract of roselle (Hibiscus sabdariffa L.) extract in producing antidiabetic compounds. The inhibition of roselle flower extract on the alpha- glucosidase enzyme was carried out in vitro. Molecular docking was also carried out to bind ligands derived from roselle flower extract's secondary metabolites to the alpha-glucosidase and alpha-amylase enzymes. Based on molecular docking, models have negative binding energies suggesting those ligands make a complex to the site receptor. Kaemferol-3-O-rutinoside and tiliroside become the most stable complex based on the lowest energy score of –9.5 and –8.1 kcal/mol for alpha-amylase and alpha-glucosidase, respectively. The highest antidiabetic activity was obtained at a 100 ppm roselle flower ethanol extract and distilled water with an inhibition value of 100.00 and 99.25%, respectively. The alpha-amylase inhibiting test, using a concentration of 2.5 mg/mL, had an inhibitory activity of 41.77%. The in vitro assessment was conducted using the meta-analysis. The meta-analysis study showed that roselle flower extract could reduce glucose levels in fasting rats better than negative controls (diabetic rats) by 61%
than those not given the roselle flower extract.
Keywords: antidiabetic, in vitro, meta-analysis, molecular docking, roselle
*Corresponding author: manguntungibaso@unsulbar.ac.id;
ISSN 0031 - 7683
Date Received: 31 Mar 2022
INTRODUCTION
Diabetes mellitus is a chronic metabolic disorder due to insulin resistance caused by complex genetic and environmental factors (Alam et al. 2021). Diabetes mellitus is characterized by hyperglycemia and glucose intolerance associated with impaired insulin secretion, peripheral sensitivity, and ultimately cell dysfunction (Ohiagu et al. 2021). The pancreas in a normal body is able to secrete insulin, but in patients with diabetes mellitus, there is a failure of insulin secretion through pancreatic cell dysfunction and insulin action failure through insulin resistance (Holt et al. 2011). Insulin resistance that occurs can trigger hyperglycemia in people with type 2 diabetes mellitus (Baynes 2015). Based on data from the International Diabetes Federation, Indonesia has diabetes alert status because it ranks 7th out of 10 countries with the highest number of diabetes patients. The prevalence of patients with diabetes in Indonesia in 2020 reached 6.2%, which means that there are more than 10.8 million people suffering from diabetes. This number will increase in 2021 to reach 19.47 million people, and this data showed an increase of 167% when compared to the number of people with diabetes in 2011, which reached 7.29 million. Diabetes mellitus is known to be associated with COVID-19 infection (Suwanwongse and Shabarek 2021; Viswanathan et al. 2021). In human monocytes, elevated glucose levels can enhance SARS-CoV-2 replication(Lim et al. 2021). This causes hyperglycemic conditions to support viral proliferation and increase the morbidity and mortality of COVID-19-infected patients (Kaminska et al. 2021).
Handling of diabetics type two is mostly done by taking antidiabetic drugs orally. These drugs have different mechanisms in controlling blood glucose levels.
Clinically, various types of oral hyperglycemic drugs used to control this disease are classified into different classes such as biguanides, sulfonylureas, thiazolidinediones meglitinides, dipeptidyl peptidase (IV) inhibitors, sodium- glucose cotransporters, and glucosidase inhibitors. Each class has a different target organ type and a different mode of action mechanism. In fact, to increase the efficacy of treatment, a combination of different antidiabetic drugs is generally used. However, long-term use of these drugs has side effects that cause chronic effects, including cardiovascular disease, lactic acidosis, hypoglycemia, gastrointestinal complaints, and others (Alam et al.
2021; Baynes 2015; Bitew et al. 2021). Furthermore, the use of herbal medicines in the treatment of diabetes began to be encouraged. Exploration of the effectiveness of antidiabetic compounds in herbal plants is one of the right ways to maximize the potential of these plants as candidates for the treatment of diabetes.
hypoglycemic activity. There are more than 800 plant species reported to be hypoglycemic (Rosemary et al.
2014). One of the plant species that is often used as a producer of antidiabetic compounds in roselle. Roselle (Hibiscus sabdariffa L.), a well-known traditional Chinese medicine, is widely grown in tropical and subtropical areas.
Roselle has been used as a medicinal in many countries such as Australia, India, Myanmar, France, Gambia, Nigeria, Greece, Saudi Arabia, Panama, Indonesia, Malaysia, Thailand, and Senegal. Bioactive compounds are documented in roselle such as alkaloids, anthocyanins, flavonoids, saponins, steroids, sterols, and tannins (Tzu et al. 2007). Those compounds can be used to treat hyperglycemia in the blood by delaying the absorption of sugar through the inhibition of several enzymes that play an important role in the pathophysiology of diabetes.
Enzymes that play a role in the pathophysiology of diabetes mellitus include alpha-glucosidase enzymes, alpha-amylase enzymes, and glucoamylase maltase enzymes (Baynes 2015; Bitew et al. 2021).
The method used to detect the ability of a compound to inhibit the enzyme is needed to assess its effectiveness.
The method used in this research is molecular docking and meta-analysis. Meta-analysis is a method of assessing the effectiveness of a compound by comparing the results of previous studies with the results of statistical processing.
The level of accuracy in meta-analytical studies is related to publication bias, and the bias is caused by the data collected from individual studies that do not represent research on a topic. According to Walker et al. (2008), a bias in the meta-analysis study affects the resulting outcome. The method for determining publication bias from our meta-analysis study had attached. Molecular docking is a method used to determine the orientation of the ligand to the receptor (protein) to form a stable complex. The preferred orientation can be used to predict the strength of the connection or the binding affinity between the ligand and the protein by utilizing the scoring function. The accuracy of molecular docking depends on the molecular docking tool used. Referring to Wang et al.
(2016), who evaluated 10 docking tools, showed that the highest accuracy that could be achieved was around 80%.
This study used autodock vina, which has an accuracy of around 50–60%, has the best scoring power (Wang et al.
2016; Boittier et al. 2020), and has low standard errors (2.85 kcal/mol) for free energies of binding prediction (Trott and Olson 2010). The use of computational-based molecular docking techniques and meta-analysis in drug discovery has developed rapidly in the development process so that it becomes a more effective and efficient technique to use (Dnyandev et al. 2021; Gouthami et al.
2021).
MATERIALS AND METHODS
Molecular Docking Simulation
Molecular docking was carried out through several stages, including the search for proteins and materials contained in roselle, preparation of proteins and materials, determination of grids and creating configuration files, and performing docking and analysis of the interaction of materials with protein residues. The search for diabetes- related proteins was carried out in the protein data bank (www.rcsb.org/) and found alpha-amylase (2QV4) and alpha- glucosidase (7KBR), whereas the search for materials contained in roselle was carried out from literature study.
3D structures of the materials were downloaded from PubChem (https://pubchem.ncbi.nlm.nih.gov/) and obtained 44 structures, including acarbose as control. Protein and material preparation was carried out using the software Chimera 1.31.1 (www.rbvi.ucsf.edu/chimera) and Avogadro (http://avogadro.cc/). Grid determination and config file creation using autodock tools software (https://ccsb.scripps.
edu/mgltools/). After the config file is formed in the .txt extension, the receptor and material in the form of PDBQT then perform docking using AutoDock Vina (Trott and Olson 2010), which produces data of binding affinity.
After getting the data of binding affinity and 11 materials were chosen; the next step was to form a complex file (a combination of protein and docked materials) using the PyMol education license software (http://www.pymol.
org/pymol). The complex file that is formed will enter the analysis stage of the interaction of the docked material with the receptor protein residue using the Protein-Ligand Interaction Profiler (Adasme et al. 2021).
Roselle Extraction
Roselle flowers are washed thoroughly and dried in the sun for 24 h. Open sun drying has the lowest processing costs and does not require expertise but still can maintain polyphenol content without any degradation (Sokač et al.
2021). Dried roselle flowers are then cut into small pieces and made into powder. The number of sieve mesh was 60 mesh. The roselle flower powder was then extracted using the maceration method using two types of solvents – namely, ethanol and distilled water. The filtrate obtained from the extraction is then filtered and stored.
Antidiabetic Activity Test
Antidiabetic activity test on roselle flower extract using the modified-glucosidase enzyme inhibition method (Kim et al. 2005), as modified by Manguntungi et al. (2020). A total of 30 µL of samples at various concentrations were mixed with 36 µL of phosphate buffer pH 6.8 and 17 µL of p-nitrophenyl-α-D-glucopyranoside. Preincubation was carried out for 5 min at 39 °C. After preincubation,
17 µL of -glucosidase (0.0045 units/mL) was added and incubated at 39 °C for 15 min. The reaction was stopped by adding 100 µL Na2CO3 (200 mM). Acarbose (100 ppm) was used as a positive control. Then the reaction results were measured using an ELISA reader at 400 nm, and the determination of alpha-glucosidase inhibition was seen from the amount of p-nitrophenol produced from p-NGP.
Meta-analysis Method
The meta-analysis of the study followed Carey et al. (2013), starting from research questions to calculating summary effects (meta-analysis). The outline consists of determining research questions, determining inclusion criteria, searching for the articles, screening articles, extracting data, and calculating meta-analysis. The research questions are as follows: how is the antidiabetic activity of roselle extract in vitro and in vivo, a means to clarify the description of research questions using PICO; population: roselle (H. sabdariffa;
intervention: extraction of roselle using various solvents;
comparison: comparing diabetic rats without treatment with diabetic rats treated with roselle extract; outcome: fasting glucose level test, pancreatic histology, and antidiabetic enzymatic test). Search for articles was done using Boolean search as follows: ("Hibiscus sabdariffa" OR "roselle") AND
"bioactive compound" AND ("antidiabetic" OR "blood glucose" OR "insulin" OR "alpha-glucosidase" OR "alpha- amylase"). Using the Scopus database, Science Direct, and Proquest, screening of meta-analysis was done using the Colandr application. The results of the screening can be seen in the following PRISMA diagram (Figure 1).
RESULTS
Molecular Docking
In this study, the molecular docking method was used to obtain a model of ligand binding to the receptor catalytic site with the AutoDock Vina Package software developed by Trott and Olson (2010). In receptor preparation, the tertiary structures of two enzymes involved in diabetes, such as alpha-amylase and alpha-glucosidase, were retrieved from the RCSB database with PDB IDs of 2QV4 and 7KBR, respectively (Figure 2).
All enzymes were then added with polar hydrogen and stored in pdbqt format. Ligand preparation was carried out and saved in pdbqt format (Figure 3). Molecular docking was done by setting the grid box parameters. These parameters were required to determine the position and rotation of the ligand to the receptor site (Table 1). Grid box size (x, y, z), grid box center (x, y, z), and Armstrong size were set using AutoDock Tools 1.5.6 developed by Moris et al. (2009). The final result of this docking
Figure 1. Prisma diagram.
Figure 2. The tertiary structure of enzymes (A) alpha-amylase and (B) alpha-glucosidase.
simulation is that the highest binding energy value is an indication that the compounds contained in roselle can inhibit enzymes involved in diabetes.
In order to analyze the stability of the complex, including the binding pocket of ligands into the moiety of receptors, molecular docking is performed by using AutoDock Vina 4 packages. In Figure 4, we show the binding energy of 11 ligands in binding with alpha-amylase and alpha- glucosidase (receptor).
Furthermore, to observe the results of hydrogen bonding between alpha-amylase and alpha-glucosidase enzyme with ligands and other components such as amino acid residues, distance D-A, and H-A can be seen in Table 2.
Antidiabetic Activity
Antidiabetic testing of roselle flower extract was carried out using two types of solvents – namely, by using ethanol and distilled water. Extraction was carried out using the maceration method so that crude extract was obtained, which was then tested for alpha-glucosidase inhibition in vitro. The extract was diluted into different concentrations to obtain comparable data on its activity. This study used 20, 40, 60, 80, and 100 ppm concentrations of roselle flower extract for each solvent. The antidiabetic assay result was shown in Figure 5. The best antidiabetic activity was obtained at highest extract concentration (100 ppm), both the ethanolic and distilled-water extracts. As shown in Figure 5, the diluted extract compared to the
Figure 3. 10 chemical structures of roselle compound and acarbose.
(A) (B) (C) (D)
(E) (F) (G) (H)
(I) (J) (K)
Figure 4. The binding energy of ligands in complex with alpha-amylase and alpha-glucosidase enzyme obtained by molecular docking.
Table 1. The identity of box size for ligand/receptor complex.
No. Enzyme Grid box size Grid box center
(x, y, z) Spacing (Å)
1 Alpha-amylase 12.875 x 47.347 x 26.189 20 x 16 x 16 1 Å
2 Alpha-glucosidase –13.98 x 26.056 x
–9.909 18 x 14 x 12 1 Å
Table 2. Hydrogen bonds between ligands and alpha-amylase enzyme obtained by molecular docking.
No. Compounds Alpha-amylase Alpha-glucosidase
Residue Distance H-A Distance D-A Residue Distance H-A Distance D-A
1 Oxalic acid 195A 3.26 3.91 451A 2.64 3.05
195A 1.92 2.89
233A 2.10 3.06
299A 2.00 2.93
2 Tartaric acid 195A 2.22 3.20 525A 2.65 3.29
197A 2.25 2.97 564A 3.10 3.52
198A 2.93 3.39 624A 3.45 4.09
299A 1.93 2.87 698A 2.40 3.10
3 5-hydroxymethylfurfural 195A 3.33 4.00 624A 2.26 3.06
195A 2.03 3.01 640A 2.43 3.23
233A 2.18 3.08
299A 3.35 4.01
4 Quercetin-3-rutinoside 59A 2.87 3.71 451A 2.21 3.01
59A 2.43 3.06 525A 2.36 2.97
63A 2.50 3.38 624A 2.81 3.72
151A 2.86 3.43 698A 1.74 2.69
195A 2.65 3.66 700A 1.88 2.87
200A 2.81 3.73
200A 2.82 3.28
235A 2.40 3.30
235A 3.58 3.96
300A 3.23 3.76
5 Kaempferol-3-O-rutinoside 62A 2.56 3.05 451A 2.23 3.02
63A 2.56 3.43 451A 1.76 2.68
195A 2.66 3.66 525A 2.34 2.96
200A 2.68 3.57 624A 2.93 3.83
235A 3.13 4.03 640A 2.22 2.70
698A 1.74 2.71
700A 1.88 2.86
6 Myricetin 62A 2.52 3.02 451A 2.01 2.92
63A 3.04 4.04 624A 2.09 3.03
101A 3.46 4.06 640A 3.25 3.68
195A 2.74 3.75 700A 2.33 3.22
197A 1.95 2.72
299A 1.86 2.79
7 N-feruloyltyramine 197A 3.37 3.78 451A 2.36 2.88
200A 3.44 3.98 624A 2.30 3.29
235A 2.86 3.80
299A 2.18 3.09
8 Tiliroside 151A 3.18 3.75 564A 2.55 3.30
195A 3.24 3.96 624A 2.02 2.98
195A 2.09 3.09 624A 2.52 3.34
299A 1.90 2.83 624A 2.32 2.98
700A 2.54 3.25
9 Capric acid 195A 3.32 3.97 624A 2.97 3.62
195A 1.95 2.92 640A 2.23 3.02
233A 2.08 3.03 698A 2.48 3.21
299A 3.32 3.95
10 Heptadecanoic acid 63A 2.73 3.26 624A 2.99 3.71
640A 3.01 3.94
698A 2.29 3.02
11 Acarbose 63A 1.90 2.80 523A 2.06 2.94
101A 2.64 3.17 527A 3.01 3.71
569A 2.99 3.82
624A 1.82 2.81
624A 3.25 3.89
640A 3.60 4.08
Table 2. Continuation.
positive control (acarbose), the ethanol extract at 80 ppm concentration was significantly different. Contrastingly, the distilled-water extract at 80 ppm concentration was equal to the positive control. This result suggested that the activity of 80 ppm ethanol extract was equal to 100 ppm of positive control. This finding was a good approach to making this roselle flower extract as a medical alternative resource. Roselle flower extraction in the research of Ademiluyi and Oboh (2013) used distilled water as a solvent (Table 3). The table above shows that the use of a concentration of 2 mg/mL roselle flower extract had an inhibitory activity of 19.21%, whereas at a concentration of 2.5 mg/mL, it had an inhibitory activity of 41.77%.
Meta-analysis
The antidiabetic meta-analysis study on roselle aimed to compare the negative control treatment (diabetic rats without roselle extract) with roselle extract treatment (diabetic rats + roselle extract) by observing the blood glucose levels of fasting rats. The results of the meta- analysis showed that roselle extract had a positive effect on control with a magnitude of "d" (combined effect size) of 0.60 or roselle extract during observation could reduce glucose levels in fasting rats better than negative controls (diabetic rats) by 60%. Test the heterogeneity of the data resulted in an I2 value of 64%, wherein the data obtained were heterogeneous; this may be due to differences in the extraction conditions, the concentration given to the test rats, and the length of observation used. The subgroup analysis test was aimed at seeing the effect of the best roselle extract in each study with the use of different extraction solutions and processes. The best roselle extract was obtained from research by Rosemary et al. (2014), wherein the effect size produced was 4,074; it was known that roselle was extracted using an ethanol solution by giving it to rats with diabetes of 600, 400, and 200 mg/kg with effect size m foreigners were 3.377 and 2.897 with 35 d of observation, respectively. The extraction process used stratified percolation, first wetting it with ethanol solution for 3 h; after that, it was inserted into the percolation tool, then ethanol solution was added for 24-h soaking.
Table 3. Inhibitory activity of roselle extract on alpha-amylase Concentration
(mg/mL)
Alpha-amylase
References
Extractor Inhibition (%) IC50 (µg/mL)
2 Distilled water 19.21 – Adisakwattana et al.
(2012)
2.5 Distilled water 41.77 –
– Distilled water – 187.9 Ademiluyi and Oboh
(2013)
DISCUSSION
Molecular Docking
From Figure 4, all models had negative binding energies suggesting those ligands make a complex to the site of the receptor. Also, Models 5 and 8 may have become the most stable complex based on the lowest energy score of –9.5 kcal/mol (blue chart); Model 5 may have become the most stable complex based on the lowest energy score of –8.1 kcal/mol (yellow chart). Further, the catalytic site of the receptor obtained by molecular docking could be predicted by evaluating the binding site of the ligand in the complex with the receptor. The binding poses and site of alpha- amylase and alpha-glucosidase, including the summary of the hydrogen bonds of ligands to the site of the receptor, are provided in Figure 4 and Table 2, respectively. Weak intermolecular interactions such as hydrogen bonding and hydrophobic interaction stabilize the ligand at the target site by altering binding affinity and drug efficacy (Patil et al. 2010). Hydrophobic interactions are the main driving force in drug-receptor or protein-ligand interaction (de Freitas and Schapira 2017). Strong H-bonds are required for the most high-affinity ligand (Rascha et al. 2018). The study by Chen et al. (2016) showed that hydrogen bonds can enhance ligand binding affinity when both donor and acceptor have either significantly stronger or significantly weaker, but in contrast to mixed strong-weak H-bonds, the pairing decreased ligand binding affinity.
From these results, the ligands (i.e. oxalic acid, tartaric acid, 5-hidroxymethylfurfural, quercetin-3- rutinoside, kaempferol-3-O-rutinoside, myricetin, tiliroside, and capric acid) participate in hydrogen bonds with the similar amino acid of 195A. This finding suggests that the region of residue 195A may be assumed as the catalytic site of alpha-amylase. The ligands (i.e. tartaric acid, 5-hidroxymethylfurfural, quercetin-3-rutinoside, kaempferol-3-O-rutinoside, myricetin, N-feruloyltyramine, tiliroside, capric acid, and heptadecanoic acid) participate in hydrogen bonds with the similar amino acid of 624A. This finding suggests that the region of residue 624A may be assumed as the catalytic site of alpha-glucosidase. The in silico test showed that the secondary metabolite extract of roselle
has antidiabetic activity. The next test carried out was testing the antidiabetic activity of roselle extract in vitro and in vivo through meta-analysis.
Antidiabetic Activity
This study had been evaluated the roselle flower extract’s biological activity using two kinds of solvents by utilizing the alpha-glucosidase enzyme inhibition test. Based on the inhibition assay, the ethanol extract gave a better effect when compared with the positive control at 80 ppm, as well as an equal effect at the highest concentration. Since ethanol has been known as a good solvent for polyphenol extraction and safe for human consumption, this study’s findings also support the data. Furthermore, polar solvents are frequently used for isolating polyphenols from plant matrices.
The bioactive compounds (phytochemicals) in roselle flower have been attributed to its therapeutic properties – with phenolic acids (especially protocatechuic acid), flavonoids, anthocyanins (delfinidine-3-sambubioside, and cyanidine-3-sambubioside), organic acids, and some polysaccharides being the most prominent (da-Costa- Rocha et al. 2014). Anthocyanins, flavonoids, organic acids (mainly citric acid, hibiscus acid, and malic acid), glycosides, and fiber are the primary phytochemicals contained in roselle flowers. On the other hand, calyxes have a similar number of organic acids and anthocyanins;
however, only flavonoids and glycosides (Guardiola and Mach 2014; Herranz-Lopez et al. 2017). Polyphenols of the flavonol and flavanol types – in simple or polymerized form, as well as a number of flavonoids – have been found in roselle flower extracts. Polyphenols and flavonoids from roselle flower have been shown to have antioxidant, antihypertensive, antimicrobial, anti-inflammatory, anticancer, and antidiabetic properties (Riaz and Chopra 2018). The main enzymes involved in carbohydrate metabolism are alpha-amylase and alpha-glucosidase.
Several studies have shown that alpha-amylase and alpha-glucosidase activity have a significant influence on blood glucose levels, and inhibiting them can significantly reduce the postprandial rise in blood glucose (Nair et al. 2013). The inhibition of alpha-amylase and alpha- glucosidase was measured in the studies (Ifie et al. 2018;
Alegbe et al. 2019). Inhibiting both enzymes could be used in diabetic therapy to lower high blood glucose levels.
Several alpha-amylase and alpha-glucosidase inhibitors isolated from medicinal plants have been found to be more potent and have fewer side effects than existing synthetic drugs. Several phenolic compounds – including caffeic acid, gallic acid, and protocatechuic acids – have been shown to have antidiabetic activity (Alegbe et al. 2019;
Matsui et al. 2006). Furthermore, to see the activity of roselle flower extract as antidiabetic, it can be presented in the forest plot table.
Meta-analysis
The roselle extract treatment had a "d" value that was significantly different from negative controls (diabetic rats) and was subsequently obtained in the study following Seung et al. (2018) – extraction using 80% ethanol solvent at 60 °C for 2 h to get roselle crude extract, then separation of the crude extract using n-hexane, chloroform, and ethyl acetate as solvents. The final stage is concentrated at 60 °C and then freeze-dried. The treatment of roselle extract given to experimental rats was 100 and 200 mg/
kg, and the resulting "d" values were 0.106 and 0.263, respectively. Furthermore, research by Alegbe et al.
(2019), the extraction produced from distilled water was partitioned by n-hexane, ethyl acetate, and n-butanol solvents for ethyl acetate solvent further fractionation was carried out using column chromatography. In this study, 15 fractions were obtained and grouped: Group I consisted of 1–5 fractions, Group II consisted of 6–10, and Group III consisted of 10–15 fractions. In this study, the value of "d" was significantly different in Group III, which can be seen in Table 4. Research conducted by Zainalabidin et al. (2018), which involved extraction using 50 mL of 99.9% methanol at a temperature of 60 ˚C for 30 min, yield a higher "d" value of 0.125 following the treatment of positive diabetic rats at 100 mg/kg.
CONCLUSION
In conclusion, molecular docking showed that all models have negative binding energies, suggesting that those ligands make a complex to the site receptor. Based on the analysis, kaemferol-3-O-rutinoside became the most stable complex based on the lowest energy score of –9.5 kcal/mol for alpha-amylase, and tilliroside became the most stable complex based on the lowest energy score of –8.1 kcal/mol for alpha-glucosidase. Based on the in vitro test, the highest antidiabetic activity (alpha-glucosidase inhibitory) was obtained at a 100 ppm roselle flower ethanol extract with an inhibition value of 100.00%, and the highest activity with distilled water was obtained at a concentration of 100 ppm with an inhibition value of 99.25%. Following the alpha-amylase inhibiting test using a concentration of 2 mg/mL, the roselle flower extract had an inhibitory activity of 19.21%, whereas at a concentration of 2.5 mg/mL, it had an inhibitory activity of 41.77%. The antidiabetic meta-analysis study showed that the roselle extract could positively affect control with a combined effect size of 0.61, or that the roselle extract could reduce glucose levels in fasting rats better than negative controls (diabetic rats) by 61% than rats that were not given the roselle extract.
Table 4. Type Subgroup analysis of the effect of roselle extract on rats blood glucose.
Figure 5. Antidiabetic activity of roselle extract using ethanol and distilled water.
ACKNOWLEDGMENTS
We are grateful to Universitas Tanjungpura, Universitas Sulawesi Barat, National Research and Innovation Agency, Sumbawa Technopark, IPB University, and the Indonesian Endowment Fund for Education (LPDP) of the Indonesian Ministry of Finance for their contribution to the initial stages of the study.
STATEMENT ON CONFLICT OF INTEREST
The authors declare that they have no conflict of interest.
NOTES ON APPENDICES
The complete appendices section of the study is accessible at https://philjournsci.dost.gov.ph
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