INTRODUCTION:

Millions of people are affected by diabetes every year, and currently, there are three important drugs, namely insulin, metformin, and sulfonylureas used for its control. However, despite the substantial side effects, many people do not benefit from taking insulin and synthetic oral hypoglycemic drugs (OHD). Herbal treatments are effective, have few or no side effects, and are fairly inexpensive. More than 800 plants have been reported as a traditional diabetes treatment¹.

Terrestrial plants that have been demonstrated antidiabetic effects are Momordica charantia², Nigella sativa³, Ocimum sanctum⁴, Abelmoschus esculentus L.⁵, Abelmoschus esculentus L.⁶, Moringa oleifera^7,8, Syzygium polyanthum⁹, Psidium guajava L¹⁰, and Muntingia calabura L.¹¹. Marine plants have also been investigated for their antidiabetic potential, including alginate extracted from brown seaweed¹² and carrageenan extracted from red algae¹³.

The selection of how to deliver these herbal ingredients to get optimal benefits for diabetics is a very important stage. Beverages, including functional drinks, are considered products that are suitable to be used to formulate herbal ingredients because drinks are products that are generally not problematic for consumption by all consumers. The important thing in the formulation of functional drinks is to get a mixture of various ingredients that produce drinks that are acceptable to consumers. However, the efficacy of lowering blood sugar levels in people with diabetes remains a major concern. As a phycocolloid material that affects the consistency of the resulting beverage, alginate was chosen as the basis for the manufacture of a functional drink in this study. To improve the performance of the antidiabetic effect of alginate drinks, the addition of extracts from plants that have antidiabetic activity can be employed. Therefore, the aim of this study was to explore the potential utilization of alginate-based functional drinks added with terrestrial plant extracts to reduce blood sugar levels in diabetics. The plant extracts added were okra (A. esculentus), moringa (M. oleifera) leaves, bay leaves (S. polyanthum), and guava (P. guajava) leaves.

MATERIALS AND METHODS:

Materials:

The raw material of Sargassum sp used to extract alginate was obtained from UD Rumput Laut Mandiri, Gunung Kidul Regency, Indonesia. Okra and bay leaves were bought from Palmerah traditional market, Jakarta Pusat, Indonesia. While, moringa, and guava leaves were supplied by the tree owners living in Jakarta, Indonesia.

Extraction of alginate:

Sargassum sp was extracted to get alginate.by soaking the seaweed in water for 30 min, then immersed in the solution of 1% HCl for 30 min before being cooked in the solution of 2% (w/v) Na₂CO₃ at 60-70^oC for one hour. The seaweed was cooked again after grinding and then filtered with a plankton net. Filtrate bleaching was carried out with a 2% NaOCl (v/v) solution, then 10% HCl solution (in 20L water) was added to precipitate alginic acid, and the filtrate was filtered using a plankton net. Furthermore, the filtrate was neutralized to pH 7-8 with 30% NaOH solution before binding the Na-alginate with 95% isopropanol and filtering to obtain the alginate fibers. The alginate fiber drying was performed at room temperature for 24 hours before being floured at a mesh size of 60¹⁴.

Preparation of functional drinks from a mixture of alginate and plant extracts:

The methods for making functional drinks from alginate and plant extracts used in this study were developed previously through preliminary research, i.e. alginate-okra extract drinks, alginate-bay leaf extract drinks, alginate-guava leaf extract drinks, and alginate-moringa leaf extract drinks.

a. Alginate - okra extract drink. Okra (120.25g) was washed with clean water, then cut into small pieces, and blanched at 80˚C for 5 min. Okra was soaked in ice water for 5 min, and mashed using a blender (Vienta; VFP1 series – HFR; made in China) by adding 372.35 ml water. After filtering using a filter cloth, the filtrate was heated at 70˚C for 5 min and added with 1.5 g stevia, 0.1g citric acid, and 0.05g salt. The mixture was blended using a homogenizer (Daihan brand; HG-15A series; made in Korea) for 10 seconds at a speed level of 30. The drink mixture was homogenized again at the same speed level for 2 min while adding 4.75g alginate and 0.75g xanthan gum. After homogenizing the drink was heated at 85˚C for 15 min and added 0.25g lychee flavour before being transferred into the bottle and pasteurized.

b. Alginate - bay leaf extract drink. Washing with water was carried out on about 8.6g of bay leaves and then boiled in 485.45ml of water for 30 min or until the water was reduced by half. The decoction of the bay leaves was then filtered using a filter cloth and the extract was poured into a beaker. Furthermore, 3.9g alginate, 0.5g xanthan gum, 1.0g stevia, and 0.05g citric acid were added to the filtrate and blended using a homogenizer (Daihan brand; HG-15A series; made in Korea) for 3min. After that, the mixture was heated at 85ºC for 15min while adding 0.5g of orange flavour and then bottling and pasteurizing.

c. Alginate - guava leaf extract drink. Guava leaves (6.55g) were washed and blanched in 500ml of 80°C water for 5 min. The guava leaves were boiled in 487.5 ml of 90°C water for approx. 30 min until the water was reduced to half. After filtration with a filter cloth, the obtained hot guava leaf extract was transferred to a beaker and the addition of 5.95g alginate, 0.25g salt, 0.5 g xanthan gum, 1.0g stevia, 0.1g citric acid, and 0.1g citric acid, and 0.5g coco pandan flavour was conducted evenly by using a homogenizer (Daihan brand; HG-15A series; made in Korea) at a speed level of 25-30 for 1-3 min. Thereafter, the drink produced was bottled and pasteurized.

d. Alginate - moringa leaf extract drink. Approx. 74.75g of moringa leaves were washed and blanched with 650ml of water at 80˚C for 5min. After that, the moringa leaves along with the blanching water were mashed using a blender (Mitzui, 061639 series, made in Korea) for 10 seconds at medium speed. Moringa leaf juice was filtered through a filter cloth. About 500ml of the juice was mixed with 2g stevia and 0.25g salt with a homogenizer (Daihan brand; HG-15A series; made in Korea) at a speed level of 28 for 10 seconds. The mixture was then added with 5g alginate and 0.15g xanthan gum and homogenized again at the same speed and duration. The mixture was heated to 85˚C and 1ml of pandan flavour was added. Finally, the obtained drink was bottled and pasteurized.

e. Alginate drink. As a control sample, an alginate drink was made by adding 5 g of alginate into 500 ml of 85^oC water until thoroughly mixed.

Analysis of product characteristics:

a. Qualitative analysis of phytochemicals:

Phytochemicals including phenolic and steroid compounds were identified qualitatively. The phenolic compounds analyzed were flavonoids, tannins, and saponins. To identify the compounds, about 5 g sample was added with distilled water, then heated for 5 min, and filtered. Furthermore, the filtrate obtained was used for the detection of flavonoids, tannins, and saponins as follows¹⁵:

Flavonoids. Mg powder, HCl solution in ethanol (1:1), and amyl alcohol were added to the filtrate. The presence of flavonoids was characterized by the formation of an orange amyl alcohol layer. Tannins: The filtrate was added with 3 drops of 10% FeCl₃. The formation of a greenish-black colour indicated the presence of tannins. Saponins: Vigorous shaking was carried out on the filtrate. The presence of saponins was characterized by the formation of stable foam. Steroids were detected by dissolving 1g sample into a hot ethanol solution and then filtering. The obtained filtrate was heated to dry, then added with 1 ml diethyl ether and further homogenized with the addition of 1 drop of concentrated H₂SO₄ and 1drop of anhydrous CH₃COOH. The presence of steroids is characterized by the formation of a green-blue colour.

b. Dietary fiber:

Two samples of about 0.5-1g were put into two different 50mL falcon tubes and transferred each sample into two 400 mL beaker glasses. After adding 40mL of MES-TRIS buffer solution and stirring evenly, 50μL of the α-amylase enzyme was poured and stirred until homogeneous which was then covered with aluminum foil. The solution was incubated in a shaking water bath at 100^oC for 30 min, then cooled to 60^oC. The gel formed at the bottom of the beaker glass was cleaned with a glass stirrer, and then the walls of the beaker glass and the glass stirrer were rinsed with 10 mL of distilled water. About 100μL of the protease enzyme was added and stirred homogeneously. After covering the top of the glass with the aluminum foil, the solution was incubated in a shaking water bath at 60^oC for 30 min. About 5mL of 0.561M HCl was added and the pH was set to pH 4.1-4.6 using 1 M NaOH or 1 M HCl. About 200μL of amyloglucosidase was added and stirred thoroughly. After covering with aluminum foil, the solution was incubated again in a shaking water bath at 60^oC for 30 min. After adding 225mL of 95% ethanol at a temperature of 60^oC and stirring homogeneously, the solution was allowed to stand at room temperature for 1 hour, then filtered with ash-free filter papers. The residue was washed with 2 x 15 mL of 78% ethanol, 2 x 15 mL of 95% ethanol, and 2 x 15 mL of acetone. The filter paper was dried in the oven at 103 ± 2^oC. Each filter paper containing residue was weighed to determine the ash weight and protein weight¹⁶.

Ash weight (A)(g) = (Dish weight + Ash) – Empty dish weight

Vp × Np × fk × 14.007

Protein weight (P) (g) = ------------------------------------

1000

(R – A – P)

Total dietary fiber content (%) = ------------------ ×100 %

Where Vp = Titration volume of 0.2 N HCl solution (mL); Np = Normality of 0.2 N HCl solution; fk = Protein conversion factor; R = Average weight of sample residue (g); A = Weight of sample ash (g); P = Sample protein weight (g); and W = Average weight of the sample (g)

c. Alfa glucosidase:

Approx. 50µL 0.1 M phosphate buffer (pH 7.0), 25µL of 0.5mM 4-nitrophenyl-D-glucopyranoside (dissolved in 0.1M phosphate buffer, pH 7.0), 10µL test sample (concentration: 500g mL^-1) and 25µL of a-glucosidase solution (1mg mL^-1 stock solution in 0.01M phosphate buffer, pH 7.0 diluted to 0.04 units mL^-1 with the same buffer, pH 7.0 immediately before testing) was completely mixed. The resulting mixture was then incubated at 37°C for 30 min. The enzymatic reaction was then halted by adding 100µL of 0.2M sodium carbonate solution. The progress of substrate hydrolysis was tracked by measuring the quantity of p-nitrophenol released into the reaction mixture at a wavelength of 410 nm using an Agilent BioTek Epoch microplate spectrophotometer (USA)¹⁷.

d. Phenolic content:

Total phenolic content was determined using the Folin-Ciocalteu method, with absorbance measured at 765 nm¹⁸. Gallic acid standard was made with a concentration range of 0-250ppm and absorbance was measured at 765 nm. For sample measurement, the sample was weighed to 0.25–2.5g, and then 0.5mL methanol, 2.5mL distilled water, and 2.5 mL Folin-Ciocalteau reagent were added. The mixture was left to stand for 5min, then 2mL of 15% Na₂CO₃ was added and vortexed, followed by incubation at 5°C for 30min, and absorbance measurement at 765nm. Total phenolic values are expressed as gallic acid equivalents (mg g^-1 dry weight).

e. Antioxidant activity:

Analysis of antioxidant activity using a modified method of scavenging activity of DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical¹⁹. 125mM DPPH solution was prepared by dissolving 2.5mg DPPH in 50ml ethanol. The ascorbic acid solution was made by dissolving 10mg in 1ml of DMSO (dimethyl sulfoxide), then sonicated and vortexed. Standard solutions of ascorbic acid were made at concentrations levels of 0 ppm, 0.78125ppm, 1.5625ppm, 3.125ppm, 6.25ppm, 12.5ppm, 25ppm, 50ppm, and 100ppm. The drink samples were prepared in the same way as for ascorbic acid. After that, each sample and ascorbic acid that had been prepared were put in as much as 100mL into a microplate, and then 100mL DPPH was added. The blank solution contained 100ml of ethanol and added 100 ml DPPH, while the negative control solution only used 100mL ethanol. Subsequently, the prepared drink sample, ascorbic acid, and control solution were incubated at ambient temperature in the dark room for 30min. Then, the absorbance was measured using an Agilent BioTek Epoch microplate spectrophotometer (USA) at a wavelength of 517nm. The value of antioxidant activity was expressed as equivalent to ascorbic acid.

Statistical Analysis:

The statistical significance of the antidiabetic potential of alginate drinks, various plant water extracts, and alginate–plant extract mixture drinks with alpha phenolic content, dietary fiber content, glucosidase inhibitor, and antioxidant capacity as dependent variables was assessed using one-way analysis of variance and Tukey's HSD (Honest Significant Difference) posthoc test. Statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS) 25 program at a significance level of 0.05(95% confidence level).

RESULT AND DISCUSSION:

Phytochemical screening:

Several phytoconstituents from different plant sources such as alkaloids, glycosides, flavonoids, terpenoids, steroids, saponins, dietary fibers, polysaccharides, etc. have been reported as effective hypoglycemic agents²⁰. This study focused on qualitatively identifying the presence of flavonoids, tannins, saponins, and steroids in alginate drinks, plant aqueous extracts, and alginate–plant extract mixture drinks.

Qualitative analysis indicated that the phytochemical compounds in alginate extracted from Sargassum sp. were mainly saponins (+) (Table 1). A previous study revealed that the aqueous extract of Sargassum duplicatum contained flavonoids, tannins, saponins, and terpenoids²¹. It was suspected that the method used to extract alginate affected its phytochemical content. Alginate extraction involving heat and drying as well as the use of NaOH, Na₂CO₃, NaOCl, and HCl probably caused the loss of phytochemicals. In addition, the alginate extraction method may not be able to optimally draw out phytochemical compounds other than saponins into the alginate, so only saponins were detected as qualitatively significant in the alginate.

Okra water extract contained tannins (+), saponins (+), and steroids (++). Those phytochemicals were also previously detected in okra aqueous extract²². However, the phytochemical compounds found in the alginate and okra extract mixture drink were only saponins (++). The tannins and steroids extracted from okra and added to the alginate during the drink's production were probably so small that they were barely detectable.

The moringa leaf aqueous extract was identified to contain flavonoids (+), tannins (+), and steroids (++). Phytochemicals previously identified in moringa aqueous extract were flavonoids, steroids, anthraquinone, saponins, alkaloids, cardiac glycosides, terpenoids, tannins, anthocyanin, and carotenoids²³ as well as phytosterols, phenols, tannins, phlobatannins, coumarins and proteins^23,24,25,26. Phytochemicals found in moringa leaves as dried leaf powder were alkaloid compounds, polyphenols, flavonoids, tannins, phenols, and quercetin²⁷. Drinks made by mixing alginate and moringa leaf extract were discovered to contain flavonoids (++), tannins (+++), saponins (+), and steroids (++). In the drink produced, there seems to be a contribution of saponins from alginate, while flavonoids and tannins showed a stronger performance than that detected in okra extract. Tannin qualitatively indicated the highest performance compared to other phytochemical compounds.

Qualitative identification of phytochemical compounds in bay leaf aqueous extract encountered tannins (+), saponins (+++), and steroids (+++). Previous investigation detected tannins and phenols in the bay leaf aqueous extract²⁸. The origin of the bay leaf affects the phytochemical content of the aqueous extract, where there are types of phytochemicals found in one area that may not be found in other areas or vice versa. For example, bay leaves from East Java also contained coniferin, bay leaves from Central Java contained juncusol and bay leaves from West Java contained retucine, while quercetin was found in bay leaves from all those areas²⁹. Phytochemical compounds detected in the functional drink made of alginate and bay leaf extract were flavonoids (+), tannins (+), and saponins (+).The processing of the drink may result in the presence of flavonoids and the absence of steroids in the final product,although this phenomenon cannot yet be explained.

Flavonoids (+), tannins (+), saponins (+++), and steroids (+++) were identified in the aqueous extract of the guava leaves. Previous studies found terpenoids, steroids, saponins, flavonoids, and phenolics^30,31 as well as proteins, fats, fixed oils, gums, and mucilages³²in aqueous guava leaf extract. A researcher reported that saponins were not identified in guava leaves³³. A drink processed using a mixture of alginate and guava leaves extracted using water had phytochemical compounds of flavonoids, tannins, and saponins. Steroids were not identified in the drinks obtained. Being previously reported that guava leaf aqueous extract did not contain steroids³³. This occurrence may also be used to explain the undetectability of steroids in alginate-based drinks involving the use of both okra and bay leaves employing water in their processing.

In general, the phytochemical content of obtained functional drinks made from alginate–plant aqueous extract mixtures showed differences in qualitative content indicator intensity and phytochemical types. Saponins identified in alginate and also detected in all aqueous extracts of plants (okra, moringa leaves, bay leaves, and guava leaves) were found in all drinks produced. Saponins were reported to have hypoglycemic effects, regulating blood sugar and preventing diabetes complications due to their antioxidant effects³⁴. Dyslipidemia associated with saponins helps reduce the risk of atherosclerosis and cardiovascular disease in diabetics. Flavonoids and tannins were identified in the drinks using moringa leaves, bay leaves, and guava leaves. Flavonoids have previously been demonstrated to prevent diabetes and its complications based on in vitro studies and animal models³⁵. Tannins were revealed to reduce blood sugar and increase body weight in the absence of insulin. Tannins can be used to treat diabetic neuropathic pain (DNP), a common chronic microvascular consequence of diabetes³⁶.

Total phenolic content:

Phenolic compounds have strong anti-inflammatory and antioxidant effects due to the specific properties of their chemical structure, which makes them promising antidiabetic drugs³⁷. Phenolic compounds have high free radical scavenging capacity, which means they have high anti-diabetic potential. Phenolic compounds prevent carbohydrate digestion and glucose absorption from the intestine by inhibiting a-glucosidase and a-amylase activities. Phenolic compounds can promote insulin production in pancreatic cells and also activate insulin receptors³⁸.

Figure 1: Phenolic content of alginate drink, plant water extracts, and alginate-plant extract mixture drinks (The different letters indicated a significant difference)

The total phenolic content of alginate was low (0.018 mg/g). Aqueous extracts of okra, moringa leaves, bay leaves, and guava leaves had total phenolic content of 0.208±0.003mg/g, 1.771±0.005mg/g, 0,194±0.001mg/g and 1.718±0.009mg/g respectively (Figure 1). This study suggests that alginate is not a potential phenolic compound source. The total phenolic content in the aqueous extracts of okra and bay leaves was low but much higher than that of alginate. Moringa leaf and guava leaf extracts have much higher phenolic content.

The plant extracts showed a significant effect on the phenolic content of functional drinks. The higher the phenolic content of the plant aqueous extract, the higher the phenol content of the drinks (Figure 1), The total phenolic contents of the alginate-based drinks processed by mixing each with okra, moringa leaves, bay leaves, and guava leaves were 0.478±0.001mg/g, 1.752±0.001 mg/g, 0.260±0.001mg/g and 1.207±0.002mg/g accordingly. The use of moringa leaf extract produced alginate functional drinks with the highest phenolic content, and the second was the one mixed with guava leaf aqueous extract.

The phenolic compounds previously encountered in moringa leaves include vitamins, flavonoids, phenolic acids, isothiocyanates, tannins, and saponins³⁹. Phenolic acids identified in moringa leaves were quercetin, gallic acid, vanillic acid, sinapic acid, 4-hydroxy benzoic acid, p-coumaric acid, caffeic acid, m-coumaric acid, 4-hydroxy-3-methoxy cinnamic acid, and syringic acid⁴⁰. While phenolic compounds detected in guava leaves were quercetin, avicularin, apigenin, guaijaverin, kaempferol, hyperin, myricetin, gallic acid, catechin, epicatechin, chlorogenic acid, epigallocatechin gallate, and caffeic acid⁴¹.

Dietary fiber:

Many studies have found a protective relationship between the higher intake of dietary fiber and the risk of type 2 diabetes mellitus⁴². Thus, an analysis of the dietary fiber content of drinks made from a mixture of alginate and plant aqueous extracts to reveal the potential ability to lower the risk of diabetes needed to be carried out.

Figure 2: The dietary fiber content of alginate drinks, plant water extracts, and alginate-plant extract mixture drinks (The different letters indicated a significant difference)

Alginate drink contained 1.23±0.03% dietary fiber, while the fiber contents of aqueous extracts of okra, moringa leaves, bay leaves, and guava leaves were 1.37±0.01%, 1.68±0.03%, 0.3±0.01%, and 1.22±0.02% respectively. Thus, alginates and plant aqueous extracts have the potential to contribute dietary fiber to drinks made by mixing both. The dietary fiber content of alginate-based drinks with various treatments of plant aqueous extract addition is displayed in Figure 2.

The dietary fiber content of alginate-based drinks mixed with okra, moringa leaves, bay leaves, and guava leaves were 1.36±0.01%, 1.54±0.02%, 1.50±0.02%, and 1.47±0.05%, accordingly. The use of alginate and plant extracts in the processing of drinks can produce products that contain dietary fiber that is beneficial for diabetics. Fiber decreases postprandial glycemia and insulin levels, which then affect plasma lipid levels in type 2 diabetes patients. Fiber prolongs glucose absorption, prolongs insulin secretion in the liver, increases insulin sensitivity at the cellular level, and binds bile acids⁴³.

Alpha glucosidase inhibitor:

The alpha-glucosidase inhibitor is an antidiabetic agent that works by inhibiting alpha-glucosidase enzyme activities. Reducing the absorption of carbohydrates from food by the intestine is a therapeutic approach for postprandial hyperglycemia⁴⁴. This study sought to find the alpha-glucosidase inhibitor from drinks made of the alginate and plant extract mixture that was developed as an alternative to treat diabetes.

Figure 3: Alfa glucosidase inhibitor of alginate drink, plant water extracts, and alginate-plant extract mixture drinks (The different letters indicated a significant difference)

Individually, alginate, as well as water extract from okra, moringa leaves, bay leaves, and guava leaves used in the development of functional drinks, showed alpha-glucosidase inhibitor activity, namely 52.18±0.46%, 69.75±0.40%, 58.31±0.60%, 89.87±0.32%, and 95.36±0.16%, respectively (Figure 3). Those results indicated that guava leaf aqueous extract had the highest alpha-glucosidase inhibitor activity, which was followed by bay leaves, okra, moringa leaves, and alginate. A similar result was reported that aqueous extracts of guava leaves showed significant inhibition of the alpha-glucosidase enzyme³¹. Inhibition of this enzyme increases the carbohydrate digestion period and reduces the absorption of glucose. While polysaccharides from guava leaves can effectively lower fasting blood glucose in diabetic model animals⁴⁵.

Alginate-based functional drinks are each mixed with okra, moringa leaves, bay leaves, and guava leaves during processing showed alpha-glucosidase inhibitor values of 82.57±0.43%, 36.63±0.12%, 97.41±0.44%, and 92.74±0.27%, respectively. A mixture of alginate and bay leaves produced a drink with the highest inhibitory activity of alpha glucoside. Observing the results of the qualitative identification of phytochemicals, it seems that the saponins which were also detected in alginate were found in all treatments of beverage products and played a significant role in diabetes treatment. The extracts of traditional Chinese medicine which had high levels of saponins were reported to demonstrate a good inhibition of a-glucosidase and a-amylase activities⁴⁶, in which saponins were also suspected to have an essential function in preventing diabetes mellitus.

Antioxidant activity:

Hyperglycemia accelerates glucose auto-oxidation to form free radicals. Free radical generation beyond the scavenging capacity of endogenous antioxidant defenses leads to macrovascular and microvascular dysfunction. Antioxidants are effective in reducing diabetes complications, and consuming natural antioxidants or taking supplements can be beneficial⁴⁷.

Figure 4: Antioxidant activity of alginate drink, plant water extracts, and alginate-plant extract mixture drinks (The different letters indicated a significant difference)

Alginate solution as a drink had a low antioxidant capacity, i.e. 3.26±0.61ppm. This value is in line with the low phenolic content of alginate drink. Phenolic compounds are more active as antioxidants than other compounds since phenolics easily release and donate hydrogen⁴⁸. Phenolic compounds are a type of phytochemicals in Sargassum⁴⁹ which is a brown seaweed generally used as raw material for alginate extraction to be reported to have strong antioxidant activity⁵⁰. Alginate extraction processes including soaking in NaOH, boiling in Na₂CO₃, bleaching with NaOCl, and drying were suspected to bring about the loss of antioxidant compounds in Sargassum due to oxidation. This study discovered that the antioxidant activity of moringa leaf water extract showed the highest (92.87±0.09 ppm), indicating to inhibit the action of free radicals better than others. However moringa leaves as powder exhibited very weak antioxidant activity²⁵. Antioxidant activities of guava leaf extract, bay leaf extract and okra extract were 84.66±0.18ppm, 69.40±0.07ppm and 69.35±0.35ppm respectively (Figure 4). A study revealed that okra extract from different fractions to greatly improve superoxide dismutase levels and tissue glucose tolerance in diabetic mice⁵¹.

Due to the low antioxidant activity, this study suggested that alginate requires higher amounts or in combination with other ingredients that have a higher antioxidant capacity for disease treatment purposes to enhance its performance. The antioxidant activity of the drinks made by mixing alginate each with okra extract, moringa leaf extract, bay leaf extract, and guava leaf extract during the boiling process was 70.77±0.26ppm, 95.64±0.00ppm, 90.71±0.18ppm, and 92.82±0.18ppm respectively, in which the use of moringa leaves demonstrated as the highest. The plant water extract could significantly increase the antioxidant activity of alginate drinks which were indicated by the higher antioxidant capacity of the obtained drinks. A previous study reported that the mixture of alginate and okra extracts showed antioxidant activity from polysaccharides and quercetin which have antihyperglycemic to anti-inflammatory characteristics⁵².

CONCLUSION:

From the perspective of phytochemical profiles, phenolic content, fiber content, alpha-glucosidase inhibitors, and antioxidant activity, all functional drinks prepared from a mixture of alginate and plant extracts, especially okra, moringa leaf, bay leaf, and guava leaf show the potential to be used for the treatment of diabetes. Concurrent use with plant extracts can improve the performance of alginate for the treatment of diabetes. Saponins found in alginate were identified in all types of drinks. The use of bay leaf and guava leaf produces alginate drinks with good alpha-glucosidase inhibitors and antioxidant capacities. Drinks made from a blend of alginate-bay leaf extract and a mixture of alginate-guava leaves could be considered to be used to treat diabetes.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

The Ministry of Education, Culture, Research and Technology of the Republic of Indonesia provided financing for this work under the PTUPT program, which the authors gratefully acknowledge. This research is part of the research collaboration activities with the University of Tsukuba, Japan under the Satreps project.

Author Contributorship:

All authors contributed equally in conducting research and writing a manuscript.

REFERENCES:

1. Prabhakar PK. Doble M. Effect of Natural Products on Commercial Oral Antidiabetic Drugs in Enhancing 2-Deoxyglucose Uptake by 3T3-L1 Adipocytes, Ther Adv Endocrinol Metab. 2011; 2(3): 103-14. https://doi.org/10.1177/2042018811411356

2. Suganya J. Radha M. Manoharan S. Vinoba. V. Francis A. Virtual Screening and Analysis of Bioactive Compounds of Momordica charantia against Diabetes using Computational Approaches. Research Journal of Pharmacy and Technology. 2017; 10(10): 3353-3360. doi: 10.5958/0974-360X.2017.00596.0

3. Sutrisna E. Azizah T. Wahyuni S. Potency of Nigella sativa linn. Seed as Antidiabetic (Preclinical Study). Research Journal of Pharmacy and Technology. 2022; 15(1): 381-4. doi: 10.52711/0974-360X.2022.00062

4. Lokhande VY. Yadav AV. In vitro Antioxidant and Antidiabetic Activity of Supercritical Fluid extract of Leaves Ocimum sanctum L. Research Journal of Pharmacy and Technology. 2018; 11(12): 5376-5378. doi: 10.5958/0974-360X.2018.00980.0

5. Zuraidah AA. Winarni D. Punnapayak H. Darmanto W. Therapeutic Effect of Okra (Abelmoschus esculentus Moench) Pods Extract on Streptozotocin-Induced Type-2 Diabetic Mice. Research Journal of Pharmacy and Technology. 2019; 12(8): 3703-3708. doi: 10.5958/0974-360X.2019.00633.4

6. Anjani PP. Damayanthi E. Rimbawan. Handharyani E. Potential of Okra (Abelmoschus esculentus L.) Extract to Reduce Blood Glucose and Malondialdehyde (MDA) Liver in Streptozotocin-Induced Diabetic Rats. J. Gizi Pangan. 2018; 13(1): 47-54. https://doi.org/10.25182/jgp.2018.13.1.47-54.

7. Thiruchelvi R. Priyadharshini S. Rajakumari K. Screening for Anti-Diabetic Peptides from Moringa oleifera leaves. Research Journal of Pharmacy and Technology. 2021; 14(11): 5886-0. doi: 10.52711/0974-360X.2021.01023

8. Watanabe S. Okoshi H. Yamabe S. Shimada M. Moringa oleifera Lam. in Diabetes Mellitus: A Systematic Review and Meta-Analysis. Molecules. 2022; 26: 3513. https://doi.org/10.3390/ molecules26123513

9. Widyawati T. Pase MA. Daulay M. Sumantri I.B. Effect of Bay Leaf Ethanol Extract on Blood Glucose Level in Patients with Type 2 Diabetes Mellitus. in The 6th International Conference on Public Health. 2019; 613-617. https://doi.org/10.26911/the6thicph-FP.05.07

10. Banu MS. Sujatha K. Beena WS. Divya N. Antihyperglycemic and Antioxidant Potentials Psidium guajava Leaf Extract and Its Isolated Fraction in Alloxan-Induced Diabetic Rats. Research Journal of Pharmacy and Technology. 2012; 5(4): 541-546

11. Sowmya S. Jayaprakash A. In-vitro Antioxidant and Antihyperglycemic Effect of Muntingia calabura L. Fruit Extracts. Research Journal of Pharmacy and Technology. 2021; 14(12): 6496-2 doi: 10.52711/0974-360X.2021.01123

12. Husni A. Pawestria S. Isnansetyo A. Blood Glucose Level and Lipid Profile of Alloxan–Induced Diabetic Rats Treated with Na-Alginate from Seaweed Turbinaria ornata (Turner) J.Agardh.”, Jurnal Teknologi. 2016; 78(4–2): 7–14. doi: 10.11113/jt.v78.8145

13. Hardoko. The Effect of Kappa-Carrageenan Consumption on Blood Glucose Level of Diabetic Wistar Rat (Ratus norwegicus) (In Indonesian with English abstract). Jurnal Teknologi dan Industri Pangan. 2006; 17(1): 67-75.

14. Yunizal. Extraction Technology of Sodium Alginate from Brown Seaweed (Phaeophyceae). in Seaweed Utilization Technology (in Indonesian). Pusat Riset Pengolahan Produk dan Sosial Ekonomi Kelautan dan Perikanan, Jakarta. 2003; 14-17.

15. Harborne JB. Phytochemical Methods: A Guide to the Modern Way of Analyzing Plants (2nd Ed.) (translated to Indonesian by Padmawinata, K.and Soediro, I.). Penerbit ITB. Bandung. 1987.

16. AOAC Official Method 991.43. Total, Soluble, and Insoluble Dietary Fiber in Foods Enzymatic Gravimetric Method, MES–TRIS Buffer. AOAC International. Washington DC. 1996. http://yiqi-oss.oss-cn-hangzhou.aliyuncs.com/aliyun/900101244/ technical_file/file_222487.pdf

17. Sancheti S. Sancheti S. Seo S.Y. Chaenomeles Sinensis: A Potent α-and β-Glucosidase Inhibitor. American Journal of Pharmacology and Toxicology. 2009; 4(1): 8-11. https://doi.org/10.3844/ajptsp.2009.8.11

18. Pourmorad F. Hossenimehr SJ. Shahabimajd N. Antioxidant Activity, Phenol, and Flavonoid Contents of Some Selected Iranian Medicinal Plants. African Journal of Biotechnology. 2006; 5(11): 1142-5. doi:10.4314/AJB.V5I11.42999

19. Salazar-Aranda R. Perez-Lopez LA. Lopez-Arroyo J. Alanıs-Garza BA. Waksman de Torres N. Antimicrobial and Antioxidant Activities of Plants from Northeast of Mexico. Evidence-Based Complementary and Alternative Medicine. 2011; Article ID 536139. https://doi.org/10.1093/ecam/nep127

20. Gaikwad SB. Mohan G.K. Rani M.S. Phytochemicals for Diabetes Management. Pharmaceutical Crops. 2014; 5(Suppl 1: M2): 11-28. doi: 10.2174/2210290601405010011

21. Septiana A.T. Asnani, A. Study of Physicochemical Properties of Brown Seaweed Extract Sargassum duplicatum Using Various Solvents and Extraction Methods (In Indonesian with English abstract). AGROINTEK. 2012; 6(1): 22-26. https://doi.org/10.21107/agrointek.v6i1.1950

22. Adekanmi AA. Adeola OH. Adekanmi U.T. Adekunle M.T. Adekanmi O.S. Adekanmi S.A. Characterization and Phytochemical Property of Okra Fruits. International Journal of Academic Engineering Research, 2020; 4(5): 34-39,

23. Nweze NO. Nwafor FI. Phytochemical, Proximate and Mineral Composition of Leaf Extracts of Moringa oleifera Lam. from Nsukka, South-Eastern Nigeria. IOSR Journal of Pharm. and Biological Sciences. 2014; 9(1): 99-103. https://doi.org/10.9790/3008-091699103

24. Paliwal R. Sharma V. Pracheta. Sharma S. Elucidation of Free Radical Scavenging and Antioxidant Activity of Aqueous and Hydro-Ethanolic Extracts of Moringa oleifera Pods. Research Journal of Pharmacy and Technology. 2011; 4(4): 566-571.

25. Hassan HE. Ahmed SH. Synergistic effect of Moringa Leaves and Antifungal on Candida albicans. Research Journal of Pharmacy and Technology. 2023; 16(3): 1369-4. doi: 10.52711/0974-360X.2023.00225

26. Jayanti RD. Wittiarika ID. Amalia RB. Winardi B. Winarsih S. Wiyasa IWA. The Effect of Moringa Leaves Aqueous Extract to Ovarian Sodium Dismutase and Apoptotic Index in Rats Treated with Depomedroxyprogesterone Acetate. Research Journal of Pharmacy and Technology. 2023; 16(5): 2103-6. doi: 10.52711/0974-360X.2023.00345

27. Cahyaningrum A. Handayani D. Soeatmadji DW. Rahayu M. Hadi S. Phytochemical Screening and Antioxidant Activity of Lombok Island Local Moringa Leaf Powder (Moringa oleifera) Predicted for Diabetes Therapy. Research Journal of Pharmacy and Technology. 2023; 16(5): 2169-6. doi: 10.52711/0974-360X.2023.00356

28. Rivai H. Yulianti S. Chandra B. Qualitative and Quantitative Analysis of Hexane, Acetone, Ethanol, and Water Extract from Bay Leaves (Syzygium polyanthum (Wight) Walp.). The Pharmaceutical and Chemical Journal. 2019; 6(3): 13-20.

29. Dewijanti ID. Mangunwardoyo W. Dwiranti A. Hanafi M. Artanti N. Short Communication: Effects of the Various Source Areas of Indonesian Bay Leaves (Syzygium polyanthum) on Chemical Content and Antidiabetic Activity. Biodiversitas. 2020; 21: 1190- 1195. https://doi.org/10.13057/biodiv/d210345

30. Nuraskin CA. Reca. Wirza. Mardiah A. Suhendra R. Faisal I. Salfiyadi T. et al Effectiveness of Guava Leaf Steep Water Against the Bacterial Growth of S. Mutans with Microdillution Method. Research Journal of Pharmacy and Technology. 2021; 14(11): 5745-8 doi: 10.52711/0974-360X.2021.00999

31. Manikandan R. Anand A.V. Kumar S. Pushpa, Phytochemical and in vitro Antidiabetic Activity of Psidium guajava Leaves. Pharmacognosy Journal. 2016; 8(4): 392-394. doi:10.5530/pj.2016.4.13

32. Venkataswamy Kumar MSN. Ramesh B. G. Ramulu A, Bharath M. Goud KS. Preparation and Evaluation of Mucilage from Fresh Leaves of Psidium guajava. Res. J. Pharma. Dosage Forms and Tech. 2018; 10(3): 125-132. doi: 10.5958/0975-4377.2018.00020.4

33. Bintarti T. Phytochemical Screening and Test of Ability as Antioxidants from Guava Leaves (Psidium guajava. L) (In Indonesian with English abstract) Jurnal Ilmiah PANNMED. 2014; 9(1): 40-44. https://doi.org/10.36911/pannmed.v9i1.341

34. El Barky AR. Hussein SA. S.A. AlmEldeen SA. Hafez YA. Mohamed T.M. Saponins and Their Potential Role in Diabetes Mellitus. Diabetes Manag. 2017; 7 (1): 148–158.

35. Hussain T. Tan B. Murtaza G. Liu G. Rahu N. Kalhoro MS. Kalhoro DH. et al Flavonoids and Type 2 Diabetes: Evidence of Efficacy in Clinical and Animal Studies and Delivery Strategies to Enhance Their Therapeutic Efficacy. Pharmacological Research. 2020; 152(February): 104629. doi:10.1016/j.phrs.2020.104629

36. Omar N. Ismail CAN. Long I. Tannins in the Treatment of Diabetic Neuropathic Pain: Research Progress and Future Challenges. Front Pharmacol. 2022; 12: 805854. https://doi.org/10.3389/fphar.2021.80585

37. Dias TR. Alves MG. Casal SP. Oliveira PF. Silva BM. Promising Potential of Dietary (Poly) Phenolic Compounds in the Prevention and Treatment of Diabetes Mellitus. Curr Med Chem. 2017; 24 (4): 334-354. doi: 10.2174/0929867323666160905150419

38. Akanbong E.P. Senol A. Devrim A.K. Phenolic Compounds for Drug Discovery: Potent Candidates for Anti-cancer, Anti-diabetes, Anti-inflammatory, and Anti-microbial. International Journal of Veterinary and Animal Research. 2021; 4(3): 115-121, https://ijvar.org/index.php/ijvar/article/view/497

39. Hassan M.A. Xu T. Tian Y. Zhong Y. Ali FAZ. Yang X. Lu B. Health Benefits and Phenolic Compounds of Moringa oleifera Leaves: A Comprehensive Review, Phytomedicine. 2021; 93: 153771. https://doi.org/10.1016/j.phymed.2021.153771

40. Mumtaz MZ. Kausar F. Hassan M. Javaid S. Malik A. Anticancer Activities of Phenolic Compounds from Moringa oleifera Leaves: In vitro and in Silico Mechanistic Study. Beni-Suef University Journal of Basic and Applied Sciences. 2021; 10: article no.12. https://doi.org/10.1186/s43088-021-00101-2

41. Kumar M. Tomar M. Amarowicz R. Saurabh V. Nair MS. Maheshwari C. Sasi M. et al Guava (Psidium guajava L.) Leaves: Nutritional Composition, Phytochemical Profile, and Health-Promoting Bioactivities. Foods. 2021; 10: 752. https://doi.org/10.3390/foods10040752

42. Jin F. Zhang J. Shu L. Han W. Association of Dietary Fiber Intake with Newly-Diagnosed Type 2 Diabetes Mellitus in Middle-Aged Chinese Population. Nutr J. 2021; 20: article no. 81,. https://doi.org/10.1186/s12937-021-00740-2

43. Chettri P. Chandran S.P. Role of Dietary Fibers in Reducing the Risk of type 2 diabetes. International Journal of Physical Education, Sports, and Health. 2020; 7 (4): 71-77. doi: 10.22271/kheljournal.2020.v7.i4b.1772

44. Feng Y. Nan H. Zhou H. Xi P. Li B. Mechanism of Inhibition of α-Glucosidase Activity by Bavachalcone. Food Sci. Technol, Campinas. 2022; 42: e123421. https://doi.org/10.1590/fst.123421

45. Luo Y. Peng B. Wei W. Tian X. Wu Z. Antioxidant and Anti-Diabetic Activities of Polysaccharides from Guava Leaves. Molecules. 2019; 24: 1343. doi:10.3390/molecules24071343

46. Dou F. Xi M. Wang J. Tian X. Hong L. Tang H. Wen A. Glucosidase and -Amylase Inhibitory Activities of Saponins from Traditional Chinese Medicines in the Treatment of Diabetes Mellitus. Pharmazie, 2013; 68: 300-304. Doi: 10.1691/ph.2013.2753

47. Bajaj S. Khan A. Antioxidants and diabetes. Indian Journal of Endocrinology and Metabolism. 2021; 16 (No Supplement 2): S267-S271. doi: 10.4103/2230-8210.104057

48. Bendary E. Francis RR. Ali HMG. Sarwat MI. El Hady S. Antioxidant and Structure-Activity Relationships (SARs) of Some Phenolic and Anilines Compounds. Annals Agricul. Sci. 2013; 58 (2): 173–81. https://doi.org/10.1016/j.aoas.2013.07.002

49. Sedjati S. Supriyantini E. Ridlo A. Soenardjo N. Santi VY. Pigmen Content, Total Phenolic Compound and Antioxidant Activity of the Seaweed Sargassum sp. Jurnal Kelautan Tropis. 2018; 21(2): 137-144. doi: https://doi.org/10.14710/jkt.v21i2.3329

50. Septiana AT. Asnani A. Antioxidant Activity of Sargassum duplicatum Seaweed Extract (In Indonesian with English abstract). Jurnal Teknologi Pertanian. 2013; 14(2): 79-86.

51. Husen SA. Wahyuningsih SPA. Ansori ANM. Hayaza S. Susilo RJK. Winarni D. Punnapayak H. et al Antioxidant Potency of Okra (Abelmoschus esculentus Moench) Pods Extract on SOD Level and Tissue Glucose Tolerance in Diabetic Mice. Research Journal of Pharmacy and Technology. 2023: 12(12): 5683-5688. doi: 10.5958/0974-360X.2019.00983.1

52. Husen SA. Setyawan MF. Syadzha MF. Susilo RJK. Hayaza S. Ansori ANM. Alamsjah MA. et al Pudjiastuti P. Awang K. Winarni DA. Novel Therapeutic effects of Sargassum ilicifolium Alginate and Okra (Abelmoschus esculentus) Pods extracts on Open wound healing process in Diabetic

Received on 16.09.2023 Modified on 19.01.2024

Research J. Pharm. and Tech 2024; 17(8):3936-3944.

DOI: 10.52711/0974-360X.2024.00611

Sample	Phytochemicals
Sample	Flavonoids	Tannins	Saponins	Steroids
Alginate drink	-	-	+	-
Green okra water extract	-	+	+	++
Alginate and green okra drink	-	-	++	-
Moringa leaf water extract	+	+	-	++
Alginate and moringa leaf drink	++	+++	+	++
Bay leaf water extract	-	+	+++	+++
Alginate and bay leaf drink	+	+	+	-
Guava leaf water extract	+	+	+++	+++
Alginate and guava leaf drink	++	+++	+++	-

Note: The (+) sign indicated the presence of the molecule while the (-) sign indicated the absence of the molecules in the fraction, triple, double, and single plus sign (+) indicated the presence of molecules in high, moderate and low concentration respectively