INTRODUCTION:

Cancer and diabetes are serious diseases whose prevalence continues to increase every year worldwide. In 2021, diabetes caused the death of 6.7 million people worldwide¹. Based on IDF data ² in 2030, diabetes sufferers will increase to 643 million people. Cancer is a metabolic disease where according to WHO the sufferers will increase from 14 million (2012) to 22 million (2032)³. Several studies explain that breast cancer is related to diabetes. One study stated that people with diabetes have a 23% greater risk of developing breast cancer than those without diabetes ⁴. High glucose levels in diabetes can support cancer development through several mechanisms, including cell proliferation and inducing resistance to apoptosis⁵. Many antidiabetic drugs have been made from herbal plants, including Dattatray et al., 2020, who succeeded in making herbal antidiabetic tablets⁶. Antidiabetic drugs have also been successfully obtained through chemical synthesis using natural flavonoids.⁷

N. alba is an aquatic plant that has many pharmacological activities. This plant contains flavonoid compounds ⁸, alkaloids ⁹ and steroids ¹⁰. N. alba leaves have antidiabetic activity ¹¹. The stem has strong antibacterial activity against S. aureus ¹² and S. pyogenes ¹³. The leaf extract has strong antifungal activity ¹⁰, and also has antiobesity activity ¹⁴. Lotus is also widely used as a cosmetic, namely polyherbal lotus oil ¹⁵. Meanwhile, R. mucronata contains alkaloid compounds ¹⁶, tannins ¹⁷, flavonoids ¹⁸. The leaf extract of R. mucronata has strong antioxidant activity¹⁹ and anti-breast cancer ²⁰. In addition, it also has antidiabetic activity ²¹. Sunita et al isolate phosphate solubilizing endophytic actinomycetes from mangrove plants ²². The milky mangrove plant, a typical Indian mangrove, contains many phenolic and tannin compounds ²³. From this explanation, it is known that traditional medicinal plants, such as N. alba and R. mucronata, offer potential advantages over synthetic drugs, including lower toxicity, cost-effectiveness, and the presence of multiple bioactive compounds that may act synergistically to enhance therapeutic outcomes.

Indonesia is an archipelago with a large water area. Mangrove plants are native to Indonesia ²⁴. Nearly 25% of all mangrove forests worldwide and nearly 50% of all mangroves in Asia are found in Indonesia²⁵. The vast wealth of mangrove forests has great potential to be developed as natural medicine. Ethanol extract of leaves of the three types of mangroves plants from Sambera Coastal Region has antidiabetic activity which can lowering blood glucose levels by 31.27%²⁶. There have been many studies examining the anti-inflammatory activity of mangroves throughout the world and their traditional use for health²⁷. The study from hari et al, 2019 showed that ethanolic extract of R. mucronata poir has significant antihyperhomocysteinemic and procoagulant factors lowering potential in hyperhomocysteinemic rats²⁸. Kumari et al., 2020 were found that mangrove plant Bruguiera gymnorhiza from India to be effectively inhibit the growth of pathogenic (S.aureus, B.subtilis, P. aeruginosa and S. typhi) ²⁹.

To the best of our knowledge, this is the first report examining the combined antidiabetic and anti-breast cancer effects of extracts derived from two aquatic plants: white lotus and mangrove. This research could provide a foundational framework for the development of natural therapeutics for breast cancer and diabetes, potentially offering an alternative to synthetic drugs with fewer adverse effects. Furthermore, these findings may support the targeted isolation and characterization of bioactive compounds responsible for the observed pharmacological activities of the plant extracts. In this study, mangrove leaf and white lotus leaf extracts were combined with a ratio of mangrove leaf concentration: white lotus respectively of 25%: 75%; 50%: 50% and 75%: 25%. The extracts were evaluated in vitro for their anti-diabetic potential through inhibition of the alpha-glucosidase enzyme, and their anti-cancer efficacy was assessed against MCF-7 human breast cancer cells. The objective was to identify the optimal combination ratio that exhibits the highest efficacy in both anti-diabetic and anti-cancer activities.

MATERIALS AND METHODS:

Materials:

White lotus leaves were taken from Kuningan, West Java, Indonesia. Meanwhile, mangrove leaves were taken from Indramayu, West Java, Indonesia. Both plants have been determined at the UPT Herbal Materia Medica Laboratory, Batu, Malang, East Java, Indonesia. With determination number 00.9.3/1743/102.20.2024 for white lotus and 00.9.3/1876/102.20.2024 fot mangrive. The tools used in this study were chemical glassware, rotary evaporator (BUCHI), analytical balance (OHAUS), 1.5 mL microtube (MCTB015), 15 mL tube (Biologix 109151 brand), 75 ml T-flask (NEST 708001), 96 well plate (NEST 701001), Biosafety Cabinet (BSC) (Thermoscientific1300 seriesa 2), Centrifuge (Thermoscientific micro CL17), CO2 Incubator (Thermoscientificseries 8000 DH), Microscope (Thermoscientific EVOSXLCore), Multimode Reader (TecanInfinite M200 PRO), FTIR. Materials The materials used in this study were Cisplatin (EDQM C2210000), Antibiotics (Sigma Aldrich P4333), DMSO (Merck D1435), PBS (Gibco 18912-014), Resazurin Sodium Salt-Powder, BioReagent (Sigma Aldrich R7017), RPMI (Gibco 11875-093), Fetal Bovine Serum (FBS) (Gibco 10270-106), Trypsin-EDTA (Gibco 25200-056), Trypan Blue (Sigma Aldrich T-8154). Phosphate buffer pH 7.0, p-nitrophenyl-a-D- glucopyranoside, a-Glucosidase (EC 3.2.1.20) type I: from Saccharomyces cereviceae, bovine serum albumin, Sodium bicarbonate, sodium carbonate, silica gel GF254 were purchased from Wako Pure Chemical Industries, Ltd (Osaka, Japan). All solvents in this study were distilled.

Extraction:

Mangrove leaves and white lotus were cleaned and dried. Then, ground to powder using a blender to obtain a weight of 5.576 Kg and 800 grams, respectively. Then, 1000 grams of mangrove leaves were macerated with 96% ethanol for 3x24 h. The maceration results were then filtered and evaporated with a rotary evaporator at 40⁰C, leaving 138 g of concentrated ethanol extract of leaves mangroves. In the same way, 400 g of dry powder of white lotus was macerated with ethanol, and concentrated yielding 26.9 g of concentrated ethanolic extract of white lotus.

Preparation of extract combination:

Mangrove leaf extract and white lotus were then combined in the following way: combination Extract A was prepared by mixing 2.5 g of mangrove extract with 7.5 g of white lotus extract. Combination Extract B consisted of an equal mixture of 5.0 g mangrove extract and 5.0 g white lotus extract. Combination Extract C was prepared by mixing 7.5 g of mangrove extract with 2.5 g of white lotus extract. Each combination yielded a total of 10 g of extract. Against the extract of mangrove leaves, white lotus leaves, combination extract A, combination extract B, and combination extract C, an antidiabetic test were then carried out on the alpha-glucosidase enzyme and an anticancer activity test on MCF-7 breast cancer cells as well as its phytochemical test.

No	Name of sample	Ratio of mangrove leaf extract: white lotus leaf extract
1	A	2.5 g : 7.5 g
2	B	5.0 g : 5.0 g
3	C	7.5 g : 2.5 g

Phytochemical Test:

Phytochemical tests were carried out by referring to the test ³⁰. Mangrove leaf and white lotus leaf extract then analyzed to determine the presence of tannin (FeCl₃ 1%), saponin (Foam test), flavonoid (Conc HCl+ Mg) and alkaloid compounds (Wagner).

Antidiabetic test:

The antidiabetic test conducted refers to³¹. 250 μL solution of p-nitrophenyl-α-D-glucopyranoside 5 mM and 400 μL of phosphate buffer pH 7 0.1 M were added to a test tube containing 100 μL (standard solution or sample solution) in DMSO with concentration variations as above. 250 μL of α-glucosidase solution (0.062 units) was added to the homogenous solution after it had been pre-incubated for 5 minutes at 37°C. The incubation was then continued for 15 minutes. One milliliter of 0.2 M Na₂CO₃ was added to stop the reaction. The absorption reading of p-nitrophenol produced at λ 400 nm was used to quantify enzyme activity. As a reference standard, quercetin has been used. Testing was conduct three replicated.

The following formula was used to measure the presentation of inhibitory activity:

(C – S) x 100

% Of Inhibition = -----------------

C= Absorbance of the blank (DMSO)
S = Absorbance of the sample (calculated as the difference in absorbance with and without enzyme treatment)

Anticancer test:

Cell culture and Positive control:

The MCF-7 HTB-22 ATCC breast cancer cells were cultivated in RPMI complete liquid culture mix, which contained 50 µL/50 mL of antibiotics and 10% fetal bovine serum (FBS). The culture incubated an incubator with 5% CO2 at 37 °C. Cisplatin was used for the test's positive control.

Cell viability assay:

The cell culture was seeded on a 96-well plate at 37°C with 5% CO₂ gas until the cell growth percentage approached 70%. The working reagent, resazurin, was administered to the cells after they had been treated with extract and cultured for 48 hours at 37°C with 5% CO₂ atmosphere. A Multimode Reader was used for the absorbance readings. In a tube, prepare 9 mL of media and add 1 mL of "Resazurin Sodium Salt Powder, BioReagent" (10 µL of reagent for 90 µL of media). Then, add 100 µL of the solution mixture to each microplate well and incubate for 1–2 hours until a color change occurs (the Resazurin Sodium Salt reagent will be reduced from the blue resazurin compound without intrinsic fluorescent value, to the red and highly fluorescent resorufin compound when it enters living cells). While the conversion value is correlated with the quantity of metabolically active cells, it is quantitatively measurable. Absorbance is measured using the resazurin and resorufin absorbance spectra. Additionally, a multimode reader is used to measure the absorbance at 570 nm (reference: 600 nm). Moreover, the IC₅₀ value indicates the concentration at which 50% of cell growth is stopped. A plot displaying the percentage of cytotoxicity to sample concentration displays the cytotoxicity 50% (IC₅₀)³².

Statistical Analysis:

One-way ANNOVA was used for statistical analysis with a confidence level of α = 0.05.

RESULT:

Phytochemical Screening of Mangrove and White Lotus Leaf Extracts

Table 1. Phytochemical constituents of R. mucronata and N. alba leaf extracts

No.	Chemical Constituents	Mangrove Leaves	White Lotus Leaves
1	Flavonoid	+	+
2	Alkaloid	+	+
3	Tannin	+	+
4	Saponin	+	+

Results of Antidiabetic Test:

Table 2. Results of Antidiabetic Test

No	Sample	IC₅₀ (µg/mL)	Category
1	Quersetin (comparative compound)	3.82 ± 0.28	Higly active
2	Mangrove	19.90 ± 0.62	Moderately active
3	White Lotus	33.60 ± 6.85	Moderately active
4	A	7.82 ± 1.30	Highly active
5	B	2.62 ± 1.93	Highly active
6	C	1.68 ± 0.70	Highly active

Inhibition graph of samples on alpha glucosidase enzyme:

Figure 1. Inhibition graph of mangrove leaves, white lotus leaves and their combination on alpha glucosidase enzyme

Results of anticancer test of samples against MCF-7 breast cancer cells:

Table 3. Results of anticancer test of samples against MCF-7 breast cancer cells

No.	Sample	IC₅₀ (µg/mL)	Category
1	Cisplatin (comparative compound)	29.36	Active
2	Mangrove	50.11	Active
3	White Lotus	405.50	Sufficiently active
4	A	485.90	Sufficiently active
5	B	148.40	Mederately active
6	C	76.26	Active

MCF-7 cancer cell viability

Figure 2. Graph of MCF-7 cancer cell viability after being given a single extract and a combination of extracts

Live cells in various sample concentrations:

Figure 3. Graph of % live cells against extract concentration. Note: p-value is shown as follows: ns (not significant), *p<0.05 indicates a significant difference

Results of cancer cell observations with a microscope

(a)

(b)

(c)

Figure 4. Results of cancer cell observations with a microscope. Morphology of MCF-7 cancer cells, negative control (without extract treatment (a). Morphological changes due to administration of extract with a concentration of 125 μg/mL: sample C (b), mangrove sample (c).

FTIR spectroscopy

DISCUSSION:

White lotus and mangrove are known to have many pharmacological activities. The combination of these two extracts in certain concentrations will increase pharmacological activity. In this study, ethanol extracts of lotus, mangrove and combined extracts were tested for cancer cell activity against MCF-7 breast cancer cells and tested for antidiabetic activity, namely by the alpha glucosidase inhibitory assay. The results of the test are as follows:

Phytochemical Test:

The results of the phytochemical test showed that the ethanol extract of mangrove leaves and white lotus contained flavonoids, alkaloids, tannins and saponins (Table 1). The presence of alkaloid content from mangrove leaves was also confirmed from research ³³ which successfully identified the presence of benzamide compounds from lotus leaves. The content of tannin compounds in white lotus leaves was confirmed from research which successfully isolated tannin compounds and other phenolic compounds from white lotus leaves ³⁴

Antidiabetic Activity Assay or In vitro Alpha-Glucosidase Inhibition Assay:

Inhibition test of alpha glucosidase activity based on in vitro enzymatic reactions. Quercetin was used as a reference standard. Table 2 and Figure 1 show the antidiabetic results from the tests.The in vitro antidiabetic activity can be determined by the inhibition of the alpha-glucosidase enzyme, as indicated by the IC₅₀ value. Antidiabetic activity can be seen from the IC₅₀ value ³⁵. As shown in Table 2, the IC₅₀ values for the mangrove leaf extract and white lotus leaf extract are 19.90 µg/mL and 33.60 µg/mL, respectively, both falling into the very strong category. Notably, the combination of these two extracts exhibits significantly enhanced antidiabetic activity compared to the individual extracts, with an IC₅₀ value below 10 µg/mL. This indicates a synergistic effect of the two extracts in terms of antidiabetic activity. What is particularly remarkable is that the IC₅₀ value for sample C is substantially lower than that of quercetin, which was used as a standard comparison compound. This finding suggests that the combination of mangrove and white lotus leaf extracts, particularly at a ratio of 75:25, holds great potential for development into a natural antidiabetic agent. When comparing combination A, B, and C, it is evident that increasing the proportion of mangrove leaf extract enhances the antidiabetic activity, likely because the mangrove leaf extract on its own exhibits superior antidiabetic effects compared to the white lotus extract. The results were further analyzed using the one-way ANOVA test, which yielded a p-value < 0.05, indicating a statistically significant difference between the mean IC₅₀values of the six samples. The graph presented in Figure 1 demonstrates the percentage of inhibition of the alpha-glucosidase enzyme for each combination of extracts, showing that a higher concentration of mangrove leaf extract leads to greater enzyme inhibition. Interestingly, the percentage inhibition for extract combination C exceeds that of the single mangrove extract.

Anticancer test:

The Resazurin technique was used to conduct the anticancer test. The concept behind this test is that the blue dye resazurin will be transformed to the red and intensely fluorescent resorufin compound when the reagent enters living cells. Since the conversion value is correlated with the quantity of metabolically active cells, it is quantitatively measurable ³⁶.

Anticancer activity can be categorized based on its IC₅₀ value³⁷. Table 5 shows that the anticancer activity of each extract is for mangrove 50.11 μg/mL with a moderate category and for lotus leaf 405.50 μg/mL with amoderate category. Meanwhile, the combination of mangrove and lotus leaf extracts gives an IC₅₀ value greater than 50 μg/mL. Figure 2 shows a graph of % cell viability against the concentration of the extract given. It can be seen that in the combination of concentrations (A-C), the greater the concentration of mangrove extract in the given combination of concentrations, the smaller the cell viability will be, where the best activity is combination C.

The reduction in the number of cells was found to limit cell proliferation and indicate disruption of cell structure. Morphological changes due to sample extract treatment were observed under a microscope (Thermoscientific EVOSXL Core). Treatment with sample extract at a dose of 125 μg/mL (Figure 4) changed the appearance of normal cell morphology. The MCF-7 stem cell line showed a regular spindle shape with smooth cell membrane boundaries. The changes found were irregular, aggregated indicating damaged cells. Cancer cells given mangrove extract (Figure 4.c) and combination extract C (Figure 4.b), showed many damaged and dead cells when compared to other extracts. This is comparable to the IC₅₀ values of these two samples, which are respectively 50.11 µg/mL and 76.26 µg/mL, both of which have the highest anticancer activity compared to other samples. Sample C contains a higher concentration of mangrove leaf extract, which is 75%, so it has higher anticancer activity compared to the combination of samples A and B. These results indicate that the combination of mangrove and lotus leaf extracts has the potential as an anti-breast cancer agent.

Next, a graph is made between the % of living cells and the concentration of the extract as shown in Figure 3. From here, it can be seen that there is a drastic decrease in living cells at a concentration of mangrove leaf extract of 125 µg/mL with a number of living cells of 45.7% as well as sample C with a number of living cells of 48.35%. Furthermore, a statistical analysis was performed using one-way ANNOVA test on each concentration of the extract tested and the results showed that there was a significant difference in the average % of living cells at each concentration tested with a p value <0.05%.

Identification of Secondary Metabolites by FTIR Spectroscopy:

The presence of secondary metabolite compounds from mangrove and lotus leaf extracts in this study was analyzed using FTIR (Figure 5). The results of the test showed the presence of alkaloid compounds contained in mangrove leaves. This is indicated by the absorption at wave number 3383.14 cm^-1 which indicates vibrational stretching absorption from the N-H group. This is supported by the absorption at 1525.69 cm^-1 which is the bending vibration absorption of the N-H group. Absorption at 1055.06 cm^-1 indicates vibrational stretching absorption from the C-O group. Absorption at 1379.10 cm^-1 indicates vibrational stretching of C-N. The presence of the amide group is known from the absorption at 1610.56 cm^-1 which is the vibrational stretching absorption of the C=O amide group. The FTIR spectral data support the presence of alkaloid compounds in mangrove leaves, alkaloids contained in mangrove leaves, this is also reinforced by the results of phytochemical tests that show positive results in alkaloid testing. Previous research ³⁸ showed that mangrove leaves contain benzamide alkaloid compounds that can be identified by LC-MS. Where, there is an m/z value of 279.2093 [M + H]⁺ and m/z 301.1956 [M + Na]⁺ which indicates the presence of a compound with a molecular weight of 278 g/mol with the molecular formula C₁₇H₁₄N₂O₂ which is identified as a benzamide alkaloid compound.

The chemical that may be in the responsibility of mangrove leaves' anticancer properties is benzoamide. The mechanism of action of benzamide as an anticancer is that benzamide is able to trigger the caspase cascade through the mitochondrial pathway and arrest cells in the G2/M phase. Compounds that can trigger apoptosis in vitro are N-substituted benzamide metoclopramide (MCA, neu-sensamide) and its structural equivalent 3-chloroprocainamide(3CPA, declopramide). Furthermore, in vivo chemotherapeutic and radiosensitizing effects of MCA and 3CPA have been demonstrated in human and murine tumor models ³⁹. Many studies have examined the antidiabetic activity of benzamide. Benzamide derivatives have shown importance in the treatment of diabetes as glucokinase activators. This enzyme plays a role in transferring ATP into glucose to produce glucose-6-phosphate which is the first stage in glycolysis. Research from⁴⁰ shows that several functional groups of benzamide can be optimized to increase benzamide activity as a glucokinase activator. Research from ⁴¹ which successfully synthesized a benzamide derivative compound then tested its antidiabetic activity against the alpha glucosidase enzyme and it was found that this compound was able to inhibit the alpha glucosidase enzyme with an IC₅₀ value of 10.13 µM.

The FTIR method was also used to determine the content of secondary metabolite compounds from white lotus in this study. From the FTIR results (Figure 5), it was found that there was absorption at a wave number of 3300-2600 cm^-1 which was very broad indicating the presence of vibrational stretching absorption from the O-H group originating from carboxylic acid. The presence of carboxylic acid can also be strengthened by the presence of vibrational stretching of C = O at a wave number of 1710.86 cm^-1. The presence of a C = C double bond in the compound structure is indicated by the presence of vibrational stretching at 1637.56 cm^-1. This FTIR spectrum also shows the presence of a C-O group indicated by absorption at 1055.66 cm^-1. From the analysis of this FTIR spectrum, the presence of phenolic compounds in white lotus leaves can be identified. The presence of tannins is also strengthened by the results of phytochemical tests which show positive results in the tannin test. Previous research has shown that white lotus leaves contain many phenolic compounds including vanillic acid, gallic acid, tannin, caffeic acid, p-coumaric acid, chlorogenic acid, ferulic acid, and brevifolin³⁴.

One promising anticancer agent is tannin. if exposed to tannin extract, breast and prostate cancer cells exhibited an increase in apoptotic activity. Black bean-derived condensed tannins prevented the proliferation of the prostate cancer cells DU 145, breast cancer cells MCF-7 and Hs578T, and colon cancer cells Caco-2. Cell cytotoxicity assays showed decreased ATP levels in cancer cells treated with tannin. Decreased ATP levels imply decreased cell proliferation and migration activities. Gross morphological examination of cells treated with tannin showed that cell death occurred through apoptosis ⁴². Tannin was able to inhibit the alpha-glucosidase enzyme with an IC50 value of 0.35 µM. Molecular docking results showed that the interaction between tannin and α-glucosidase was mainly driven by hydrogen bonds, electrostatic interactions, and hydrophobicity ⁴³.

CONCLUSION:

This study indicate that mangrove leaves (R. mucronate) and white lotus leaves (N. alba) contain secondary metabolites, including flavonoids, alkaloids, saponins, and tannins. FTIR analysis confirmed the presence of alkaloids in mangrove leaves and phenolic compounds in white lotus leaves. Notably, the mangrove leaf extract exhibited superior antidiabetic and anticancer activities compared to the lotus leaf extract, with IC₅₀ values of 19.90 ± 0.62 µg/mL and 50.11 µg/mL, respectively. The optimal combination of extracts was identified as a 75:25 ratio of mangrove to lotus (combination C), yielding IC₅₀ values of 1.68 ± 0.70 µg/mL (highly active) for antidiabetic activity and 76.26 µg/mL (active) for anticancer activity. These results suggest that the synergistic combination of N. alba and R. mucronata extracts holds significant potential for development as natural agents for antidiabetic and anticancer therapies.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

We would like to thank to Direktorat Riset, Teknologi, dan Pengabdian kepada Masyarakat (DRPTM) of the Ministry of Research, Technology and Higher Education Indonesia for funding this research through the Penelitian Fundamental scheme.

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Received on 25.11.2024 Revised on 10.06.2025

Accepted on 17.10.2025 Published on 13.01.2026

Available online from January 17, 2026

Research J. Pharmacy and Technology. 2026;19(1):364-371.

DOI: 10.52711/0974-360X.2026.00053

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