INTRODUCTION:

Black turtle bean (Phaseolus vulgaris L.) is a legume variety of black bean, derived from the Fabaceae family¹. It is cultivated in the hilly regions and is popularly consumed in the tropical and sub-tropical countries². Their rich history dates back to ancient civilizations, originating from the Mesoamerica and the Andes³. In India, black turtle bean has been the staple food for tribal population and is also used in the preparation of food for traditional festivals⁴.

Nutritionally, black turtle bean is a powerhouse, packed with essential nutrients. They are an excellent source of complex carbohydrates and plant protein, low in fat and rich in dietary fiber⁵. They have a good one-to-one ratio of proteins and fiber which is unique and unparallel with other foods⁶. Additionally, they have comprehensive profile of micronutrients with higher folate and minerals namely, calcium, magnesium, potassium and iron⁵. Black turtle bean possesses a diverse array of phytochemicals, owing to their dark seed coat⁷. Previous studies have highlighted the promising health benefits of black turtle bean (Phaseolus vulgaris L.) in various in vitro models. Priyadarshini and Beatrice⁷ reported significant anticancer activity of black turtle bean extracts against breast and colorectal adenocarcinoma cells in a pre-clinical study. Similarly, Abdulrahman et al.⁸ demonstrated strong antioxidant and antidiabetic properties, supporting its role in oxidative stress reduction and glycemic control. Tan et al.⁹ further observed that phenolic compounds in black turtle beans exhibited considerable inhibitory effects on α-amylase, α-glucosidase, and lipase, suggesting potential applications in the dietary management of diabetes and hyperlipidemia.

Despite their rich nutritional and phytochemical profile and promising health benefits, black turtle bean remains underutilized⁷. This could primarily be due to the limited awareness of the bean, and to our knowledge, only few studies have investigated its multifunctional health benefits in the Indian context. Hence, the present study aimed to study the anti-cholesterol, anti-hypertensive, anti-ulcer and anti-microbial activities of raw and cooked black turtle bean. This study will bring new and basic insights into the suitability of black turtle bean as a functional food for managing lifestyle-related disorders.

MATERIALS AND METHODS:

Sample collection and extract preparation:

Black turtle bean (Phaseolus vulgaris L.) was procured from a farmer in Yercaud hills, Salem district, Tamil Nadu, India. The beans were washed, dried and cleaned to remove stones or chaff of plant parts. Aqueous extracts from raw and cooked black turtle beans were prepared to investigate the in vitro anti-cholesterol and anti-hypertensive activities. To prepare the raw extract, raw black turtle beans were coarsely ground and kept in water bath with distilled water (1:10w/v) for 10minutes. The mixture was centrifuged, filtered and stored in glass bottle. To prepare the cooked extract, raw black turtle beans were soaked in water (1:5 w/v) overnight for 8 hours and pressure-cooked for a duration of 12minutes. Homogenization of the cooked beans was done by blending with distilled water and the blended mixture was centrifuged, filtered and stored in glass bottle for further analysis.

Anti-cholesterol activity:

The anti-cholesterol activity of raw and cooked black turtle bean was evaluated using cholesterol esterase inhibition assay as per the procedure given by Pietsch and Gu¨tschow¹⁰. Briefly, pancreatic cholesterol esterase (lyophilized CEase) was dissolved in 1mL of sodium phosphate buffer (100mM, pH 7.0) and separated into 200µL aliquots. One aliquot was thawed and diluted to a concentration of 5µg/mL using the same buffer. The raw and cooked extracts of black turtle bean were taken at concentrations of 20, 40, 60, 80, 100 and 120µg/mL and added to the reaction mixture (50µL). One mL of Triton X-100 (5%, w/w), 50μL of p-nitrophenyl butyrate (p-NPB) (0.05M in acetonitrile), and 850μL of assay buffer (100mM sodium phosphate, 100mM NaCl, pH 7.0) are added to the reaction mixture. This mixture was thoroughly mixed for 5minutes at 25^oC and the reaction was initiated by adding 50μL of the CEase solution (5 μL). The resultant mixture was then incubated at 25^oC for 15minutes and the absorbance was measured using spectrophotometer at a wavelength of 405nm. Simvastatin was used as the standard. The percentage of inhibition was calculated using the formula: (A-B)/A x 100, where A and B are the absorbance without and with extracts, respectively.

Anti-hypertensive activity:

The anti-hypertensive activity of raw and cooked black turtle bean was evaluated using ACE inhibition assay as described by Tutor et al.¹¹. Briefly, the assay mixture was prepared with Sodium borate buffer (0.05M, pH 8.2) containing 0.3 M NaCl. The raw and cooked extracts of black turtle bean were taken at concentrations of 20, 40, 60, 80, 100 and 120µg/mL and added to the buffer solution. A substrate solution of 5mM hippuryl-histidyl-leucine (HHL) was prepared and ACE enzyme extract was added to assay mixture and was kept for pre-incubation at 37^oC for 10 minutes. After pre-incubation, additional HHL substrate was added to initiate ACE enzyme reaction and was kept for further incubation for 30 minutes at 37^oC. After incubation period, 50 µL of HCl (1 M) was added to halt the enzymatic activity and 100µL of pyridine and 50µL of benzenesulfonyl chloride was added to develop yellow colour. The absorbance was measured at 410nm using a spectrophotometer. Captopril was used as the standard. The percentage of inhibition was calculated using the formula:

Percentage of inhibition = 1- Abs inhibited – Abs initial

Abs uninhibited

Anti-ulcer activity:

The anti-ulcer activity as assessed using urease inhibition assay was carried as per the procedure given by Weatherburn¹² with slight modifications. Aqueous extracts of raw and cooked black turtle beans was taken at concentrations of 20μg/mL, 40μg/mL, 60μg/mL, 80 μg/mL, 100μg/mL, and 120μg/mL and was incubated with urease enzyme and buffer solution containing urea, EDTA and phosphate buffer (pH 8.2) for 60 minutes at 37°C. Phenol reagent (1% phenol and 0.005% sodium nitroprusside) and alkali reagent (5% NaOH and 0.1% NaOCl) were added to each tube after the incubation period and the tubes were kept for further incubation for 30minutes. The absorbance of the reaction mixture was measured at 625nm using a spectrophotometer. The percentage inhibition of urease activity is calculated using the formula: 100 - (test OD / Control OD) × 100.

The ATPase inhibition assay method was carried to assess the anti-ulcer activity of raw and cooked black turtle beans as per the procedure given by Gupta et al.¹³. Briefly, a reaction mixture of goat parietal cell extract (300μg) was pre-incubated with the raw and cooked black turtle bean extract at varying concentrations (20 μg/mL, 40μg/mL, 60μg/mL, 80μg/mL, 100μg/mL, and 120μg/mL) for 60 minutes at 37°C. A substrate of 2mM ATP (200μL), 2mM magnesium chloride (200μL) and 10mM potassium chloride (200μL) were added to the mixture to initiate reaction and kept for additional 30 minutes incubation at 37°C. After the incubation period, ammonium molybdate (4.5%) and perchloric acid (60%) were added to terminate the reaction and the mixture was centrifuged for 10minutes at 2000rpm. The supernatant (1mL), Millipore water (4mL), 2.5% of ammonium molybdate (1mL) and ANSA (0.4 mL) were added and the absorbance was measured at 660nm using spectrophotometer. The percentage of enzyme inhibition was using the formula:

Percentage of inhibition = Activity (control) - Activity (test) × 100

Activity (control)

Anti-microbial activity:

The anti-microbial activity of raw and cooked black turtle bean was assessed using well diffusion method for bacterial strains namely Bacillus subtilis, E. coli, Pseudomonas aeruginosa and staphylococcus aureus and fungal strains such as Candida albicans and Aspergillus niger. The anti-bacterial assay was carried out using the procedure described by Sivaraj et al.¹⁴ and the anti-fungal assay was carried out as per the procedure outlined by Dhanalakshmi et al.¹⁵ and Pandian et al.¹⁶.

Briefly, nutrient agar was prepared as a growth medium for the bacterial strains, while potato dextrose agar was prepared as the growth medium for the fungal strains. Under aseptic conditions, in a wooden box with short-wave UV light, the respective agar plates were poured, covered and inverted after solidification. The microorganisms were streaked on to the plates with solidified growth medium and wells were created using a previously sterilized 8 mm cork borer. The aqueous extract of raw and cooked black turtle beans was poured in to the wells of the streaked plates at varying concentrations (25μg/mL, 50μg/mL, 75μg/mL, 100 μg/mL). Tetracycline was the standard/reference compound for bacterial strains and amphotericin B for fungal strains. The plates were labelled and incubated at 37°C for 24hours to facilitate bacterial and fungal growth. At the end of the incubation period, if present, the zone of inhibition around the wells were measured.

Statistical Analysis:

The statistical analysis of the collected data was carried out using SPSS (Version 25.0). The significance level (p value) was set as <0.05. The in vitro assays were carried out in triplicates and descriptive analysis followed by one way ANOVA and tukey test were done¹⁷.

RESULT:

The results of the anti-cholesterol and anti-hypertensive activities of raw and cooked black turtle beans are presented in figure 1 and 2, respectively. The results of the anti-ulcer activity of raw and cooked black turtle bean as assessed by its ability to inhibit the activity of urease and ATPase are presented in figure 3 and 4, respectively. The half maximal inhibition potential (IC₅₀) for anti-cholesterol, anti-hypertensive and anti-ulcer activities of standard, raw and cooked black turtle beans are presented in table 1. The results of the anti-microbial activity of raw and cooked black turtle beans against bacteria and fungi are also discussed as follows.

Anti-cholesterol activity:

The anti-cholesterol activity of raw and cooked black turtle bean as assessed by its ability to inhibit the activity of cholesterol esterase enzyme showed a concentration dependent inhibition (figure 1). At 20 µg/mL concentration, the inhibition percentage of raw and cooked black turtle bean was found to be 2.58± 0.19% and 3.37±0.06% respectively, and at 120µg/mL concentration, the inhibition percentage increased significantly to 26.38±0.16% and 31.74±0.12% (p<0.001).

Figure 1. Cholesterol esterase inhibition activity of raw and cooked black turtle beans

Table 1 shows that the IC₅₀ for anti-cholesterol activity of raw and cooked black turtle beans is in the following order: standard (58.91±1.61µg/mL) >cooked black turtle bean (78.00±0.74µg/mL) >raw black turtle bean (82.29±0.14µg/mL). Lower IC₅₀ value indicates greater inhibitory potential. It can be confirmed that the cooked black turtle bean has higher ability to inhibit the activity of cholesterol esterase enzyme than the raw black turtle bean and comparable to the standard, thereby exhibiting excellent anti-cholesterol activity.

Anti-hypertensive activity:

The ability of raw and cooked black turtle bean to inhibit the activity of angiotensin-converting enzyme (ACE) were found to be concentration dependent (figure 2). At 20µg/mL concentration, the inhibition percentage of raw and cooked black turtle bean was found to be 5.11±0.12% and 6.25±0.23% respectively, and at 120 µg/mL concentration, the inhibition percentage increased significantly to 47.23±0.29% and 52.36± 0.08%, respectively (p<0.001).

Figure 2. ACE inhibition activity of raw and cooked black turtle beans

From table 1, it can be observed that IC₅₀ for anti-hypertensive activity of raw and cooked black turtle beans is in the following order: standard (24.34±0.00 µg/mL) >cooked black turtle bean (77.76±1.59µg/mL) > raw black turtle bean (127.03±0.81µg/mL). This shows that the cooked black turtle bean has higher ability to inhibit the activity of angiotensin-converting enzyme than the raw black turtle bean and comparable to the standard, thereby exhibiting good anti-hypertensive activity.

Anti-ulcer activity:

The anti-ulcer activity of raw and cooked black turtle bean showed a concentration dependent inhibition against urease (figure 3). At 20µg/mL concentration, the inhibition percentage of raw and cooked black turtle bean was found to be 2.58±0.07% and 5.02±0.06% respectively, and at 120µg/mL concentration, the inhibition percentage increased significantly to 30.27± 0.20% and 37.39±0.21% (p<0.001).

Figure 3. Urease inhibition activity of raw and cooked black turtle beans

Table 1 shows that the IC₅₀ for anti-ulcer activity of raw and cooked black turtle beans is in the following order: standard (29.81±0.00µg/mL) > cooked black turtle bean (160.46±0.91µg/mL) > raw black turtle bean (198.23± 1.35µg/mL). It can be confirmed that the cooked black turtle bean has higher ability to inhibit the activity of urease enzyme than the raw black turtle bean and comparable to the standard, thereby exhibiting excellent anti-ulcer activity.

The ability of raw and cooked black turtle bean to inhibit the activity of ATPase were found to be concentration dependent (figure 4). At 20µg/mL concentration, the inhibition percentage of raw and cooked black turtle bean was found to be 8.89±0.07% and 15.47±0.43% respectively, and at 120µg/mL concentration, the inhibition percentage increased significantly to 44.45±0.23% and 60.03±0.26%, respectively (p<0.001).

Figure 4. ATPase inhibition activity of raw and cooked black turtle beans

From table 1, it can be observed that IC₅₀ for anti-ulcer activity of raw and cooked black turtle beans as assessed by its ability to inhibit ATPase activity is in the following order: standard (51.92±2.07µg/mL) > cooked black turtle bean (93.80±0.42µg/mL) > raw black turtle bean (160.06±40.61µg/mL). This shows that the cooked black turtle bean has higher ability to inhibit the activity of ATPase enzyme than the raw black turtle bean and comparable to the standard, thereby exhibiting excellent anti-ulcer activity.

Table 1. Half maximal inhibition potential (IC50) for anti-cholesterol, anti-hypertensive and anti-ulcer activities of standard, raw and cooked black turtle beans

Sample	Anti-cholesterol activity (µg/mL)		Anti-hypertensive activity (µg/mL)		Anti-ulcer activity (µg/mL)
Sample	Cholesterol esterase inhibition activity	p value	ACE inhibition activity	p value	Urease inhibition activity	p value	ATPase inhibition activity	p value
Standard	58.91 ± 1.61^a	0.000	24.34 ± 0.00^a	0.000	29.81 ± 0.00^a	0.000	51.92 ± 2.07^a	0.000
Raw Black Turtle Bean	82.29 ± 0.14^b		127.03 ± 0.81^b		198.23 ± 1.35^b		160.06 ± 40.61^b
Cooked Black Turtle Bean	78.00 ± 0.74 ^c		77.76 ± 1.59^c		160.46 ± 0.91^c		93.80 ± 0.42^ac

Different superscripts indicate significant difference at p<0.01

Anti-microbial activity:

The anti-microbial activity of raw and cooked black turtle bean was assessed for its ability to inhibit bacteria and fungi. It was found that the raw and cooked black turtle beans did not have any inhibitory effect against bacteria (Bacillus subtilis, E. coli, Pseudomonas aeruginosa and staphylococcus aureus) and fungi (Candida albicans and Aspergillus niger) when compared to the standard.

DISCUSSION:

Inhibition of cholesterol esterase enzyme activity is crucial in limiting the absorption of dietary cholesterol, as they produce cholesterol and free fatty acids by hydrolysing cholesteryl esters, making it readily absorbable into the blood through the intestine¹⁸. Therefore, reduction in cholesterol levels accounts to lowered risk of cardiovascular diseases¹⁹. To our knowledge this is the first study to report the in vitro cholesterol esterase inhibition activity of raw and cooked black turtle beans. According to Li et al.²⁰, the proposed mechanism behind the cholesterol esterase inhibitory potential could be attributed to the polyphenols, especially, procyanidin B2 and tannin acid.

ACE is a key component in the renin-angiotensin-aldosterone system, that is responsible for elevated blood pressure levels, as they convert angiotensin-I to angiotensin-II^21,22. Treatment regiments targeting the inhibition of ACE will help to lower blood pressure levels. Consistent with the results of the in vitro anti-hypertensive activity of black turtle beans in the present study, published literature on black turtle bean also confirms their anti-hypertensive activity via the inhibition of ACE^23,24. The ACE inhibitory activity of raw and cooked black turtle beans could be attributed chiefly to its phenolic compounds and flavonoids²⁵. Zhang et al.²³ reported that protocatechuic acid (anthocyanin), a type of polyphenol belonging to flavonoid family, possess higher inhibitory activity against ACE. This compound is abundantly present in black turtle bean²⁴. Tawalbeh et al.²⁶ states that legume peptides produce ACE inhibitors which bind to the active ACE sites, inhibiting ACE activity and thereby lowering blood pressure levels.

Ulceration is defined as the formation of sores or disruption of mucosal integrity of the gastrointestinal tract. It is often chronic in nature and is associated with cancer and other diseases^27,28. H.pylori is reported to be the main culprit behind the onset of ulceration²⁹, that survives well in the acidic environment by secreting urease, an enzyme that breaks down urea into ammonia and carbon dioxide which in turn, enables them to cause ulceration³⁰. Thus, inhibition of the activity of urease enzyme will thus expose the bacterium to the acidic environment, thereby leading to destruction of H.pylori and lowering ulceration. Another mechanism by which gastric ulceration can be controlled is through the inhibition of ATPase enzyme, that functions as gastric proton pump, leading to lower production of gastric acid and reduced risk of ulceration^31,32,33. To our best knowledge, no study was found to assess the urease and ATPase inhibitory activities of black turtle bean. The present study confirms that raw and cooked black turtle beans have the potential to inhibit the activity of urease and ATPase, thereby exhibiting anti-ulcer activity. The anti-ulcer activity is primarily attributed to the flavonoids^34,35, which is abundantly present in raw and cooked black turtle beans⁷. Further, saponins are also reported to inhibit urease enzyme³⁶.

With respect to the anti-microbial activity of black turtle bean, the present study did not find any inhibitory potential against Bacillus subtilis, E. coli, Pseudomonas aeruginosa, Staphylococcus aureus, Candida albicans and Aspergillus niger. Although, there is no anti-microbial activity in the aqueous extract of raw and cooked black turtle beans, this does not disprove the possible potential altogether in black turtle beans in other methods of extraction.

In the present study, it is notable that the in vitro cholesterol and blood pressure levels lowering effect and anti-ulcer activity of black turtle bean was present in both raw and cooked beans, but the effect was more pronounced in cooked black turtle beans. This could be due to the diverse phytochemical profile of cooked black turtle beans than the raw beans. Priyadarshini and Beatrice⁷ reported that cooked black turtle bean contained 16 phytochemical compounds when compared to the raw beans with 13 compounds, primarily comprising of 3-phenyl-6,7-dicarboxyindan-1-one, quercetin and myricetin. Higher phytochemicals in the cooked black turtle beans could be due to the retention of soaked water for cooking black turtle beans that contain the leached compounds. In line with this, Teixeira-Guedes et al.³⁷ also reported that cooking black bean (boiling and pressure cooking) and retention of the cooking water led to retention of total phenol and flavonoid content and increased the anti-oxidant capacity. Further, cooking in itself induces the release of bound phenolics, leading to high polyphenol contents³⁸.

CONCLUSION:

The role of black turtle bean (Phaseolus vulgaris L.) as an anti-cholesterol, anti-hypertensive and anti-ulcer agent seems to be promising in the treatment of dyslipidemia, obesity, hypertension and ulcer. Consumption of cooked black turtle bean and utilization in the food industry will be advantageous as the cooked extract had higher potential than the raw extract of black turtle bean. In order to understand the possible mechanisms, a thorough mechanistic investigation of the compounds present in raw and cooked black turtle beans responsible for these activities is essential to completely understand the mechanisms of action. Future studies are recommended to explore the anti-microbial effect of black turtle beans using crude and purifies extracts from other solvents at varying concentrations.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

The authors would like to thank United Board for Christian Higher Education in Asia (UBCHEA) for funding the project that introduced us to the black turtle bean, which was a key focus of the present study. The authors are deeply grateful to the Principal of Women’s Christian College, Chennai for their support and encouragement. We would also like to thank Apex Biotechnology Training and Research Institute for their assistance with the analysis.

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Received on 30.05.2024 Revised on 14.06.2025

Accepted on 30.12.2025 Published on 01.07.2026

Available online from July 04, 2026

Research J. Pharmacy and Technology. 2026;19(7):3281-3287.

DOI: 10.52711/0974-360X.2026.00467

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