Evaluation of inhibitory effect of the Abelmoschus esculentus flavonoid extract on digestive enzymes of citrus brown snail

doi:10.21203/rs.3.rs-1606470/v1

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Evaluation of inhibitory effect of the Abelmoschus esculentus flavonoid extract on digestive enzymes of citrus brown snail

https://doi.org/10.21203/rs.3.rs-1606470/v1

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Objective: In this study, we extracted the flavonoid contents of Abelmoschus esculentus L. as the main bioactive components in the okra plant. Then, an ultraviolet colorimetric method and a high-performance liquid chromatography (HPLC) were applied to measure the total flavonoid content of okra extract.

Materials and methods: the inhibitory effect of okra extract on α-amylase and α-glucosidase was evaluated on the digestive enzymes from citrus brown snail (Caucasotachea lencoranea).

Results: The results showed high content of rutin, chlorogenic acid, and catechin with remarkable inhibitory effects on α-glucosidase and α-amylase.

Conclusion: Thus, studies related to an identifying the bioactive compounds and correlating them to their biological activities are beneficial for further research to explore the potential of A. esculentus as a source of therapeutic flavonoids.

High-performance liquid chromatography

Abelmoschus esculentus

α-amylase

α-glucosidase

Flavonoids

As a flowering plant, Abelmoschus esculentus (L.) Moench (okra or Bamie) belongs to the Malvaceae family. The okra plant is an African-native edible plant that can be planted in many hot and humid or subtropical regions worldwide, such as Southeast Asia and the Mediterranean. In traditional medication, okra treats gastritis typically. Among many pharmacological features, antioxidant, antidiabetic, neuroprotective, anti-fatigue, and antihyperlipidemic activities are mainly highlighted[1]. Also, it is worth noting that the okra plant contains high contents of flavonoids, polyphenols, and polysaccharides. Flavonoids and polyphenols hold robust antioxidant properties[2, 3] and originate from the okra seeds, while its covering extract scarcely showed such responses. Luo et al. (2018) found flavonoids content of 788.56 mg/g in okra flower extract to be the highest compared to other parts using high-performance liquid chromatography (HPLC). Also, the quercetin glucopyranoside content was reported at 122.13 mg/g of the flower extract[4]. Isoquercitrin is one of the frequent-mentioned substances in the okra extract with advanced bioavailability compared to quercetin, demonstrating in vitro and in vivo chemo-protective special effects touching oxidative tensions, allergic responses, cardiovascular syndromes, diabetes, and cancer[5]. Specifically, isoquercitrin can trigger the inhibition of the urinary bladder and the progress of pancreatic tumors[6, 7], along with the suppression of malignant colon cells[8]. In 2021, Peter et al. determined the optimal extraction conditions for extracting high content of polyphenols and flavonoids from okra fruits quantified using the Folin-Ciocalteu and aluminum chloride colorimetric methods respectively[9]. New studies, then, are needed to identify additional polyphenolic compounds and determine their antidiabetic effects.

Using as traditional herbal medicine or food, native communities have been utilized okra fruits as curry[10], fresh vegetables[11], aiding digestion[12], managing gonorrhea, anemia, and urethrorhea[13], and diabetes mellitus[14]. The excellent source of bioactive compounds in okra fruits and seeds makes it an effective traditional medicine. These phytochemicals were considered volatile oils, glycosides, alkaloids, and polysaccharides polyphenols which are the most prominent and universal secondary metabolites herbal class[9]. Free or sugar-bonded flavonoids such as myricetin[15] and quercetin and its derivatives[16] are also the sub-class of polyphenol isolated from the okra fruits and seeds. The widespread bioactivities of polyphenols with high structural diversity include antioxidant and antidiabetic. According to Shen et al. flavonoids extracted from the okra fruits showed an α-glucosidase and α-amylase inhibitory effect on concentration dependency[17]. Besides, oligomeric proanthocyanidins extraction moiety, active polyphenolic compounds from okra seeds indicated α-glucosidase and α-amylase inhibitory activity[18]. Likewise, phenolic- and flavonoid-rich extraction of okra seeds ensured exceptional antioxidant activity in vitro [18]. At the same time, Myricetin exhibited a plasma glucose concentration decrease and insulin sensitivity improvement in glucose rats with obesity problems[20]. Giving in hand the antidiabetic activity of polyphenolic compounds, it could be supposed that the antidiabetic activity of okra extracts could be due to the presence of these compounds. Then, the diverse action mode of polyphenols with antidiabetic activity could potentially be helpful for type 2 diabetes mellitus due to numerous organs’ participation in its existence[21].

Regardless of the above studies, the lack of studying the inhibition properties of okra plant extract on enzymes can be sensed. In this study, we aimed to investigate the effects of flavonoids extraction from A. esculentus on α-glucosidase and α-amylase enzymes produced from citrus brown snail (Caucasotachea lencoranea ).

2.1. Collecting plant samples

The okra plant was collected in the western region of Iran from Qasr-Shirin, Kermanshah. The climate of this desert region is warm, and it is a suitable place for the growth of this plant. Okra germinates in mid-spring and can be harvested in mid-summer. Okra fruit was collected in the mentioned season and placed in sterile containers to dry completely, it was entirely pulverized by a milling machine, and finally, the powdered sample was kept in the refrigerator at 4°C.

2.2. Preparation of flavonoid-rich extracts

Fresh samples of okra plant powder (60 g) were poured into 400 mL of 70% ethanol and placed on a shaker for 24 hours and mixed thoroughly. Using ethanol as an extraction solvent offers the highest concentration of phenolic materials reported by Ramic et al. [22] and Rincon et al. [23]. After 24 hours, the plant extract was filtered on Whatman paper, and the plant tissue was discarded. The resulting ethanol extract was washed three times with 100 mL of chloroform in a decanter funnel, and the chloroform phase was discarded at each stage. The ethanolic extract was washed three times with 50 ml of ethyl acetate. The ethanol phase placed at the bottom of the separating funnel was separated and collected. This extract was dried at 45 ° C utilizing a rotary evaporator, then located in an oven at 42°C for 72 hours to completely evaporate the solvent and stored in the refrigerator until use.

2.3. Determination of Total Flavonoid and quercetin Content

The total flavonoid (TF) content was calculated using spectrophotometry[24]. A sample extract of 10, 20, 30, and 40 µL was mixed with 1500 µL water. Then, AlCl₃ (150 µL, 10%) and 75 µL of 0.5% sodium nitrate were added with 1500 µL deionized water, respectively. The mixture was incubated in the dark for 5 minutes. The total content of flavonoids was measured afterward using a microplate reader (510 nm). A calibration curve was plotted using quercetin standard solution as quercetin equivalents (QE) per dry extract (gram). The quantitative analysis of polyphenols was done using a liquid chromatography apparatus (waters 2695 (USA) and a PDA Detector waters 996 ‎‎(USA)) for the HPLC analysis‎ (15 cm×4.6 mm with pre-column, Eurospher 100-5 C₁₈ analytical column provided by waters (Sunfire) reversed phase matrix (3.5 µm) (Waters), and elution carried out in a gradient system with methanol as the organic phase (solvent A) and distilled water (solvent B) with the flow-rate of 1 mL min^− 1 (195–400 nm wavelength)[25]. Data acquisition and integration were performed with Millennium 32 software.

2.4. Preparation of animal samples

Equal sizes of citrus brown snail (Caucasotachea lencoranea) were collected from the garden campus of Mazandaran University in October and kept in isolated containers. The snails were fasted for three days to complete their digestion and release their shells. To dissect the snails, after separating the oyster with a sharp scissor, the gastrointestinal gland, located at the end of the helical part of the body, was separated with several vertical grooves [26]. For every 5 grams of the isolated digestive gland, distilled water (20 mL, 4°C) was added and homogenized using a homogenizer at 4°C. The homogenized mixture was then centrifuged at a rate of 16,000 rpm. After this step, the supernatant was kept as an enzyme source at -20°C.

2.5. Determination of α-amylase enzyme activity

The determination of enzyme activity was done by considering the Bernfeld method [27]. Enzyme solution (250 µL) was incubated with 500 µL 1% solvable starch solution (dissolved in the Tris-HCl buffer 0.1 M pH 4.0) for 15 minutes at 40°C. Then, 1 mL of 3,5 dinitro salicylic acid (DNSA) was poured into stopping the substrate-enzyme reaction and was boiled in a water bath for ten minutes. Test tubes were kept for cooling after boiling. Then the enzyme activity was detected with a microplate reader at 540 nm.

2.6. Determination of α-glucosidase activity

The determination of enzyme activity was performed using Siegentsler [28] and Low et al. [29]. In this method, initially, 15 µL of enzyme solution was mixed with 65 µL of Tris-HCl buffer 0.1 M (pH 6.0). Then, 20 µL of 4-Nitrophenyl α-D-glucopyranoside (10 mM) was added and incubated for 15 minutes at 40°C. Then the reaction was ended by adding 100 µL of sodium hydroxide (1 N). The amount of hydrolysis of the substrate at intervals of 3, 6, 9, 12, 15 minutes was measured by a microplate reader at 405 nm. All tests were performed in 3 replications.

2.7. Evaluation of α-amylase inhibition

The α-amylase inhibition evaluation was carried out using the method reported by Tan with minor modifications [30]. Typically, flavonoid-rich okra extract powder (0.1 g) was dissolved in 2 mL of phosphate buffer pH 6.9 and 2 mL of ethanol. Then, the extract was mixed with α-amylase solution (Table 1), and incubated at 40°C for 15 min with continuous shaking. Then 500 µL of soluble starch (1%, w/v) was added to the mixture. Afterward, 3,5-dinitrosalicylic acid (DNSA) reagent was added to the mix and incubated in a boiling water bath for 7 min. Finally, the absorbance of the blend was measured at 540 nm. Acarbose standard (5 mg dissolved in 6 mL DMF under ultrasonic conditions for 10 min) and quercetin (5 mg dissolved in 6 mL DMF) was used as the positive controls (Tables 2 and 3). Results were expressed as inhibition (%) of the α-amylase activity [17].

Table 1

The studied variables on the effect of okra extract on α-amylase activity
Entry	Okra extract (µL)	α-amylase (µL)	Incubation time (min)	Phosphate buffer (µL)
1	-	250	-	250
2	20	250	15	230
3	30	250	15	220
4	40	250	15	210
5	50	250	15	200
6	60	250	15	190
7	70	250	15	180
8	-	-	-	500
9	20	-	-	480
10	30	-	-	470
11	40	-	-	460
12	50	-	-	450
13	60	-	-	440
14	70	-	-	430

Table 2

The studied variables on the effect of acarbose on α-amylase activity
Entry	Acarbose (µL)	α-amylase (µL)	Incubation time (min)	Phosphate buffer (µL)
1	-	250	-	250
2	25	250	15	225
3	50	250	15	200
4	100	250	15	150
5	200	250	15	50
6	400	250	15	50
7	-	-	-	500
8	25	-	-	475
9	50	-	-	450
10	100	-	-	400
11	200	-	-	300
12	400	-	-	100

Table 3

The studied variables on the effect of quercetin on α-amylase activity
Entry	Quercetin ‎ (µL)	α-amylase (µL)	Incubation time (min)	Phosphate buffer (µL)
1	-	250	-	250
2	25	250	15	225
3	50	250	15	200
4	100	250	15	150
5	200	250	15	50
6	400	250	15	50
7	-	-	-	500
8	25	-	-	475
9	50	-	-	450
10	100	-	-	400
11	200	-	-	300
12	400	-	-	100

2.8. Evaluation of α-glucosidase inhibition

The evaluation of α-glucosidase ‎inhibition was performed as the previously mentioned procedure [30]. Usually, the extract powder of flavonoid-rich okra (0.02 g) was dissolved in phosphate buffer (2 mL) and ethanol (1 mL). Then, the extract was mixed with α-glucosidase solution (Table 4) and incubated at 40°C for 15 min with continuous shaking. At that time, 20 µL of p-nitrophenyl-α-D-glucopyranoside (pNαG, 10 mM) dissolved in the phosphate buffer was added to the mixture. The absorbance of the mixture was restrained at 405 nm. Acarbose standard (5 mg dissolved in 6 mL DMF under ultrasonic conditions for 10 min) and quercetin (5 mg dissolved in 6 mL DMF) was used as the positive controls (Tables 2 and 3). Results were stated as inhibition (%) of α-glucosidase activity[17].

Table 4

The studied variables on the effect of okra extract on the α-glucosidase activity
Entry	Okra extract (µL)	α-glucosidase ‎ (µL)	Incubation time (min)	Phosphate buffer (µL)
1	-	15	-	65
2	10	15	15	55
3	15	15	15	50
4	20	15	15	45
5	25	15	15	40
6	30	15	15	35
7	35	15	15	30
8	40	15	15	25
9	-	-	-	80
10	10	-	-	70
11	15	-	-	65
12	20	-	-	60
13	25	-	-	55
14	30	-	-	50
15	35			45
16	40			40

Table 5

The studied variables on the effect of acarbose on the α-glucosidase activity
Entry	Acarbose (µL)	α-glucosidase ‎ (µL)	Incubation time (min)	Phosphate buffer (µL)
1	-	15	-	65
2	2.5	15	15	62.5
3	5	15	15	60
4	10	15	15	55
5	20	15	15	45
6	40	15	15	25
7	-	-	-	80
8	2.5	-	-	77.5
9	5	-	-	75
10	10	-	-	70
11	20	-	-	60
12	40	-	-	40

Table 6

The studied variables on the effect of quercetin on the α-glucosidase activity
Entry	Quercetin ‎ (µL)	α-glucosidase ‎(µL)	Incubation time (min)	Phosphate buffer (µL)
1	-	15	-	65
2	2.5	15	15	62.5
3	5	15	15	60
4	10	15	15	55
5	20	15	15	45
6	40	15	15	25
7	-	-	-	80
8	2.5	-	-	77.5
9	5	-	-	75
10	10	-	-	70
11	20	-	-	60
12	40	-	-	40

Preceding studies have exposed phenolic derivatives of rutin, quercetin-3-O-gentiobioside, hydroxyl cinnamic, and catechin found in okra extracts[31, 32]. Therefore, some phenolic compounds, including rutin, chlorogenic acid, and catechin, were chosen and investigated in okra extract. The total content of phenolic compounds in okra extract was recognized in Fig. 1. The three flavonoid glycosides, catechin (flavan-3-ol), chlorogenic acid (polyphenol and the ester of caffeic acid and quinic acid), and rutin glycoside were utilized as the standard phenolic mixture (Fig. 1a). The sharp peaks of these structures, especially catechin and rutin, are shown in Fig. 1b and c, which are the flavonoid components in A. esculentus extract[4, 33].

All parts of the okra plant, including seeds, skins, roots, leaves, flowers, and fruits, are used broadly as traditional medicines due to their anti-cancer, anti-inflammatory, and anti-diabetes features due to their flavonoid sources[4]. The amount of total flavonoids was determined using an aluminum chloride reagent. Quercetin was used as a standard in this experiment. Methanol was used as a quercetin solvent. The standard diagram was drawn based on adsorption against different concentrations of quercetin. The quantitative analysis results showed in Fig. 2. The increase in the quercetin content at the stages from 0.02 to 0.1 µg/µL led to an increase in absorbance intensity (R² = 0.991) (Fig. 2a). In comparison, the ‎accumulation pattern of the extract was similar to that of quercetin. ‎Besides, the contents of extract derivative increased from 0.1 to 0.5µg/µL (IC50 = 0.0099 µg/µL, R² = 0.999) [34].

Enzymes that break oligosaccharides and disaccharides down into monosaccharides are α-glucosidase and α-amylase[35]. Also, the binding capacities and inhibitory activities of α-amylase and α-glucosidase were previously investigated [36]. Consequently, the inhibition of these enzymes can be understood as significant approach to counter the alterations of metabolic processes related to hyperglycemia and diabetes[37]. Some other reports showed the strong inhibitory effect of okra extract on α-glucosidase and α-amylase inhibition [38, 39]. Nevertheless, changing special inhibitory effects on α-amylase and α-glucosidase by different concentrations of okra extract have infrequently been explored. Figures 3a, b showed the inhibition effect of acarbose and quercetin compared to okra flavonoid-rich extract on the α-amylase enzyme, respectively. The IC₅₀ values of inhibitory effects on α- amylase varied from 11.5 µg/mL, 32.19 µg/mL, and 0.402 mg/mL according to acarbose, quercetin, and okra flavonoid extract, respectively. On the other hand, the IC₅₀ values of inhibitory effects on α-glucosidase were found at 8.4 µg/mL, 74 µg/mL, and 3.25 mg/mL for acarbose, quercetin, and okra flavonoid extract, respectively. The change patterns of inhibitory effects on α-glucosidase and α-amylase of okra fruit were similar to Shen et al.[17]. Furthermore, compared with the acarbose standard, the okra flavonoids showed extraordinary inhibitory effects on α-amylase (IC₅₀ = 11.5 and 8.4 µg/µL) and α-glucosidase (IC₅₀ = 0.402 and 3.25 mg/mL). Then, okra flavonoid-rich extract might be utilized as an excellent anti-hyperglycemic agent.

Herein, we investigated the simple extraction of total flavonoids and measured the three main flavonoid glycosides of rutin, chlorogenic acid, and catechin in the okra extract. Furthermore, the okra extract showed remarkable inhibitory properties on digestive enzymes of α-amylase and α-glucosidase, which recommended okra as a good candidate for further industrial and medical applications.

Conflict of interest

The authors declare that there is no conflict of interest.

Ethics approval and consent to participate

“Not Applicable”.

Funding

“No funding was obtained for this study”.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

[email protected]

Acknowledgment

The authors acknowledge the financial and technical support provided by the University of Mazandaran.

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No competing interests reported.

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Evaluation of inhibitory effect of the Abelmoschus esculentus flavonoid extract on digestive enzymes of citrus brown snail

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Version 1

Abstract

Figures

1. Introduction

2. Experimental

2.1. Collecting plant samples

2.2. Preparation of flavonoid-rich extracts

2.3. Determination of Total Flavonoid and quercetin Content

2.4. Preparation of animal samples

2.5. Determination of α-amylase enzyme activity

2.6. Determination of α-glucosidase activity

2.7. Evaluation of α-amylase inhibition

2.8. Evaluation of α-glucosidase inhibition

3. Results And Discussion

4. Concluding Remarks

Declarations

References

Additional Declarations

Status:

Version 1

Entry	Okra extract (µL)	α-amylase (µL)	Incubation time (min)	Phosphate buffer (µL)
1	-	250	-	250
2	20	250	15	230
3	30	250	15	220
4	40	250	15	210
5	50	250	15	200
6	60	250	15	190
7	70	250	15	180
8	-	-	-	500
9	20	-	-	480
10	30	-	-	470
11	40	-	-	460
12	50	-	-	450
13	60	-	-	440
14	70	-	-	430

Entry	Acarbose (µL)	α-amylase (µL)	Incubation time (min)	Phosphate buffer (µL)
1	-	250	-	250
2	25	250	15	225
3	50	250	15	200
4	100	250	15	150
5	200	250	15	50
6	400	250	15	50
7	-	-	-	500
8	25	-	-	475
9	50	-	-	450
10	100	-	-	400
11	200	-	-	300
12	400	-	-	100

Entry	Quercetin ‎ (µL)	α-amylase (µL)	Incubation time (min)	Phosphate buffer (µL)
1	-	250	-	250
2	25	250	15	225
3	50	250	15	200
4	100	250	15	150
5	200	250	15	50
6	400	250	15	50
7	-	-	-	500
8	25	-	-	475
9	50	-	-	450
10	100	-	-	400
11	200	-	-	300
12	400	-	-	100

Entry	Okra extract (µL)	α-glucosidase ‎ (µL)	Incubation time (min)	Phosphate buffer (µL)
1	-	15	-	65
2	10	15	15	55
3	15	15	15	50
4	20	15	15	45
5	25	15	15	40
6	30	15	15	35
7	35	15	15	30
8	40	15	15	25
9	-	-	-	80
10	10	-	-	70
11	15	-	-	65
12	20	-	-	60
13	25	-	-	55
14	30	-	-	50
15	35			45
16	40			40

Entry	Okra extract (µL)	α-amylase (µL)	Incubation time (min)	Phosphate buffer (µL)
1	-	250	-	250
2	20	250	15	230
3	30	250	15	220
4	40	250	15	210
5	50	250	15	200
6	60	250	15	190
7	70	250	15	180
8	-	-	-	500
9	20	-	-	480
10	30	-	-	470
11	40	-	-	460
12	50	-	-	450
13	60	-	-	440
14	70	-	-	430

Entry	Acarbose (µL)	α-amylase (µL)	Incubation time (min)	Phosphate buffer (µL)
1	-	250	-	250
2	25	250	15	225
3	50	250	15	200
4	100	250	15	150
5	200	250	15	50
6	400	250	15	50
7	-	-	-	500
8	25	-	-	475
9	50	-	-	450
10	100	-	-	400
11	200	-	-	300
12	400	-	-	100

Entry	Quercetin ‎ (µL)	α-amylase (µL)	Incubation time (min)	Phosphate buffer (µL)
1	-	250	-	250
2	25	250	15	225
3	50	250	15	200
4	100	250	15	150
5	200	250	15	50
6	400	250	15	50
7	-	-	-	500
8	25	-	-	475
9	50	-	-	450
10	100	-	-	400
11	200	-	-	300
12	400	-	-	100

Entry	Okra extract (µL)	α-glucosidase ‎ (µL)	Incubation time (min)	Phosphate buffer (µL)
1	-	15	-	65
2	10	15	15	55
3	15	15	15	50
4	20	15	15	45
5	25	15	15	40
6	30	15	15	35
7	35	15	15	30
8	40	15	15	25
9	-	-	-	80
10	10	-	-	70
11	15	-	-	65
12	20	-	-	60
13	25	-	-	55
14	30	-	-	50
15	35			45
16	40			40

Entry	Okra extract (µL)	α-amylase (µL)	Incubation time (min)	Phosphate buffer (µL)
1	-	250	-	250
2	20	250	15	230
3	30	250	15	220
4	40	250	15	210
5	50	250	15	200
6	60	250	15	190
7	70	250	15	180
8	-	-	-	500
9	20	-	-	480
10	30	-	-	470
11	40	-	-	460
12	50	-	-	450
13	60	-	-	440
14	70	-	-	430

Entry	Acarbose (µL)	α-amylase (µL)	Incubation time (min)	Phosphate buffer (µL)
1	-	250	-	250
2	25	250	15	225
3	50	250	15	200
4	100	250	15	150
5	200	250	15	50
6	400	250	15	50
7	-	-	-	500
8	25	-	-	475
9	50	-	-	450
10	100	-	-	400
11	200	-	-	300
12	400	-	-	100

Entry	Quercetin ‎ (µL)	α-amylase (µL)	Incubation time (min)	Phosphate buffer (µL)
1	-	250	-	250
2	25	250	15	225
3	50	250	15	200
4	100	250	15	150
5	200	250	15	50
6	400	250	15	50
7	-	-	-	500
8	25	-	-	475
9	50	-	-	450
10	100	-	-	400
11	200	-	-	300
12	400	-	-	100

Entry	Okra extract (µL)	α-glucosidase ‎ (µL)	Incubation time (min)	Phosphate buffer (µL)
1	-	15	-	65
2	10	15	15	55
3	15	15	15	50
4	20	15	15	45
5	25	15	15	40
6	30	15	15	35
7	35	15	15	30
8	40	15	15	25
9	-	-	-	80
10	10	-	-	70
11	15	-	-	65
12	20	-	-	60
13	25	-	-	55
14	30	-	-	50
15	35			45
16	40			40