INTRODUCTION:

A sedentary lifestyle is becoming more common as technology becomes more convenient, leading to increasingly more common metabolic disorders such as hypertension and diabetes¹.

According to WHO statistics, about 7% of the entire population in Indonesia had diabetes in 2016². Diabetes is caused by the body’s inability to regulate blood glucose levels due to insulin resistance³. Insulin resistance can be caused by several factors ranging from genetics to lifestyle. Among these factors, oxidative stress is believed to be important in causing diabetes^4,5.

Diabetes is a metabolic issue that requires a lifetime of treatment and lifestyle adjustments⁶. One of the drugs used to treat diabetes is acarbose. Acarbose lowers blood glucose by inhibiting the α-glucosidase enzyme⁷. This enzyme breaks down the 1,4-α bond of polysaccharides into glucose⁸. This property can also be found naturally in herbal plants and has antioxidative properties that help prevent and treat diabetes^9-12.

Some herbal medicine is believed to have a positive effect in treating and preventing diabetes^13-15. Senna alexandrina is one of such plants used in African countries as a laxative and antidiabetic drug^16-18. Its leaves are often used in tea and as a supplement^19,20. Studies also have proven its antioxidative and antidiabetic properties^21-24. Different results between studies and household applications are expected as studies tested the properties of S. alexandrina using solvents not used in household settings^21,22. This study determined the antioxidative and antidiabetic properties of S. alexandrina leaves using ethanol, ethanol 50%, and water solvents.

MATERIALS AND METHODS:

Materials:

The dried Senna alexandrina leaves were employed as a sample in this investigation, with ethanol and water as solvents. The experiment was carried out in vitro, and the sample was macerated and separated into three groups to make three extracts. An evaporator was used to help the extraction process, and an LC-MS/MS was used to predict the compounds in the extracts. The plant extract samples were also used to test antioxidant and antidiabetic properties, DPPH and the α-glucosidase enzyme were used, respectively, and a spectrophotometer was used to quantify the results.

Extraction:

Dried S. alexandrina leaves were grounded into a fine powder and divided into three groups weighing 20g. The three groups were soaked in ethanol, ethanol-water mixed at 1:1, and boiling water by following Yuniarto et al. (2018) method with some modifications²¹. The solution was then filtered after 24 hours and further concentrated using an evaporator to produce a dry extract, and the process was repeated until three times the extraction process was done.

Phytochemical screening:

The phytochemical screening was done to analyze quantitatively secondary metabolite compounds such as alkaloids, flavonoids, tannins, saponins, and terpenoids in S. alexandrina leaf extracts in various solvents²⁵.

LC-MS/MS Analysis:

LC-MS/MS was used to profile the bioactive compounds from S. alexandrina leaf extracts. A portion of the extract was set aside to be tested using the LC-MS/MS machine. Acetonitrile was used as the solvent, and the process was done using 10-40V. In predicting the compound, UNIFI software was used²⁶.

Phenolic Content Determination:

Phenolic content was determined using the Folin-Ciocalteu reagent. A standard curve was made using gallic acid to determine the content of the extract. A gallic acid solution was diluted into six concentrations (10, 20, 40, 60, 80, and 100g/mL) to generate a standard curve..Phenolic content was measured by mixing the sample with a 0.4mL Folin-Ciocalteu reagent. The solution was left for 4-8minutes, and added 4 mL of Na₂CO₃ 7%. The results were calculated with a spectrophotometer at λ = 744nm²⁷.

Flavonoid content:

Flavonoid content was measured by comparing the sample with a quercetin standard curve. The curve was made using quercetin with six different concentrations (10, 20, 40, 60, 80, and 100µg/mL). The measurement began with mixing the sample with 3 mL methanol, 0.2 mL AlCl₃ and 0.2mL potassium acetate 1M. The solution was diluted until 10mL was achieved and was let for 30 min. The results were measured at 431nm²⁸.

Antioxidant assay:

The antioxidant assay was done on a 96-well plate in triplicates. A portion of the extract was diluted in 1 mL DMSO, and a further 40µL of the solution was diluted with 160µL DMSO to reduce the color saturation to make a 200g/mL concentration. The assay was done by comparing the blank solution with the sample. The blank solution well was filled with 160µL DMSO and 40µL DDPH, and the sample well was filled with 155µL DMSO, 40µL DPPH, and 5µL sample. The results were read at 517nm. L-ascorbic acid was used as control. The best result was further obtained to calculate the IC₅₀ value. The inhibition percentage was calculated using the following formula²⁹.

Antidiabetic assay:

Antidiabetic assay was done on a sample well in triplicates using the α-glucosidase enzyme. The extract was prepared similarly to the antioxidant assay to decrease the color saturation. The sample concentration was 200µg/mL. The assay was done by using background and blank well. The background test well was divided into two groups, one for blank and one for the sample well. The background test well was filled with 70µL buffer solution and 25µL substrate. Background test and blank test wells were added with 5 µL DMSO, and for a test well was added with 5µL sample, respectively. Moreover, the blank test well was filled with 45µL buffer, 25µL substrate, and 5µL DMSO., while the sample well was filled with 45µL buffer solution, 25µL substrate, and 5µL extract sample. After 5 min, all wells except the background were filled with 25µL of the enzyme. The test was let for 30 minutes before adding 100µL of sodium bicarbonate. The result was read at 400nm. The extract with the best result was tested to find its IC₅₀ value³⁰. The results were calculated using the following formula:

AB = Blank absorbance

AB0 = Blank background absorbance

AT = Sample/test absorbance

AT0 = Sample/test background absorbance

RESULT:

Extraction yield:

Extraction of dried S. alexandrina leaves produced different yield percentages. Ethanol solvent had the highest yield (14.82%), followed by ethanol 50% (12.9%) and water (12.2%) solvents, respectively. Extraction results can be seen in table 1.

Table 1. S. alexandrina leaves extract yields

S. No	Sample Weight (g)	Solvent	Extract Weight (g)	Yield (%)
1	20	Ethanol	2.96	14.8
2	20	Ethanol 50%	2.58	12.9
3	20	Water	2.44	12.2

Phytochemical screening:

Most of the tests for phytochemical screening of S. alexandrina leaves showed a positive result; the only negative results came from the tannin screen on water solvent. The details are listed in table 2.

Table 2. Qualitative phytochemical screening

No.	Solvent	Alkaloid	Flavonoid	Tannin	Saponin	Terpenoid
1.	Ethanol	+	+	+	+	+
2.	Ethanol 50%	+	+	+	+	+
3.	Water	+	+	-	+	+

Phenolic and flavonoid content:

Similar results came from phenolic and flavonoid content testing regarding the order in which extract had the most content. Ethanol-water mixed extract showed the most content of phenolic and flavonoid content. Ethanol extract showed the second-best phenolic and flavonoid content, and water extract showed the worst result for phenolic and flavonoid content. Results from phenolic and flavonoid content can be found in table 3. The standard curve used to calculate the phenolic content can be found in figure 1 and the standard curve used to calculate flavonoid content can be found in figure 2.

Table 3. Phenolic and flavonoid content

No	Solvent	Phenolic content (µg GA eq/mL)	Flavonoid content (µg Q eq/mL)
1	Ethanol	37.49±0.48	58.04±0.37
2	Ethanol 50%	39.52±2.07	81.19±2.04
3	Water	25.18±0.16	11.19±1.67

Each solvent has a different compound prediction and yield percentage. This is due to the different polarities of the solvents employed. Creating a solution adheres to the rule of like dissolves like, which means polar solvent will solute the polar compound more easily and the non-polar solvent-solute non-polar compound more easily. Due to the solvent nature, it is impossible to solute all the compounds contained using a single solvent³¹. Some studies suggested adding water as a polar solvent to ethanol, a non-polar solvent, to increase the number of polyphenols contained in the solution^{32, 33}. The results in this study for phenolic and flavonoid content showed that ethanol 50% solvent had the most phenolic and flavonoid content. However, the results showed yield percentage of ethanol was the highest among the three solvents.

Figure 1. A) Gallic Acid Standard Curve, and B) Quercetin Standard Curve.

LC-MS/MS compound prediction:

Results showed that each extract contains 5-6 active compounds. The process used acetonitrile as solvent and UNIFI software to predict the results. The compounds found are listed in table 4 and figure 2.

Figure 2. LC-MS/MS chromatogram profiles of bioactive compounds from Senna alexandrina leaf extract using various solvents: A) Extract in ethanol; B) Extract in ethanol 50%; C) Extract in water.

Antioxidant capacity:

All extracts of S. alexandrina leaves showed antioxidant properties. Ethanol-water mixed extract showed the best antioxidant properties at 55.1%, ethanol extract at 53.5%, and water extract at 37.6%. Ethanol 50% extract was tested further to find its IC₅₀ value. The IC₅₀ value of antioxidant activity from ethanol 50% extract was 160.50µg/mL. The results are shown in figure 3.

Figure 3. Antioxidant capacity of S. alexandrina leaves extract in three different solvents.

Antidiabetic activity:

The test results showed that all extracts had α-glucosidase inhibition properties. Ethanol 50% had the best inhibition, followed by water extract and ethanol extract. The results of the testing can be found in figure 4. Ethanol 50% extract showed an IC₅₀ value was 33.151 µg/mL.

Figure 4. Antidiabetic properties of S. alexandrina leaves extract in three different solvents.

DISCUSSION:

The results of the antioxidant activity differ from Yuniarto et al., which reported that extract using water solvent had better antioxidant activity than extract using ethanol solvent²¹. This is unique due to there are some modifications in the extraction method.

Antioxidant activity is related to the phenolic^34-36 and flavonoid^33,37 compounds. Phenolic and flavonoid content results showed the most abundant in ethanol 50% extract, followed by ethanol and water extracts. The IC₅₀ value of antioxidant power from ethanol 50% extract was 160.5µg/mL. We measured quercetin as a standard compound and found the IC₅₀ value was 5.8 µg/mL. It means the antioxidant properties of S. alexandrina leaf extract using ethanol 50% solvent are not as effective as quercetin.

S. alexandrina showed good α-glucosidase inhibition activity. The ethanol 50% extract showed an IC₅₀ value of 33.2µg/mL. Yuniarto et al. used acarbose to compare their study with an IC₅₀ value of 36.2µg/mL⁹. Therefore, ethanol 50% extract showed better α-glucosidase inhibition than acarbose. Yuniarto et al. also found that ethanol S. alexandrina had better α-glucosidase than water, which is different from the result found in this study²¹.

Antidiabetic properties are found to be related to total flavonoid content³⁷. The efficacy associated with the α-glucosidase inhibitory activity is the phytochemical properties of plants that affect one enzyme more than the other, as it is clear that the plant contains a variety of phytochemicals such as flavonoids, polyphenols, and carotenoids^38-42. The α-glucosidase is an enzyme located on the brush border of the microvilli of the small intestine⁴³. This enzyme catalyzes the last step in carbohydrate metabolism, and it converts oligosaccharides into monosaccharides before they are absorbed into the bloodstream, alleviating postprandial blood glucose elevations⁴⁴.

The most abundant compound predicted in the ethanol 50% extract were torachrysone-8-O-β-D-glucopyranoside, oroxin B, and 3-O-[β-D-glucopyra-nosyl-(12)]-β-D-glucopyranosyl-kaempferol. The other compounds predicted were 7-Hydroxy-1-methoxy-2-methoxyxanthone, rhamnetin, and rubilactone. Among these compounds, torachrysone-8-O-β-D-glucopyranoside⁴⁵ and oroxin B⁴⁶ have antioxidative effects. Torachrysone-8-O-β-D-glucopyranoside was also found to have a good α-glucosidase inhibition activity⁴⁷. These compounds were also found to be the most abundant in the ethanol 50% extract compared to ethanol-only and water-only extract. Rhamnetin may also contribute to the antioxidant and antidiabetic effect as it is commonly found in other plants with antioxidative and antidiabetic effects, such as Anacardium occidentale and Anthemis kotschyana^48,49.

CONCLUSION:

Extraction of dried S. alexandrina leaves produced different % yields. Most of the tests for phytochemical screening of S. alexandrina leaves showed positive results. Ethanol-water mixed extract showed the most content of phenolic and flavonoid content. Ethanol extract had the second highest phenolic and flavonoid concentration, whereas water extract had the lowest phenolic and flavonoid content. The antioxidant capacity of ethanol 50% extract was 160.5g/mL. The ethanol 50% extract was inhibited the most, followed by the water extract and the ethanol extract. Phenolic and flavonoid contents were highest in ethanol 50% extract, followed by ethanol and water extracts. S. alexandrina showed good α-glucosidase inhibition activity. Antidiabetic properties are found to be related to total flavonoid content. Antidiabetic activity is also related to total polyphenols. The most abundant compounds predicted in the ethanol 50% extract were torachrysone-8-O-β-D-glucopyranoside, oroxin B, and 3-O-[β-D-glucopyra-nosyl-(12)]-β-D-glucopyranosyl-kaempferol.

CONFLICT OF INTEREST:

The authors have no conflicts of interest in this matter.

ACKNOWLEDGMENTS:

This research was supported by PDUPT and PUTI grants, DRPM Universitas Indonesia.

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Received on 11.12.2021 Modified on 06.06.2022

Research J. Pharm. and Tech 2022; 15(12):5835-5840.

DOI: 10.52711/0974-360X.2022.00985

Extract	Predicted Compound	Retention Time (min)	Molecular Weight
Ethanol	Oroxin B	4.27	595.17
	3-O-[β-D-Glucopyranosyl-(12)]-β-D-glucopyranosyl-kaempferol	4.91	611.16
	7-Hydroxy-1-methoxy-2-methoxyxanthone	4.91	287.06
	Torachrysone-8-O-β-D-glucopyranoside	5.96	409.15
	Stearidonic acid	9.46	277.22
	Daturametelin H	10.99	621.31
Etanol 50%	Oroxin B	4.27	595.17
	3-O-[β-D-Glucopyranosyl-(12)]-β-D-glucopyranosyl-kaempferol	4.91	611.16
	7-Hydroxy-1-methoxy-2-methoxyxanthone	4.91	287.06
	Rhamnetin	4.97	317.07
	torachrysone-8-O-β-D-glucopyranoside,	5.96
	Rubilactone	8.62	271.06
Water	Trigonelin	1.19	138.06
	Oroxin B	4.27
	Digiprolactone	5.99	197.12
	Isorhamnetin	7.53	317.07
	Stigmast-4-en-6β-ol-3-one	10.42	429.37