INTRODUCTION:

Oxidative stress is a condition of increased production of free radicals or reactive oxygen species in the body due to a malfunctioning of the body's oxidant defense system, which controls the balance between oxidants antioxidants in cells. This condition can cause tissue damage and various diseases, such as diabetes, inflammation, cancer, atherosclerosis, cardiovascular, heart stroke Alzheimer's, Parkinson's, arthritis, immunological incompetence, neurodegenerative disorders others^1,2,3,4.

Diabetes mellitus (DM) is currently a common disease with a worldwide prevalence of 4%. Its majority will continue to increase, and in 2030, it is estimated that the world's population suffering from diabetes will reach more than 230 million⁵. DM occurs due to disruption of glucose metabolism, causing reduced insulin production.

The hormone insulin is produced by beta cells in the pancreas gland; this hormone works to lower blood glucose levels⁶. The insulin produced by pancreatic beta cells is used to help the entry of glucose into the cells. Glucose Transporter Type 4 (GLUT 4), which is found on the cell membrane, helps insulin deliver glucose to the cells, then glucose is metabolized into ATP. If it is little or no insulin produced, glucose will not enter the cells and continue to be in the bloodstream, resulting in hyperglycemia^7,8.

Various drugs have been widely applied to control blood glucose levels in DM, one of which is a dipeptidyl peptidase (DPP IV) inhibitor⁹. Sitagliptin is a drug that has been used as a potential inhibitor of DPP IV but has side effects on the upper respiratory tract, so it is necessary to seek alternative drugs¹⁰. Various plants have been reported to contain compounds that can help treat various diseases such as diabetes. These plants have been studied in vitro to inhibit DPP IV, such as Petroselinum crispum, Ipomoea batatas (L). (purple), Cajanus cajan (L). Millsp., And white tea leaves^11,12,13. Another plant that has potential as an antidiabetic and antioxidant drug is R. nasutus. Preparations of the roots, stems, and leaves of R. nasutus have been used in traditional medicine to treat diabetes¹⁴. R. nasutus leaves and stem bark extract have been reported to have alpha-glucosidase inhibitory activity^15,16, but DPP IV inhibitory activity from R. nasutus stem bark has not been reported.

METHODS:

Simplicia Setup:

Plant identity checks were carried out by Herbarium Bogoriense Botany field of the Indonesian Institute of Sciences Biology Research Center, Cibinong, Bogor Regency, West Java. The stem bark samples (Figure 1) were obtained from the researchers' private plants grown in the city of Bogor. The stem bark that is sampled is one year old. The stems were washed with water, and then the stem bark was separated from the stems and dried in the open air without sunlight for two weeks. Simplicia was weighed and mashed using a blender. Simplicia powder is stored separately in dry, closed, identified containers and protected from direct sunlight until extraction is carried out.

Extraction of Simplicia:

The simplicia powder of the stem bark of R. nasutus was weighed as much as 150g, then added 500mL of 70% ethanol solvent. The mixture was sonicated using a vibrating ultrasonic probe for 30 minutes at room temperature with an amplitude of 0.6m.

Phytochemical Screening:

Phytochemical screening was carried out on the crude ethanol extract of the stem bark of R. nasutus using procedures that have been reported in previous studies. Phytochemical screening includes tests for alkaloids, flavonoids, phenols, saponins, tannins, glycosides, and sterols-triterpenoids^17,18,19,20.

DPPH Method Antioxidant Test:

Amount of 5mg of stem bark extract was dissolved with methanol pa in a 5mL measuring flask, resulting in a sample solution with a concentration of 1,000mg/L. Solution pipette 40; 80; 160; 320; and 640µL, then each was put into five 5mL measuring flasks, then added 1 mL of DPPH 39mg/L solution, then measured with methanol pa, and homogenized (sample concentrations 8, 16, 32, 64, and 128mg/L). The solution was incubated for 30 minutes at room temperature (25^oC), then the absorption of the solution was measured using a visible light spectrophotometer at a wavelength of 515nm²¹. The work is carried out in three repetitions. The same work is done for the BHT comparators by pipetting 10, 20, and 40µL of BHT solution of 1,000mg/L (BHT concentrations of 2, 4, and 8mg/L).

Antioxidant activity was measured as a decrease in DPPH solution uptake due to the addition of the sample. The absorption value of the DPPH solution on the sample is called the percent inhibition (% inhibition) with the following equation:

(A_blank - A_sample)

% Inhibition = --------------------------------- x 100 %

A_blank

Details:

A_blank = Absorbance without sample

A_sample = Absorbance of the sample

The calculated value is entered into a linear equation (Y = bX + a) with the ppm concentration (mg/L) and the % inhibition value. The IC₅₀ value is obtained from the calculation when the % inhibition is 50%.

Antioxidant Test of the FRAP Method:

The method used refers to Benzie and Strain with several modifications²². A total of 5mg of stem bark extract was dissolved with ethanol pa in a 5mL measuring flask, resulting in a sample solution with a concentration of 1,000mg/L. Solution pipette 10; 20; 30; 40; and 50µL, then each was put into five 5mL measuring flasks, then 0.4mL of 0.001M citric acid was added; 0.2mL of 0.002 M FeCl₃ solution; 0.4mL o-phenanthroline 0.2%, then filtered with distilled water, and homogenized (sample concentrations 2, 4, 6, 8, and 10mg/L). The solution was incubated for 35 minutes at 37^oC, the absorption of the sample solution was measured using a visible light spectrophotometer at a wavelength of 550nm. The work is carried out in three repetitions. The same work is carried out for the gallic acid comparators by means of pipetting 1.25; 2.50; and 3.75µL of gallic acid solution of 1,000mg/L (concentrations of gallic acid 0.25; 0.50; and 0.75mg/ L).

Reducing activity can be calculated with the following equation:

(A_sample – A_blank)

% Reduction Power = ---------------------- x 100 %

A_sample

Details:

A_blank = Absorbance without sample

A_sample = Absorbance of the sample

The calculated value is entered into a linear equation (Y = bX + a) (X-axis) and the% inhibition (Y-axis). The IC₅₀value is obtained from calculations when the% reduction power is 50%.

DPP IV Inhibitor Activity Test:

The DPP IV inhibition activity test was carried out on a blank solution, control blank solution, sitagliptin solution as a positive control, and stem bark extract as sample/inhibitor (Table 1). The reaction was started by adding 50µL of the diluted substrate solution to all the wells used, then incubated for 30 minutes at 37°C. The plate cover is removed, and fluorescence read using an excitation wavelength of 350-360nm and an emission wavelength of 450-465nm²³.

Table 1. Testing for inhibition of DPP IV activity

Hole Plate	Buffer Tris-HCl	DPP-IV	Solvent	Inhibitor	Substrate
Blank	30 µL	10 µL	10 µL	-	50 µL
Control Blank	40 µL	-	10 µL	-	50 µL
Sitagliptin	30 µL	10 µL	-	10 µL	50 µL
Inhibitor (sample)	30 µL	10 µL	-	10 µL	50 µL

The % inhibition of DPP-IV activity can be calculated by the following equation:

(initial activity – inhibitor)

% Ibhibition = ------------------------------------- x 100

Initial activity

With the initial activity is a blank that has been subtracted from the blank control.

RESULT AND DISCUSSION:

Phytochemical Screening:

Table 2. shows the results of phytochemical screening on ethanol extract of R. nasutus stem bark. Positive results for the presence of steroid glycosides, alkaloids, flavonoids, phenolics, and tannins. The steroid glycosides and alkaloids' content gave stronger results than other compounds, while the sterol triterpenes showed negative test results. Steroid glycosides have been reported to have a substantial effect on heart rate and acting as a diuretic, and inhibiting alpha-glucosidase enzyme activity^24,25,26. Alkaloids have been reported to have various pharmacological activities, such us antihyperglycemic, antimalaria, anticancer, and antibacterial activities, so that they are widely used as natural healing drugs^25,26,27,28. Tannins have been reported to have high immunomodulatory and antibacterial activity^28,29. Phenolic compounds have been studied to have several biological effects such as inhibiting alpha-glucosidase enzyme activity, anti-carcinogenic, anti-inflammatory, antioxidant, and the ability to scavenge free radicals. Flavonoids have antioxidant activity, inhibit alpha-glucosidase enzyme activity, and others^25,26,30.

(A) (B)

Figure 1. The stem bark samples (A)and (B) ethanolic extracts of the stem bark of R. nasutus

Table 2. The results of phytochemical screening of the ethanol extract of the stem bark R. nasutus

Secondary Metabolite	Test Results
Alkaloids: Dragendrof Meyer	++ +
Flavanoids	+
Phenolic	+
Saponin	-
Tannin	+
Steroid Glycosides	+++
Triterpenes Sterol	-

DPPH Method Antioxidant Activity:

DPPH compound are stable free radical. The antioxidants contained in plant extracts will neutralize DPPH radicals by giving electrons to DPPH, resulting in a change in the color of the solution from purple to yellow, or the intensity of the purple colour of the solution is reduced. The test solution's colour intensity was measured by the visible spectrophotometric method at a wavelength of about 520nm^31,32. Reduction in colour intensity indicates an increased ability of antioxidants to trap free radicals³³. The reaction between DPPH and antioxidants will reduce DPPH and antioxidant radicals^32,33.

The antioxidant activity test results with DPPH are expressed as % inhibition (Table 3), which is then linked to a series of sample or standard concentrations to produce a curve, as show in Figure 3. The regression equations for BHT and ethanol extract of R. nasutus stem bark obtained was y = 6.0274x+24.113 and y = 0.4591+8.3744. From this equation, the IC₅₀ value for BHT was 4.299±0.004mg/L and for the ethanol extract of R. nasutus stem bark was 90.668±0.084mg/L. In general, BHT has a better DPPH inhibitory activity than the ethanol extract of R. nasutus stem bark, but the antioxidant activity of the extract of R. nasutus stem bark was included in the strong category because the IC₅₀ value in the range of 50-100mg/L³⁴.

Table 3. The results of the DPPH method antioxidant activity test

Sample	Concentration (mg/L)	% Inhibition	IC₅₀ (mg/L)
BHT	2	34.16±0.04	4.299±0.004
	4	51.23±0.01
	8	71.33±0.04
Ethanolic Extract	8	7.03±0.02	90.668±0.084
	16	13.87±0.02
	32	26.41±0.01
	64	45.40±0.01
	128	63.03±0.01

Figure 2. Graph of the relationship between concentration and % inhibition for IC₅₀ determination (A) BHT and (B) ethanolic extracts of R. nasutus stem bark

Antioxidant Activity of the FRAP Method:

The FRAP method is used to test for antioxidants in plants³⁵. The reagent used in this study and which can give a specific colour is 1,10-phenanthroline³⁶. The ferric ion will be reduced by antioxidants to produce ferrous ions, and then these ferrous ions will react with 1,10-phenanthroline to form an orange-red complex. The colour does not depend on acidity in the pH range 2-9 and is stable for a long time³⁷. This complex compound can be read for its absorbance at 510nm³⁶.

The antioxidant activity test results using the FRAP method are expressed as % reduction power (Table 4), which is then connected with a standard concentration series or sample to produce a curve, as shown in Figure 3. The regression equation for gallic acid and ethanol extract of R. nasutus stem bark was y = 28.886x+ 28,647 and y = 2.7675x+25.352. From this equation, the IC₅₀ value for gallic acid was 0.74±0.004mg/L and for the ethanol extract of R. nasutus stem bark was 8.91±0.023mg/L. In general, gallic acid has a better reducing ability to Fe³⁺ than ethanol extract of R. nasutus stem bark, but reduction power of ethanol extract is included in the very strong category because IC₅₀ value is less than 50mg/L³⁴.

Table 4. The results of the FRAP method antioxidant activity test

Sample	Concentration (mg/L)	% Reduction Power	IC₅₀ (mg/L)
Gallic Acid	0.25	36.38±0.05	0.74±0.004
	0.5	42.06±0.05
	0.75	50.83±0.16
Ethanolic Extract	2	30.66±0.03	8.91±0.023
	4	35.49±0.02
	6	43.85±0.09
	8	47.42±0.05
	10	52.37±0.05

Figure 3. Graph of the relationship between concentration and % reduction power for IC₅₀ determination (A) gallic acid and (B) ethanol extract of R. nasutus stem bark

The results showed that the ethanol extract of the stem bark of R. nasutus has the potential as an alternative source of natural antioxidants. The content of phenolic compounds in plants has a direct correlation to antioxidant activity^38,39,40,41. Phenolic compounds can donate protons so that free radicals will become stable radicals. The formation of stable radicals is due to resonance in the aromatic ring so that the electrons will be delocalized^42,43,44. Antioxidant compounds in plants will also undergo oxidation-reduction reactions with FRAP reagents. Consequently, they can trap and neutralize free radicals, triplet oxygen or peroxide decomposition, and quenching singlet^45,46.

Potential Inhibition of DPP IV Activity:

The DPP IV inhibitory activity was measured quantitatively by the presence or absence of the extract, and sitagliptin was used as a positive control¹³. The results of the digging can be seen in Table 5.

Table 5. The results of the DPP IV activity inhibition test

Sample	Final Concentrate (µg/mL)	% Inhibition
Sitagliptin	100	85.45±2.258
ethanolic extract	100	52.19±1.667

The inhibitory activity of DPP IV by sitagliptin produced in this study was 85.45±2.258%, while ethanol extract of R. nasutus stem bark was 52.19±1.667%. These results indicate that the ethanol extract has the potential as an inhibitor DPP IV activity. The mechanism of action of DPP IV inhibitor is to increase glucagon like peptide-1 (GLP-1) levels, increase insulin secretion, and inhibit the release of glucagon^47,48,49. DPP IV is well known for the inactivation of the incretin hormone GLP-1 and gastric inhibitory polypeptide (GIP)⁵⁰. DPP IV inhibitors enhance exogenous and endogenous GLP-1 and GIP action by blocking N terminal degradation. Patients with type 2 diabetes have decreased incretin response, which results in reduced insulin secretion, increased postprandial glucagon levels, and increased postprandial glucose. DPP-IV inhibitors extend the half-life and increase the concentration of circulating (active) incretin. Increased incretin levels lead to increased inhibition of glucagon, which in turn increases insulin secretion, reduces gastric emptying, and lowers blood glucose levels. Therefore, DPP-IV inhibitors may improve glucose tolerance by increasing the incretin effect in patients with type 2 DM⁵⁰. It has been reported that the inhibitory activity of DPP IV is due to the polyphenol content in plants¹².

CONCLUSION:

From the phytochemical test results on the ethanol extract of the stem bark of R. nasutus it was found that there were steroid glycosides, phenolic, alkaloids, and tannins. The antioxidant activity test using the DPPH and FRAP methods gave an IC₅₀valuesof 90.668±0.084 mg / L and 8.91±0.023 mg / L, respectively. The presence of polyphenol compounds is thought to have a role in the high antioxidant activity. In addition, the ethanol extract of the stem bark can inhibit the activity of DPP IV with a % inhibition of 52.19±1.667. It can be concluded that the ethanol extract of R. nasutus stem bark has potential as a source of antioxidants and antidiabetes.

ACKNOWLEDGEMENT:

This work was supported by Doctoral Dissertation Research Grant 2021 [Nomor: NKB-295/UN2.RST/HKP.05.00/2021] from Ministry of Research and Technology/National Research and Innovation Agency, Indonesia.

CONFLICT OF INTEREST:

The authors declare that they have no conflict of interest.

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Received on 17.10.2021 Modified on 20.05.2022

Research J. Pharm. and Tech 2023; 16(3):1187-1192.

DOI: 10.52711/0974-360X.2023.00197