The Antioxidant and Anti-Diabetic Chemical Compounds in Rhinacanthus nasutus (L.) Kurz Leaf Extract Were Determined Using UHPLC-MS/MS QTOF
Candra Irawan1,2, Berna Elya1*, Muhammad Hanafi3,4, Fadlina Chany Saputri1
1Faculty of Pharmacy Universitas Indonesia, Depok, West Java 16424, Indonesia.
2Department of Food Nanotechnology, Politeknik AKA Bogor, Bogor, West Java 16154, Indonesia.
3Research Center for Pharmaceutical Ingredients and Traditional Medicine,
National Research and Innovatioan Agency-BRIN, Kawasan Puspiptek, Tangerang Selatan 15134 Indonesia.
4Faculty of Pharmacy Universitas Pancasila, Jakarta Selatan 12640, Indonesia.
*Corresponding Author E-mail: berna.elya@farmasi.ui.ac.id
ABSTRACT:
Background: Rhinachantus nasutus (L.) Kurz (Rnz) is a plant that has the potential to be an antioxidant as well as an anti-diabetic medication. Traditional medicine has used preparations made from the roots, stems, and leaves of Rnz to treat diabetes. Aim: This study aimed determine the antioxidant and anti-diabetic potential of Rnz leaf extract by inhibiting the activity of dipeptidyl peptidase IV (DPP IV). Method: Rnz leaf samples were extracted using Ultrasound-Assisted Extraction (UAE). Following that, the antioxidant capacity was determined by trapping 2,2-diphenyl-1-picrylhydrazyl (DPPH), ferric reduction antioxidant power (FRAP), and its potential as an antidiabetic was determined by inhibiting DPP IV activity. Results: The extraction yield of ethanol from Rkz leaves using the UAE method was 8.36%. The IC50 and EC50 values for the antioxidant capacity determined using the DPPH and FRAP methods were 97.34 ± 0.07 mg/L and 8.69 ± 0.02 mg/L, respectively. In addition, ethanolic leaf extract has potential as an antidiabetic through the mechanism of inhibition of DPP IV activity with an inhibition value of 43.05 ± 1.2%. The determination of compounds in the ethanolic extract of Rnz leaves using UHPLC-MS/MS QToF succeeded in identifying 65 compounds, with several compounds reported to be active as antidiabetic. These compounds are 1,3,7-trihidroxy-6-methoxy-4,5-diisoprenylxanthone (9), palmitic acid (10), 3,5-di(ethylthio)isothiazole-4-carboxaide (12), 4,8-dihydroxy-6-methoxy-3-methyl-3,4-dihydro-1H-isochromen-1-one (15), glaucine (20), 5-isopropyl-3,8-dimethylazulene-1-carbaldehhyde 1-(4-nitrophenyl)hydrazone (21), 2,5-anhydro-6-[(2-chloro-5-ethoxybenzoyl)amino]-4,6-dideoxy-4-(4-phenyl-1-piperazinyl)-D-galactitol (27), 2-(4-methylphenyl)-4-phenyl-2,3-dihydro-1,5-benzothiazepine (30), 1-palmitoylglycerol (32), (2S)-2-({1-[(4-fluorophenyl)ethyl]indazole-3-carbonyl)amino)-3-methylbutanoic acid (43), and ethyl 2-[(6-oxo-6H-benzo[c]chromen-3-yl)oxy]acetate (45). Conclusion: the ethanolic extract of the leaves of Rnz from the UAE has the potential as a source of antioxidants and antidiabetics.
KEYWORDS: Antidiabetic, Antioxidant, Dipeptidyl peptidase IV, Rhinachantus nasutus, UHPLC-MS/MS QToF.
INTRODUCTION:
Diabetes mellitus (DM) is a chronic metabolic illness that can have a substantial influence on a patient's quality of life, both in terms of their socioeconomic situation as well as their health1. DM is characterised by high blood glucose levels2,3. In accordance with the findings of the International Diabetes Federation (IDF), 643 million people will be living with diabetes in the year 2030, and 783 million people will have the disease in the year 20454. Obesity and a lack of regular physical activity are the primary contributors to the development of Type 2 DM (T2DM), which accounts for approximately 90% of all diabetes cases5. The rise in blood sugar levels that occurs in T2DM is the result of impaired insulin action and secretion. This impairment can be prominent in insulin secretion disorders, or it can be predominant in insulin resistance6,7. Oral hypoglycemic medications that inhibit alpha-glucosidase (aGl) and DPP IV have been shown to be helpful in patients with T2DM8,9.
Plants with medicinal properties are immensely advantageous to both the health of individuals and the well-being of communities. The presence of numerous bioactive compounds in these plants contributes to their medicinal value. Synthetic drugs that have been widely used are generally based on single molecules, but herbal-based drugs contain many bioactive compounds in their formulations. Due to their mechanism of action, which involves additive or synergistic actions at a single or multiple target sites involved in physiological processes, these plant-based medicines may exercise beneficial effects10,11. In contrast to synthetic medications, which can cause undesirable side effects, natural herbal formulations are inexpensive and have few or no side effects10,11,12.
Rnz is a plant that has the potential to be an antioxidant as well as an anti-diabetic medication. Traditional medicine has used preparations made from the roots, stems, and leaves of Rnz to treat diabetes13. Rnz leaves and stem bark extracts were shown to have ΔGi inhibitory activity14,15; besides that, stem bark extracts have been reported to have activity as DPP IV inhibitors16, but the DPP IV inhibitory activity of Rnz leaves has not been reported.
Accurate characterization of chemical compounds that have antioxidant activity and are active as antidiabetics in Rnz leaves is very important. The most appropriate analysis method for the entire characterization is LC/MS-QToF technology17,18,19,20,21. Therefore, this study aims to determine chemical compounds that have the potential as antioxidants and antidiabetics through the mechanism of inhibition of DPP IV and inhibition of ΔGi activity in the leaf extract of Rnz. In order to identify the chemical components found in the extract, LC-MS/MS QToF was utilised.
Simplia Setup:
Determining the identity of plants used in research has been reported in previous studies16. The leaf samples used were six months old. The treatment of samples and sample sources is the same as in previous studies.
Extraction of Simplicia:
Rnz leaves simplicia powder was weighed at approximately 50 g, put into a beaker, and added to 400 mL of 70% ethanol. Then it was sonicated for 30 minutes at room temperature with an ultrasonic probe vibrating at an amplitude of 0.6 m. The resulting extract was then subjected to solvent evaporation, and the resulting weight was measured. The percent yield value was then computed.
Structural Elucidation by UHPLC MS/MS QToF:
Five microliters of extract sample were injected into a Waters Acquity UPLC system and separated at 0.6 milliliters per minute using an HSS T3 column with a gradient from solvent X (0.1 percent HCOOH in distilled water) to solvent Y (0.1 percent HCOOH in CH3CN). Elution was conducted out in a linear gradient at 1 percent of solvent X for 0.5 minute, then from 1 percent to 35 percent of solvent X for 16 minutes, then from 35 to 100 percent of solvent X for 2 minutes, and finally a linear gradient at 99 percent of solvent Y for 2 minutes. The column eluent was injected into a Waters Xevo G2 Q-ToF-MS with an electrospray source and operated in either positive or negative ionization mode, as well as a data-dependent MS/MS mode (15–40 V ramp). The cone and capillary voltages were set to 40 V and 2.0 kV for positive and negative ionization modernization modes, respectively. The N2 desolvation temperature was set to 550 degrees Celsius, and the source temperature was set to 120 degrees Celsius, with a flow rate of 1000 liters per hour. The mass spectra were identified using the spectrum database for organic compounds in the Univy and MzCloud applications.
Evaluation of Antioxidants with the DPPH and FRAP Methods:
The ethanol extract of Rnz stem bark was tested for antioxidant activity by the DPPH and FRAP methods, referring to our previously reported study. Extract sample weights, sample concentration series, and positive control concentration series used were similar to our previous reports; only the sample types were different16.
Evaluation of DPP IV Activity Inhibition:
Evaluation of DPP IV inhibitory activity was conducted on blanks solutions, control blanks, positive controls (sitagliptin), and inhibitors (Rnz leaf extract) with compositions as shown in Table 1. Each treatment was incubated for 30 minutes at 37°C. Next, the fluorescence of the solution was measured at excitation wavelengths of 350–360 nm and emission wavelengths of 450–465 nm 22.
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 |
The following equation can be used to determine the % inhibition of DPP-IV activity.
(Initial activity – Inhibitor)
% Inhibition = ----------------------------------- x 100%
Initial activity
Initial activity = Blank - Control blank
RESULTS AND DISCUSSION:
Ultrasound-Assisted Extraction:
The use of 70% ethanol as a solvent during the UAE of Rnz leaves produced a crude extract with a weight of 5.8520 grammes and a yield of 8.36%.
Chemical Compounds by UHPLC-MS/MS QToF:
The LC-MS/MS QToF method was used to structure the chemical compounds in the ethanolic extract of the leaves of Rnz. The identified compounds are 65. Figure1 depicts the chromatogram of the Rnz leaf ethanolic extract, and Table 2 lists the constituents of the extract.
Figure 1. Chromatogram of ethanolic extract of Rnz leaf, (A) positive and (B) negative ion modes as determined by LC-MS/MS QtoF
Table 2: The content of chemical compounds in the leaf extract of Rnz
|
No. |
RT |
EM |
Monoisonic Massa |
Compound Name |
SI (%) |
|
1 |
0.29 |
(+) |
425.19621 |
1-{[(2R,3S,4R,5S)-3,4-Dyhydroxy-5-{2-[(2R)-2-(methoxymethyl)-1-pyrrolidinyl]-2-oxoethyl}tetrahydro-2-furanyl]methyl}-3-(3-fluorophenyl)urea |
100 |
|
2 |
0.37 |
(+) |
363.16952 |
[(3S)-3-{5-[4-(Dimethylamino)phenyl]-1,3,4-oxadiazol-2-yl}-1-pyrrolidinyl](3-pyridinyl)methanone |
100 |
|
3 |
0.42 |
(-) |
169.03098 |
2-(2-thienylmethylidene)hydrazine-1-carboxamide |
92.9 |
|
4 |
0.70 |
(+) |
490.07629 |
4-[(1E)-2-{4-[bis(2-sulfamoyethyl)aino]phenyl}diazen-1-yl]benzene-1-sulfonamida |
93.6 |
|
5 |
0.77 |
(+) |
355.13545 |
N1-tricyclo[5.2.1.0~2,6~]dec-4-en-8-yl-5-nitroindoline-1-carbothiomide |
100 |
|
6 |
1.52 |
(+) |
1602.73011 |
(2R,3R,4R,5R)-3-{[(2S,3R,4S,5S,6S)-4-{[(2S,3R,4R)-3,4-dihydroxy-4-(hydroxymethyl)oxolan-2-yl]oxy}-5-{[2S,3R,4S,5S)-3,5-dihydroxy-4-{[(2S,3R,4S,5S)-3,4,5-tryhydroxyoxan-2-yl]oxy}oxan-2-yl]oxy}-3-hydroxy-6-methyloxan-2-yl}oxy}-5-hydroxy-4-{[(2S,3R,4R,5R,6S)-3,4,5-tryhidroxy-6-methyloxan-2-yl]oxy}oxan-2-yl(4aR,5R,6aS,6bR,10R,11S,12aR,14bS)-5,11-dihydroxy-2,2,6a,6b,9,9,12a-heptaethyl-10-{[(2S,3R,4S,5S)-3,4,5-trihydroxy-6-({[(2S,3R,4S,5S)-3,4,5-trihidroxyoxan-2-yl]oxy}ethyl)oxan-2-yl]oxy}-1,2,3,4,4a,5,6 |
99 |
|
7 |
1.73 |
(+) |
442.09970 |
1-Butyl-3-[5-(tert-butyl)-1,3,4-thiadiazol-2-yl]-5-(3,4-dichlorophenyl)-4-hydroxyimidazolidin-2-one |
96.6 |
|
8 |
4.83 |
(-) |
372.04007 |
7-Chloro-4,6-dimethoxy-3a,12a-dihydro-5H-furo[3`,2`:4,5]furo[3,2-b]xathen-5-one |
100 |
|
9 |
5.43 |
(+) |
410.17294 |
1,3,7-Trihidroxy-6-methoxy-4,5-diisoprenylxanthone |
96.7 |
|
10 |
5.54 |
(-) |
256.24023 |
Palmitic acid |
90.3 |
|
11 |
6.12 |
(-) |
450.11621 |
Astilbin |
98.3 |
|
12 |
6.15 |
(+) |
219.97987 |
3,5-di(ethylthio)isothiazole-4-carboxaide |
99.5 |
|
13 |
6.33 |
(-) |
382.04555 |
2-[3-chloro-2-hydroxy-4-methoxy-6-(methoxycarbonyl)phenoxy]-6-hydroxy-4-methylbenzoic acid |
99.9 |
|
14 |
6.52 |
(+) |
240.04226 |
Alizarin |
100 |
|
15 |
7.20 |
(+) |
224.06847 |
4,8-Dihydroxy-6-methoxy-3-mmethyl-3,4-dihydro-1H-isochromen-1-one |
95.4 |
|
16 |
7.22 |
(+) |
315.09291 |
1-(2,3-Dihydro-1-benzofuran-5-ylsulfonyl)-1,2,3,4-tetrahydroquinoline |
100 |
|
17 |
7.48 |
(-) |
246.15773 |
Carbamazepine-d10 |
93.8 |
|
18 |
7.50 |
(+) |
238.12051 |
1-(3-ethyl-2,4-dihydroxy-6-methoxyphenyl)butan-1-one |
96.4 |
|
19 |
7.67 |
(-) |
178.06299 |
4-Methoxycinnamic acid |
97.9 |
|
20 |
8.00 |
(+) |
355.17836 |
Glaucine |
99.9 |
|
21 |
8.18 |
(+) |
361.17903 |
5-Isopropyl-3,8-dimethylazulene-1-carbaldehhyde 1-(4-nitrophenyl)hydrazone |
99.9 |
|
22 |
8.36 |
(+) |
336.09977 |
3-hydroxy-8-methoxy-3-methyl-1,2,3,4,7,12-hexahydrotetraphene-1,7,12-trione |
98.6 |
|
23 |
8.43 |
(+) |
383.17327 |
(3S,4R)-4,5-dihydroxy-3-methoxy-4-(4-mmethoxyphenyl-6-(3-methylbut-2-en-1-yl)-1,2,3,4-tetrahydroquinolin-2-one |
96.8 |
|
24 |
8.59 |
(-) |
298.14164 |
Methyl 2-(1,3,5-trihydroxy-4a-methyl-8-oxo-decahydronaphthalen-2-yl)prop-2-enoat |
100 |
|
25 |
8.63 |
(+) |
294.11034 |
7-ethoxy-6-(1,2,3-trihydroxy-3-ethylbutyl)-2H-chromen-2-one |
100 |
|
26 |
8.87 |
(+) |
238.03315 |
5-chloro-2-tetrahydro-1H-pyrrol-1-yl-1,3-benzothiazole |
92.4 |
|
27 |
9.67 |
(+) |
475.18740 |
2,5-Anhydro-6-[(2-chloro-5-ethoxybenzoyl)amino]-4,6-dideoxy-4-(4-phenyl-1-piperazinyl)-D-galactitol |
100 |
|
28 |
9.97 |
(-) |
457.98704 |
2,4-dichloro-N`-({[4-phenyl-5-( trifluoromethyl)- 2- thienyl]carbonyl} oxy) benzenecarboximidamide |
98 |
|
29 |
10.43 |
(+) |
245.99061 |
2-Chlorothioxanthone |
93.7 |
|
30 |
10.79 |
(+) |
329.12382 |
2-(4-Methylphenyl)-4-phenyl-2,3-dihydro-1,5-benzothiazepine |
99.5 |
|
31 |
10.92 |
(+) |
436.13695 |
Phlorizin |
100 |
|
32 |
11.30 |
(-) |
330.27701 |
1-Palmitoylglycerol |
100 |
|
33 |
11.47 |
(-) |
342.13147 |
(4E)-2-(acetyloxy)-6,7-dihydroxy-7-(6-oxo-3,6-dihydro-2H-pyran-2-yl)hept-4-en-3-yl acetate |
99.7 |
|
34 |
12.13 |
(-) |
430 |
Ononin |
100 |
|
35 |
12.15 |
(+) |
258.05282 |
Norlichexanthone |
99.9 |
|
36 |
12.27 |
(+) |
399.16819 |
Colchicine |
91.5 |
|
37 |
13.04 |
(+) |
432.21480 |
Schisandrin |
91.5 |
|
38 |
13.28 |
(+) |
307.21474 |
Betaxolol |
100 |
|
39 |
13.55 |
(+) |
365.17150 |
Cyclohexyl{(3S)-3-[5-(trifluotomethyl)-1H-benzimidazol-2-yl]-1-pyrrolidinyl}methanone |
100 |
|
40 |
14.53 |
(+) |
198.08921 |
5,8-dihydroxy-10-methyl-5,8,9,10-tetrahydro-2H-axecin-2-one |
97.8 |
|
41 |
14.73 |
(+) |
324.20893 |
Cannabinol monomethylether |
96.3 |
|
42 |
14.75 |
(-) |
312.30283 |
Arachidic Acid |
90.8 |
|
43 |
14.95 |
(+) |
369.14887 |
(2S)-2-({1-[(4-Fluorophenyl)ethyl]indazole-3-carbonyl)amino)-3-methylbutanoic acid |
100 |
|
44 |
15.50 |
(+) |
504.08452 |
Hypericin |
92.7 |
|
45 |
15.52 |
(-) |
298.08412 |
Ethyl 2-[(6-oxo-6H-benzo[c]chromen-3-yl)oxy]acetate |
93 |
|
46 |
15.59 |
(+) |
207.08954 |
4-morpholinobenzoic acid |
92.1 |
|
47 |
15.68 |
(+) |
250.14164 |
2-Methylbutyl beta-D-glucopyranoside |
92.2 |
|
48 |
15.94 |
(+) |
341.16271 |
N-methylhernagine |
90.6 |
|
49 |
16.39 |
(+) |
273.10011 |
Dimethyl 4-methyl-5-phenyl-1H-pyrrole-2,3-dicarboxylate |
93.4 |
|
50 |
16.43 |
(+) |
382.14481 |
Loratadine |
93.6 |
|
51 |
16.58 |
(+) |
496.08531 |
(1S,3R,4R,5R)-1,3-dihydroxy-4,5-bis(3,4,5-trihydroxybenzoyloxy)cyclohexane-1-carboxylic acid |
99.6 |
|
52 |
16.60 |
(-) |
280.13107 |
4,6-dihydroxy-5a-methyl-3-methylidene-2-oxo-dodecahydronaphtho[1,2-b]furan-9-carbaldehyde |
100 |
|
53 |
16.74 |
(-) |
416.16825 |
(2R,3R,4R,5R,6S)-2-{[(2R,3S,4S,5R,6R)-6-(benzyloxy)-3,4,5-trihydroxyoxan-2-yl]methoxy}-6-methyloxane-3,4,5-triol |
99.5 |
|
54 |
17.02 |
(+) |
732.32313 |
Cyclo(glycyltryptophylprolylglycylvalyl glycyl-β-hydroxytyrosyl |
100 |
|
55 |
17.14 |
(+) |
636.09626 |
(2S,3R,4S,5S,6R)-4,5-dihydroxy-2(3,4,5-trihydroxybenzoyloxy)-6-[(3,4,5- trihydroxybenzoyloxy)ethyl]oxan-3-yl 3,4,5-trihydroxybenzoate |
99.4 |
|
56 |
17.33 |
(+) |
650.40300 |
(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl(4aS,6aS,6bR,9R,10R,11R,12aR)-10,11-dihydroxy-9-(hydroxyethyl)-2,2,6a,6b,9,12a-hexamethyl-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4a-carboxylate |
97.1 |
|
57 |
17.54 |
(+) |
650.40300 |
1-O-[(2alpha,3beta,5xi,9xi,18xi)-2,3,19-Trihydroxy-28-oxours-12-en-28-yl]-beta-D-glucopyranose |
97.6 |
|
58 |
17.72 |
(+) |
490.16864 |
(1S,4aS,7aR)-7-ethyl-1-{[(2S,3R,4S,5S,6R)-3,4,5-trihydroxyethyl)oxan-2-yl]oxy}-4a-{[(2S,3R,4R,5R,6S)-3,4,5-trihydroxy-6-methyloxan-2-yl]oxy}-1H,4aH,5H,7aH-cyclopenta[c]pyran-5-one |
91.3 |
|
59 |
17.82 |
(+) |
432.10565 |
5-Hydroxy-3-(4-hydroxyphenyl)-7-{[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy}-4H-chromen-4-one |
90.9 |
|
60 |
18.01 |
(-) |
575.31077 |
alpha-Ergocryptine |
90.1 |
|
61 |
18.26 |
(+) |
547.20536 |
Colchicoside |
93.4 |
|
62 |
18.29 |
(-) |
722.50979 |
1,2-Dipalmitoylphosphatidylglycerol |
100 |
|
63 |
18.42 |
(+) |
584.29853 |
(1S,2S,3aR,4S,5S,9R,11R,13aS)-4,9,11-tris(acetyloxy)-3a-hydroxy-2,5,8,8,12-pentammethyl-1H,2H,3H,3aH,4H,5H,8H,9H,10H,11H,13aH-cyclopenta[12]annulen-1-yl benzoate |
98.1 |
|
64 |
18.52 |
(-) |
554.30054 |
Cinnamyldenafil |
90 |
|
65 |
18.59 |
(+) |
242.03459 |
beta-Lapachone |
99.6 |
Evaluation of Antioxidants with the DPPH Method:
The results of antioxidant evaluation using the DPPH method were expressed in % inhibition, as shown in Table 3. These results are then connected to a series of sample or standard concentrations to produce a curve. The positive control, which was a BHT compound, had a regression equation of y = 6.0248x+24.12, and the ethanol extract of Rnz leaves had a regression equation of y = 0.459x+5.9144. Based on this equation, the value for the IC50 concentration of BHT was 4.30±0.009 mg/L, and the concentration of the ethanol extract of Rnz leaves was 97.34±0.07 mg/L. Based on these findings, the antioxidant activity of RnZ leaf extract belongs to the strong group. This was related to the IC50 values obtained in the range of 50–100 mg/L23.
Table 3: The outcomes of the DPPH method's evaluation of antioxidant activity
|
Sample |
Concentration (mg/L) |
% Inhibition |
IC50 (mg/L) |
|
BHT |
2 |
34.16 ± 0.05 |
4.30 ± 0.009 |
|
4 |
51.24 ± 0.09 |
||
|
8 |
71.31 ± 0.03 |
||
|
Ethanolic extract |
8 |
5.78 ± 0.03 |
97.34 ± 0.07 |
|
16 |
12.62 ± 0.01 |
||
|
32 |
20.66 ± 0.02 |
||
|
64 |
42.52 ± 0.01 |
||
|
128 |
60.31 ± 0.07 |
Evaluation of Antioxidants with the FRAP Method:
The results of antioxidant evaluation using the FRAP method were expressed in % reduction power, as shown in Table 4. These results are then connected to a series of sample or standard concentrations to produce a curve. The positive control, which was a gallic acid compound, had a regression equation of y = 28.886x + 28.654, and the ethanol extract of Rnz leaves had a regression equation of y = 4.9122x + 7.3208. Based on this equation, the value for the EC50 concentration of gallic acid was 0.74±0.03 mg/L, and the concentration of the ethanol extract of Rnz leaves was 8.69±0.02 mg/L. Based on these results, gallic acid has a better ability to reduce Fe3+ than the ethanol extract of Rnz leaves; however, the reducing power of the ethanol extract is in the very strong category because the EC50 value is less than 50 mg/L23.
Table 4: The results of the FRAP method antioxidant activity test
|
Sample |
Concentration (mg/L) |
% Reduction Power |
EC50 (mg/L) |
|
Gallic Acid |
0.25 |
36.38 ± 0.1 |
0.74 ± 0.03 |
|
0.5 |
42.06 ± 0.02 |
||
|
0.75 |
50.82 ± 0.1 |
||
|
Ethanolic extract |
2 |
15.71 ± 0.08 |
8.69 ± 0.02 |
|
4 |
27.95 ± 0.07 |
||
|
6 |
38.26 ± 0.06 |
||
|
8 |
46.47 ± 0.05 |
||
|
10 |
55.58 ± 0.2 |
The results of evaluating antioxidant activity using the DPPH and FRAP methods show that the ethanol extract of RnZ leaves can be a source of natural antioxidant raw materials. The presence of compounds 9, 12, 14, 16, 17, 21, 26, 30, 42, 46, 49, 60, 65, and 66 in the leaf extract was revealed by LC-MS/MS-QToF analysis. These compounds have been reported to have strong antioxidant properties.
Compounds 9 and 30 are xanthone analogues. Xanthone analogues have been known to have strong antioxidant activity24. Compound 12 belongs to the flavonoid groups. Antioxidant abilities: this compound is associated with the presence of a phenolic group, which can donate hydrogen atoms to free radicals, making them less reactive25,26,27. Compound 14 is a hydroxy benzoic acid derivative. P-hydroxybenzoic acid has been reported to have the ability to act as a strong antioxidant28. Compounds 16, 26, 46, and 60 are coumarin derivatives (2H-chromen-2-one). Coumarin has been reported to have strong antioxidant activity29. Compound 17 is a derivative of tetrahydroquinoline. The DPPH method and total reducing power (TRP) assays have revealed that this compound's derivatives have high antioxidant activity30.
Compound 21 is a type of alkaloid known as an isoquinoline alkaloid. Compound 21 isolated from N. arvensis has been shown to have antihyperlipidemic, antidiabetic, antioxidant, antiobesity, antiviral, cytotoxic, immunoregulatory activity, and antiplatelet effects31. Compound 42 is a derivative of cannadiol compounds, which have been shown to have potent antioxidant properties32. Compound 49 is an aporphine alkaloid group which has been reported to have strong antibacterial and antioxidant activity33. Compound 65 is a cinnamic acid derivative, and it has been reported that cinnamyl has strong antioxidant abilities34. Compound 66 is a derivative of ortho-naphthoquinone. These natural compounds have anti-inflammatory, anti-obesity, antifungal, antibacterial, neuroprotective, nephroprotective, and wound healing properties35.
Evaluation of DPP IV Activity Inhibition:
Evaluation of DPP IV inhibitory activity was conducted in vitro using the substrate glysine-proline-7-amido-4-methylcoumarin (Gly-Pro-AMC). Gly-Pro-AMC converts to Gly-Pro and AMC. Its inhibitory activity was based on the level of AMC, which was determined by measuring the fluorescence using a microplate reader. The inhibitory activity of DPP IV was measured quantitatively in the presence or absence of the extract, and sitagliptin was used as a positive control36.
Table 5: The results of a test for the inhibition of DPP IV activity
|
Sample |
Final Concentrate (µg/mL) |
% Inhibition |
|
Sitagliptin |
100 |
85.54 ± 0.9 |
|
Ethanolic Extract |
100 |
43.05 ± 1.2 |
The inhibitory activity of DPP IV by sitagliptin produced in this study was 85.54 ± 0.9%, while the ethanolic extract of the leaves had an inhibitory activity of 43.05 ± 1.2% in succession. These results indicate that the ethanolic extract of the leaves of Rkz has the potential as a DPP IV inhibitor. According to the reports, the inhibitory activity of DPP IV is due to the content of polyphenols in plants, such as flavonoids37. In addition, reports exist of Rkz leaf extract being active as an antidiabetic through an aGl inhibition mechanism14.
DPP IV and aGl inhibitory activity was linked to Rkz leaf extracts containing compounds 9, 10, 12, 15, 20, 21, 27, 30, 32, 43, and 45. Compounds 9 and 30 are xantone derivatives. aGl activity in xanthone analogues has been studied18. Compounds 10 and 43 are saturated fatty acids. It has been reported that fatty acids have the potential for antidiabetic T2DM38. Compound 12 is a member of the flavonoid group and has been reported to be active in aGl inhibition39. Compound 15 is thought to be active as an antidiabetic. Alizarin-2-methyl ether isolated from the root of M. officinalis exhibits potential anti-diabetic properties40. Compound 20 is a cinnamic acid derivative. Cinnamic acid derivatives have been reported to have aGl inhibitory activity. The compound 4-methoxycinnamic acid has been reported to have 10 times stronger activity than cinnamic acid41. Compound 21 is an isoquinoline alkaloid group. Glaucine isolated from N. arvensis has been reported to have antidiabetic activity31. Compound 27 is a benzothiazole derivative. Benzothiazole derivatives are a class of heterocyclic compounds with a variety of pharmacological properties, including anti-diabetic and anti-inflammatory properties42. Compound 32 is an alkaloid group and has been reported to be active in aGl inhibition39. Compound 45 is a naphthodianthrone derivative which has been reported to have an interesting protective effect against the harmful consequences of lipid dysregulation in the context of T2DM43,44.
CONCLUSIONS:
Evaluation of antioxidant activity using the DPPH and FRAP methods, respectively, gave IC50 and EC50 values of 97.34±0.07 mg/L and 8.69±0.02 mg/L. In addition, ethanolic leaf extract has potential as an antidiabetic through the mechanism of inhibition of DPP IV activity with an inhibition value of 43.05 ± 1.2%. The determination of compounds in the ethanolic extract of Rkz leaves using UHPLC-MS/MS QToF succeeded in identifying 65 compounds, with several compounds reported to be active as antidiabetic. These compounds are (9), (10), (12), (15), (20), (21), (27), (30), (32), (43), (45). In conclusion, the ethanolic extract of the leaves of Rkz from the UAE has the potential to be an antioxidant and antidiabetic raw material.
Acknowledgement:
This research was realized thanks to the 2021 Doctoral Dissertation Research Grant provided by the Ministry of Research and Technology and the National Research and Innovation Agency in Indonesia.
The authors have stated that there are no potential conflicts of interest.
1. Cho NH. Shaw JE. Karuranga S. Huang Y. da Rocha Fernandes JD. Ohlrogge AW. Malanda B. IDF Diabetes Atlas: Global Estimates of Diabetes Prevalence for 2017 and Projections for 2045. Diabetes Research and Clinical Practice. 2018; 138: 271-81. doi.org/10.1016/j.diabres.2018.02.023
2. Kumari MS. Babu MK. Sulthana R. Srinivas M. Prasanthi C. Diabetes Mellitus: Present Status and Drug Therapy Updates. Research Journal of Pharmacy and Technology. 2014; 7(1): 84-94.
3. Aphale P. Sharma D. To Study and Compare the Efficacy of LM Potency and Centesimal Potency of Homoeopathic Medicine Acid Phosphoricum in Management of Type-2 Diabetes Mellitus. Research Journal of Pharmacy and Technology. 2023; 16(4): 1689-4. doi.org/10.52711/0974-360X.2023.00277
4. International Diabetes Federation. IDF Diabetes Atlas. 10th edition. 2021.
5. Goyal RI. Jialal I. Type 2 Diabetes. 2023, StatPearls Publishing. https://www.ncbi.nlm.nih.gov/books/NBK513253
6. Galicia-Garcia U. Benito-Vicente A. Jebari S. Larrea-Sebal A. Siddiqi H. Uribe KB. Ostolaza H. Martín C. Pathophysiology of Type 2 Diabetes Mellitus. International Journal of Molecular Sciences. 2020; 21(17): 6275. doi.org/10.3390/ijms21176275
7. Ormazabal V. Nair S. Elfeky O. Aguayo C. Saloon C. Zuñiga FA. Association Between Insulin Resistance and the Development of Cardiovascular Disease. Cardiovascular Diabetolog. 2018; 17(1): 122. doi.org/10.1186/s12933-018-0762-4
8. Bhat N. Kumar A. Kumar P. Pai A. A Comprehensive Review on the Antidiabetic Activity of Oxadiazole Derivatives. Research Journal of Pharmacy and Technology. 2023; 16(6): 2771-2775. doi.org/10.52711/0974-360X.2023.00455
9. Ankita. Bhardwaj K. Khurana N. Sutte A. Khatik G. Identification of Dipeptidyl Peptidase-4 (DPP-4) Inhibitors as Potential Antidiabetic Agents Using Molecular Docking Study. Research Journal of Pharmacy and Technology. 2020; 13(11): 5257-5262. doi.org/10.5958/0974-360X.2020.00919.1
10. Parasuraman S. Thing GS. Dhanaraj SA. Polyherbal Formulation: Concept of Ayurveda. Pharmacognosy Reviews. 2014; 8(16): 73-80. doi.org/10.4103/0973-7847.134229
11. Yuan H. Ma Q. Cui H. Liu G. Zhao X. Li W. Piao G. How Can Synergism of Traditional Medicines Benefit from Network Pharmacology?. Molecules. 2017; 22(7): 1135. doi.org/10.3390/molecules22071135
12. Grover JK. Yadav S. Vats V. Medicinal Plants of India with Anti-diabetic Potential. Journal of Ethnopharmacology. 2002; 81(1): 81-100. doi.org/10.1016/s0378-8741(02)00059-4
13. Siripong P. Kanokmedakul K. Piyaviriyagul S. Yahuafai J. Chanpai R. Suchirawat S. Oku N. Antiproliferative Napthaquinone Esters from Rhinacanthus nasutus Kurz. Roots on Various Cancer Cells. Journal of Traditional of Medicines. 2006; 23: 166-172. doi.org/10.11339/jtm.23.166
14. Shah MA. Khalil R. Ul-Haq Z. Panichayupakaranant P. α-Glucosidase Inhibitory Effect of Rhinacanthins-Rich Extract from Rhinacanthus nasutus Leaf and Synergistic Effect in Combination with Acarbose. Journal of Functional Foods. 2017; 36: 325-331. doi.org/10.1016/j.jff.2017.07.021
15. Irawan C. Elya B. Hanafi M. Saputri FC. Application of Ultrasound-Assisted Extraction on the Stem Bark of Rhinachantus nasutus (L.) Kurz, Total Phenolic, and its Potential as Antioxidant and Inhibitor of Alpha-Glucosidase Enzyme Activity. Pharmacognosy Journal. 2021; 13(5): 1-7. doi.org/10.5530/pj.2021.13.164
16. Irawan C. Elya B. Hanafi M. Saputri FC. Potential Ethanol Extract of Rhinachantus nasutus (L.) Kurz Stem Bark as Antioxidant and Inhibitor of Dipeptidyl Peptidase IV (DPP IV) Activity. Research Journal of Pharmacy and Technology. 2023; 16(3):1-6. doi.org/10.52711/0974-360X.2023.00197
17. Kallepalli P. Annapurna MM. Separation, Identification and Quantification of Process Related Impurities and Stress Degradants of Olaparib by LC-ESI-Q-TOF-MS. Research Journal of Pharmacy and Technology. 2018; 11(8):3718-3726. doi.org/10.5958/0974-360X.2018.00682.0
18. Abha S. Anchal C. Kumar SR. Amanpreet K. Isolation and Identification of Two Triterpenoids from Ethyl Acetate Extract of Bark of Boehmeria rugulosa. Research Journal of Pharmacy and Technology. 2021; 14(6):2919-2923. doi.org/10.5958/0974-360X.2018.00682.0
19. Chaudhari BP. Daniel K. A Validated Ultra Performance Liquid Chromatography Method for Simultaneous Estimation of Diacerein and Aceclofenac in Bulk and Pharmaceutical Formulation. Research Journal of Pharmacy and Technology. 2022; 15(4):1467-1. doi.org/10.52711/0974-360X.2022.00243
20. Kusnanda AJ. Dharma A. Armaini. Syafrizayanti. Chaidir Z. Carotenoid Profile of Freshwater Microalgae Mychonastes racemosus AUP1 and its Antioxidant Properties. Research Journal of Pharmacy and Technology. 2023; 16(1):404-410. doi.org/10.52711/0974-360X.2023.00069
21. Mathew J. Neethu MV. RP-HPLC Hyphenated with ESI-Q-TOF–MS for the Detection of Potential Degradants in Dolutegravir. Research Journal of Pharmacy and Technology. 2023; 16(3):1012-6. doi.org/10.52711/0974-360X.2023.00169
22. Cayman Chemical Company. DPP (IV) Inhibitor Screening Assay Kit. 2017; Cayman Chemical Company: AnnArbor, MI, USA.
23. Jun M. Fu HY. Hong J. Wan X. Yang CS. Ho CT. Comparison of Antioxidant Activities of Isoflavones from Kudzu Root (Pueraria lobate Ohwi). Journal of Food Science. 2006; 68(6):2117-2122. doi.org/10.1111/j.1365-2621.2003.tb07029.x
24. Santos CMM. Freitas M. Fernandes E. A Comprehensive Review on Xanthone Derivatives as Alpha-Glucosidase Inhibitors. European Journal of Medicinal Chemistry. 2018; 5(157):1460-1479. doi.org/10.1016/j.ejmech.2018.07.073
25. Petacci F. Freitas SS. Brunetti IL. Khalil NM. Inhibition of Peroxidase Activity and Scavenging of Reactive Oxygen Species by Astilbin Isolated from Dimorphandra mollis (Fabaceae, Caesalpinioideae). Biological Research. 2010; 43(1): 63-74.
26. Chigurupati S. AlGobaisy YK. Alkhalifah B. Alhowail A. Bhatia S. Das S. Vijayabalan S. Antioxdiant and Antidiabetic Potentials of Curcubita pepo Leaves Extract from the Gulp Region. Rasayan Journal of Chemistry. 2021; 14(4): 2357-2362. doi.org/doi.org/10.31788/RJC.2021.1446455
27. Olugbuyiro JAO. Banwo AS. Adeyemi AO. Taiwo OS. Akintokun OA. Phytochemical Constituents, Antioxidant and Antimicrobial Activities of Eugenia uniflora linn. Leaf. Rasayan Journal of Chemistry. 2018; 11(2): 798-805. doi.org/10.31788/RJC.2018.1121823
28. Manuja R. Sachdeva S. Jain A. Chaudhary J. A Comprehensive Review on Biological Activities of p-Hydroxy Benzoic Acid and its Derivatives. International Journal of Pharmaceutical Sciences Review and Research. 2013; 22(2): 109-115.
29. Kostova I. Bhatia S. Grigorov P. Balkansky S. Parmar VS. Prasad AK. Saso L. Coumarins as Antioxidants. Current Medicinal Chemistry. 2011; 18(25): 3929-3951. doi.org/10.2174/092986711803414395
30. Farooq S. Mazhar A. Ghouri A. Ul-Haq I. Ullah N. One-Pot Multicomponent Synthesis and Bioevaluation of Tetrahydroquinoline Derivatives as Potential Antioxidants, α-Amylase Enzyme Inhibitors, Anti-Cancerous and Anti-Inflammatory Agents. Molecules. 2020; 25(11): 1-28. doi.org/10.3390/molecules25112710
31. Ma C. Wang J. Chu H. Zhang X. Wang Z. Wang H. Li G. Purification and Characterization of Aporphine Alkaloids from Leaves of Nelumbo nucifera Gaertn and Their Effects on Glucose Consumption in 3T3-L1 Adipocytes. International Journal of Molecular Sciences. 2014; 15(3): 3481-3493. doi.org/10.3390/ijms15033481
32. Atalay S. Karpowicz IJ. Skrzydlewska E. Antioxidative and Anti-inflammatory Properties of Cannabidiol. Antioxidants. 2020; 9(21):1-20. doi.org/10.3390/antiox9010021
33. Masnon FF. Hassan NPS. Ahmad F. Aporphine Alkaloids of Cinnamomum mollissimum and Their Bioactivities. Natural Product Communications. 2014; 9(1):31-32.
34. Hussein MA. El-Gizawy HAE. Gobba NAEK. Mosaad YO. Synthesis of Cinnamyl and Caffeoyl Derivatives of Cucurbitacin-Eglycoside Isolated from Citrullus colocynthis Fruits and Their Structures Antioxidant and Anti-inflammatory Activities Relationship. Current Pharmaceutical Biotechnology. 2017; 18(8): 677-693. doi.org/10.2174/1389201018666171004144615
35. Gomes CL. Sales VAW. Melo CG. Silva RMF. Nishimura RHV. Rolim LA. Neto PJR. Beta-Lapachone: Natural Occurrence, Physicochemical Properties, Biological Activities, Toxicity and Synthesis. Phytochemistry. 2021; 186(112713). doi.org/10.1016/j.phytochem.2021.112713
36. Amin MS. Saputri FC. Mun`im A. Inhibition of Dipeptidyl Peptidase 4 (DPP IV) Activity by Some Indonesia Edible Plants. Pharmacognosy Journal. 2019; 11(2): 231-236. doi.org/10.5530/pj.2019.11.36
37. Ekayanti M. Sauriasari R. Elya B. Dipeptidyl Peptidase IV Inhibitory Activity of Fraction from White Tea Ethanolic Extract (Camellia sinensis (L.) Kuntze) ex Vivo. Pharmacognosy Journal. 2018; 10(1): 190-193. doi.org/10.5530/pj.2018.1.32
38. Savych A. Marchyhshyn S. Basaraba R. Determination of Fatty Acid Composition Content in the Herbal Antidiabetic Collections. Pharmacia. 2020; 67(3): 153-159. doi.org/10.3897/pharmacia.67.e51812
39. Hanafi. Irawan C. Rochaeni H. Sulistiawaty L. Roziafanto AN. Supriyono. Phytochemical Screening, LC-MC Studies and Antidiabetic Potential of Methanol Extracts of Seed Shells of Archiendron bubalinum (Jack) I.C Nielsen (Julang Jaling) from Lampung, Indonesia. Pharmacognosy Journal. 2018; 10(6): s77-s82. doi.org/10.5530/pj.2018.6s.15
40. Liu Q. Kim SB. Ahn JH. Hwang BY. Kim SY. Lee MK. Anthraquinones from Morinda officinalis Roots Enhance Adipocyte Differentiation in 3T3-L1 Cells. Natural Product Research. 2012; 26(18): 1750-1754. doi.org/10.1080/14786419.2011.608676
41. Ernawati T. Radji M. Hanafi M. Mun’im A. Yanuar A. Indonesia. Cinnamic Acid Derivatives as α-Glucosidase Inhibitor Agents. Indonesian Journal of Chemistry. 2017; 17(1): 151-160. doi.org/10.22146/ijc.23572
42. Kumar S. Dubey B. A Review on Emerging Benzothiazoles: Biological Aspects. Journal of Drug Delivery and Therapeutics. 2022; 12(4-S): 270-274. doi.org/10.22270/jddt.v12i4-s.5549
43. Liang C. Hao F. Yao X. Qiu Y. Liu L. Wang S. Yu C. Song Z. Bao Y et al Hypericin Maintians PDX1 Expression via the Erk Pathway and Protects Islet β-Cells Against Glucotoxicity and Lipotoxicity. International Journal of Biological Sciences. 2019; 15(7): 1472-1487. doi.org/10.7150/ijbs.33817
44. Liang C. Li Y. Bai M. Huang Y. Yang H. Liu L. Wang S. Yu C et al Hypericin Attenuates Nonalcoholic Fatty Liver Disease and Abnormal Lipid Metabolism via the PKA-Mediated AMPK Signaling Pathway In vitro and In vivo. Pharmacological Research. 2020; 153(104657). doi.org/10.1016/j.phrs.2020.104657
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Received on 05.09.2023 Revised on 13.07.2024 Accepted on 18.11.2024 Published on 28.01.2025 Available online from February 27, 2025 Research J. Pharmacy and Technology. 2025;18(2):495-501. DOI: 10.52711/0974-360X.2025.00075 © RJPT All right reserved
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