Flavanone compounds of Cyperus rotundus L:

FTIR Profile and Alfa Glucosidase Enzyme Inhibition Assay

 

Laeli Fitriyati1, Laela Hayu Nurani2*, Dwi Utami2, Wahyu Widyaningsih2

1Pharmacy Study Program, Faculty of Health Sciences,

Muhammadiyah Gombong University, Kebumen, Indonesia.

2Department of Pharmacy, Faculty of Pharmacy,

Universitas Ahmad Dahlan, Umbulharjo, Yogyakarta, Indonesia.

*Corresponding Author E-mail: laela.farmasi@pharm.uad.ac.id

 

ABSTRACT:

In Indonesia, the prevalence of diabetes mellitus is expected to reach 19.5 million by 2021. The enzyme α glucosidase contributes to diabetes mellitus. Sedge grass (SG) contains flavanone chemicals that reduce blood glucose levels by blocking the α-glucosidase enzyme. This study analyzes the α-glucosidase enzyme's inhibitory effect and the flavanone chemicals' FTIR profile in fractionated SG tuber extract. It also examines the characterization of the active fraction. This experimental study employed samples of 96% ethanol extract. The study began with maceration of SG tubers in 96% ethanol, followed by fractionation with ethanol: water (7:3) (sample I), ethyl acetate (sample II), and hexane (sample III). Testing was done in vitro with three groups. The fraction was examined for inhibition of the α-glucosidase enzyme using the UV-Vis spectrophotometric technique and IC50 parameters with the reference substance acarbose. The active fraction was characterized using an FTIR spectrophotometer. The data were analyzed using the ANOVA test. The IC50 values for acarbose groups I, II, and III were 25.8894 ± 0.03223 ppm, 9.4731 ± 0.0424 ppm, and -34.2001 ± 14.2534 ppm, respectively. Groups I and II significantly limit the activity of the α-glucosidase enzyme, while Group III does not. The characterization results suggest that the functional group resembles the flavanone group of chemicals. The active fraction of SG tubers contains functional groups nearly identical to normal functional groups, particularly flavanone compounds.

 

KEYWORDS: Flavanone, Cyperus rotundus L, α-glucosidase enzyme, FTIR.

 

 

INTRODUCTION: 

A metabolic disease called diabetes mellitus (DM) is characterized by elevated blood sugar levels1. Oxidative stress damages pancreatic beta cells2. Diabetes is caused by insulin secretion and resistance, as well as degradation of pancreatic β-cell function, which leads to metabolic problems3.

 

Type 2 DM patients are frequently treated with oral hypoglycemic medication therapy (OHO). Natural α-glucosidase inhibitors are used for oral antidiabetic therapy (OHO). This can be used as a safe and effective alternative to new medications for diabetic patients4.

 

The study aims to identify natural α-glucosidase inhibitors as an alternative therapy option for diabetes. Cyperus rotundus L. is also widely recognized as a possible antioxidant5. Sedge grass tubers are an alternative herbal therapy with the potential to treat this illness. Flavonoids are chemical entities with the configuration C6-C3-C6 (two phenyl rings connected by a propane bridge). They are mostly classified as flavanones, chalcones, flavonols, and isoflavones. Because of their broad spectrum of biological actions, both synthetic and natural flavonoids have attracted a lot of research6. A novel flavanone, 7,8-dihydroxy-5,6-methylenedioxyflavone, from sedge grass (Cyperus rotundus L.), displays antioxidant activity with an IC50 value of 62.82 ppm5,7. Flavanone molecules have antioxidant activity, which has been linked to anti-diabetes because antioxidant substances can inhibit the detrimental effects of oxidation in the body by donating electrons to oxidizing compounds1.

 

The use of antioxidants in diabetic patients has an impact on reducing the prevalence of complications associated with the pathological process of diabetes mellitus, which is produced by oxidative stress2. To investigate the potential of sedge grass as an alpha-glucosidase inhibitor, the researchers examined sedge grass tubers using the FTIR (Fourier Transform Infrared) method. This involved extraction, fractionation, and in vitro testing for enzyme inhibitory activity against α-glucosidase.

 

MATERIALS AND METHODS:

Materials and equipment:

SG tubers were collected from Argosari Village, Ayah District, Kebumen Regency. The following materials were used: 96% ethanol, distilled water, ethyl acetate, alpha-glucosidase enzyme from Saccharomyces cerevisiae, DMSO (Dimethyl sulfoxide), p-nitrophenyl-α-D-glucopyranoside (PNPG) 20Mm Sigma Aldrich, phosphate buffer pH 7, NaHCO3 0.2M, and aluminum foil, filter paper, beaker glass, Erlenmeyer, measuring cup, and the Nicolet iS 10 FT-IR Spectrometer trademark is used in this research for Fourier transform Infrared Spectroscopy (FTIR). Nicolet iS 10 FT-IR Spectrometer is a widely used tool for infrared spectroscopy study.

 

Procedure:

This study conducted plant determination to ensure the accuracy of a plant that will be used in research published by Ahmad Dahlan University's Biology faculty under the number 287/Lab.Bio/B/VI/2023. Afterwards, SG was collected in Ayah Beach in Kebumen, Central Java.

 

Extraction and fractionation:

Simplicia extracts SG tubers through a maceration procedure. A total of 100grams of SG tuber simplicia were harvested and macerated in 1000mL of 96% ethanol solvent for three consecutive 24-hour periods. The resulting macerate was filtered using Whatmann filter paper no. 1 and then evaporated at 49°C using a rotary evaporator to separate the solvent from the macerate. The evaporation process was conducted in a water bath to yield a thick extract8.

 

The extracted products were divided into 10gram portions and diluted in a mixture of 100mL water and ethanol (7:3 ratio). Subsequently, 100mL of n-hexane was added, and the resulting mixture was separated using a separating funnel. Ethyl acetate solvent (100 mL) was then introduced, and the liquid was separated again using a separating funnel. This procedure yielded three products: ethanol, n-hexane, and ethyl acetate. The resulting fractions were separated from the solvents using a rotary evaporator and then evaporated in a water bath until a viscous fraction was obtained8.

 

FTIR Analysis for Active Flavone Compounds:

The sample was dissolved and deposited onto the surface of the KBr cell, which was cupped until the liquid produced a thin layer of capillary film before being placed in the cell holder. The sample spectra were obtained at wavenumbers 4000-450 cm-1.

 

α-glucosidase Inhibitory Activity Assay:

The extracts and SG tuber fractions were dissolved in 8 mg of DMSO. The standard solution of flavonone molecules was diluted to concentrations of 200, 100, 50, 25, 10, and 5ppm. Similarly, the SG tuber extract was prepared in the same six concentrations. In each tube, 5 µL of sample was mixed with 495µL of pH 7 phosphate buffer and 250µL of 20mM PNP-α-D-glucopyranoside. The solution was homogenized and incubated at 37℃ for 5 minutes. Next, add 250µL of α-glucopyranosidase enzyme and incubate for an additional 15 minutes. The enzymatic process was halted by adding 1mL of 2M NaHCO3.

 

The resulting p-nitrophenol concentration was measured at 405nm. Additionally, sample preparation was conducted without the presence of α-glucopyranosidase and with the inclusion of an acarbose comparison solution (200mg dissolved in phosphate buffer up to 2%)9,10.

 

RESULT:

Preparation of Simplicia:

A total of 250grams of simplicia underwent extraction through the maceration method utilizing a 96% ethanol solvent. The resulting extract exhibited characteristics of being brown in color, viscous, and sticky, and weighed 35.19grams, which corresponded to a yield of 17.59% for simplicia.

 

Extraction and Fractionation:

The simplicia powder underwent a three-day extraction process through maceration with stirring. Subsequently, the macerate was concentrated using a rotary evaporator at a temperature of 50°C. The resulting extract was then fractionated using water-ethanol, ethyl acetate, and n-hexane.  The results of the extraction that had been carried out previously were taken in 10grams and then dissolved in 100mL of water: ethanol with a ratio of (7:3) and added with 100mL of n-hexane. The mixture was separated using a separating funnel. Next, 100mL of ethyl acetate was added. Then separated using a separating funnel. Then 3 results were obtained from this fractionation, namely water: ethanol, n-hexane, and ethyl acetate. After obtaining this fraction, it is then separated from the solvent using a rotary evaporator. The yield values of the SG tuber fraction can be found in Table 1.

 

Table 1. Quercetin Solution Absorbance Measurement Results

Extract

Yield (%)

Ethanol

17.59

Fraction

Yield (%)

Ethanol: water ( 7:3)

32.30

Ethyl acetate

6.7

n-hexane

10.5

 

FTIR Analysis for Active Flavone Compound:

The results of the FTIR spectrophotometer analysis of the extract of SG tuber in Figure 1 show that there are similarities with the functional groups of flavanone group compounds, including having a bound -OH functional group, CH3 stretch, CH2 stretch, C=O, C=C, aliphatic C-H, -C-O-C (ether), cyclic C-OH, and substituted aromatic rings.

 

 

X= transmittance (%); Y= wavenumber (cm-1)

Figure 1. FTIR Spectrum of SG Tuber Extract

 

α-glucosidase Inhibitory Activity Assay:

The inhibitory action of the α-glucosidase enzyme was tested utilizing the principle of the enzyme, which hydrolyzes p-nitrophenyl-α-D-glucopyranoside to create the yellow molecule p-nitrophenol and glucose4. Increasingly, the clearer the yellow color in the test, the less glucose produced11, as seen in Figure 2.

 

Figure 2. Enzymatic Reaction of α-glucosidase

 

 

DISCUSSION:

The simplicia powder was extracted by maceration method for 3 days by stirring so that all the ingredients were completely dissolved in the liquid solvent. Stirring could increase the contact between the sample and the solvent so that the extraction process would be more complete. Ethanol was chosen because the polar nature of the flavonoid compounds tends to dissolve in polar solvents12. The maserate obtained was then concentrated using a rotary evaporator and the temperature was maintained at <50oC because flavonoids are unstable at high temperatures. They have a conjugated aromatic system which is easily damaged at temperatures >50oC13. The yield of the extract obtained from the maceration process was 17.59%. The higher the yield value, the higher the content of bioactive compounds that can be extracted and the more extracts produced14.

 

The resultant extract was then subjected to a fractionation procedure in which it was separated using a separating funnel. According to the polarity level, the fractionation procedure of SG tuber extract yields three fractions: ethanol: water (7:3), n-hexane, and ethyl acetate. Table 2 shows the prediction of the peak of the FTIR spectrum of SG tuber extract. Flavanones have a saturated C ring with three double bonds at positions 2 and 315.

 

Table 2. Prediction of the Peak of FTIR Spectrum of SG Tuber Extract

SG Tuber Extract

References (cm-1)

Prediction of Functional Groups

3318.96

3200-3450

O-H (bound) strain

1641.12

1647-1740

C-O strain

1042.31

1050-1260

-C-O-C-eter

 

Figure 3 depicts the FTIR spectrophotometer analysis of pure flavanones, which reveals the existence of bonded -OH functional groups, C-O stretches, aromatic rings, and carbon bonds. Flavanones are distinguished by the presence of a hydroxyl group at position 5 in the flavanone molecule, which plays a significant role in the reaction of the saturated C ring; it has three double bonds between positions 2 and 3, as illustrated in Figure 4.

 

Figure 3. The FTIR spectrum of pure flavanone compounds, the x-axis shows the transmittance value and the y-axis shows the wavelength

Table 3. The percentage of inhibition of α-glucosidase enzyme by acarbose, ethanol fraction: water (7:3), ethyl acetate fraction and n-hexane fraction

 

Sample

Inhibition percentage value

1%

(33.6668 µg/ml)

0.5%

(16.8334µg/ml)

0.25%

(8.4167µg/ml)

0.125%

(4.2083µg/ml)

0.0625%

(2.1042 µg/ml)

0.03125%

(1.0521 µg/ml)

Acarbose

60.43±0.03553

44.74±0.02918

41.38±0.03601

32.19±0.00639

26.74±0.01729

-

Group I

57.43±0.07472

42.16±0.00957

28.66±0.06273

22.77±0.00957

9.32±0.01913

5.59±0.00957

Group II

98.38±0.4586

79.28±0.3829

71.23±4.9127

55.46±1.2764

13.42±0.4318

8.48±0.3442

Group III

88.36±4.2069

79.45±16.3200

76.04±1.5264

75.66±0.3350

69.22±11.4145

62.22±5.6634

 

 

Figure 4. Flavanone Structure

 

Kusumawati et al. (2021) compared α-glucosidase activity to that of acarbose, a chemical known to inhibit α-glucosidase11. Table 3 displays the results of the α-glucosidase activity inhibition test for samples and acarbose

 

Table 3 shows that the proportion of inhibition of α-glucosidase enzyme activity increased as sample concentration rose. This event was triggered by the sample's high concentration; the bigger number of secondary metabolite chemicals present in each sample, the stronger the inhibition at 33.6668ppm. The ethyl acetate fraction (33.6668ppm) showed the strongest inhibitory capacity compared to other samples, with a percentage value of 98.38±0.4586%. The IC50 value of acarbose control is 24.3961ppm, which is considered to have a strong inhibitory potential. The IC50 value for the ethanol : water fraction is 25.8894 ppm, indicating that it has a considerable inhibitory potential. The ethyl acetate fraction has a significant inhibitory potential (IC50 = 9.4731ppm), while the n-hexane fraction has no inhibitory potential (IC50 = -34.2001ppm) as shown in Figure 5.

 

Figure 5. IC50 values for acarbose, ethanol fraction: water (7:3), ethyl acetate fraction and n-hexane fraction against the α-glucosidase enzyme

Inhibitors of alpha glucosidase are useful in the treatment of hyperglycemia because they delay carbohydrate digestion, reducing postprandial plasma glucose levels16. Alpha glucosidase inhibitors compete with the enzyme alpha glucosidase, which is necessary for carbohydrate digestion. In the small intestine, alpha glucosidases hydrolyze complex carbohydrates to provide glucose and other monosaccharides. As a result, postprandial blood sugar levels rise, resulting in hyperglycemia. As a result, inhibiting the alpha glucosidase enzyme is necessary for controlling postprandial blood glucose levels17. Inhibiting these enzyme systems contributes to a slower rate of glucose digestion. Because the carbohydrates are not broken down into glucose molecules, the body absorbs less of them. In diabetics, short-term treatment with these inhibitors helps lower elevated blood glucose levels. Thus, decreasing hyperglycemia is the most recent and widespread therapy method for Diabetes Mellitus18.

 

CONCLUSION:

The study concluded that SG tuber extract contains functional groups that are thought to be related to the flavanone group of chemicals. The 7:3 ethanol-water fraction inhibited α-glucosidase with an IC50 value of 25.8894ppm ±0.032, whereas the ethyl acetate fraction had an IC50 value of 9.473 ppm ±0.042 and the n-hexane fraction had an IC50 value of -34.2001ppm ± 14.25. The ethanol-water and ethyl acetate fraction groups had the highest α-glucosidase inhibitory efficacy.

 

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

 

ACKNOWLEDGMENTS:

The authors would like to thank the Ministry of Research and Technology through the 2023 Doctoral Dissertation Research (PDD) scheme with contract number 181/E5/PG.02.00.PL/2023 which was awarded to Dr. Wahyu Widyaningsih, M.Sc., Pharmacy Laboratory, Muhammadiyah University Gombong, Faculty of Pharmacy, Ahmad Dahlan University Yogyakarta who has assisted with FTIR testing and α-glucosidase activity.

 

REFERENCES:

1.      Nuralifah N, Fitrawan LOM, Parawansah P, Trisetya M. Histopatologi Organ Pankreas Tikus DM tipe 2 yang diberi Ekstrak Etanol Daun Gedi Merah (Abelmoscus manihot L. Medik). Journal Syifa Sciences and Clinical Research. 2022; 4(1). doi:10.37311/jsscr.v4i1.13566

2.      Prawitasari DS. Diabetes Melitus dan Antioksidan. Keluwih: Jurnal Kesehatan dan Kedokteran. 2019; 1(1): 47-51. doi:doi.org/10.24123/kesdok.v1i1.2496

3.      Ozougwu J, Obimba KC, Belonwu CD, Unakalamba CB. The pathogenesis and pathophysiology of type 1 and type 2 diabetes mellitus. Academic Journals. 2013; 4: 46-57.

4.      Sumarlin LO, Sukandar D, Pratiwi L. Aktivitas Penghambatan α-Glukosidase Campuran Ekstrak Daun Namnam (Cynometra cauliflora L.) dan Madu Kaliandra. al Kimiya: Jurnal Ilmu Kimia dan Terapan. 2019; 6(2): 87-94. doi:10.15575/ak.v6i2.6577

5.      Lydia J, Sudarsanam D. Total phenol and total tannin content of Cyperus rotundus L. and its medicinal significance. Research Journal of Pharmacy and Technology. 2012; 5(12): 1500-1502.

6.      Bhat KI, Kumari R, Kumar A, Kumar P. Synthesis of Some Novel Flavanones and Evaluation of Antioxidant Activities. Research Journal of Pharmacy and Technology. 2019; 12(5): 2141-2144. doi:10.5958/0974-360X.2019.00355.X

7.      Anandea FA. Uji Antioksidan Dan Uji Sitotoksisitas Ekstrak Umbi Rumput Teki (Cyperus Rotundus L.) Dengan Metode DPPH Dan MTT Assay Pada Sel Leukemia HL-60. Sarjana. Universitas Brawijaya; 2019. Accessed February 26, 2024. https://repository.ub.ac.id/id/eprint/175911/

8.      Putri ASHP, Da’i M. Uji Sitotoksik Fraksi Dan Ekstrak Etanol Rumput Teki (Cyperus Rotundus) Terhadap Sel Kanker Payudara (MCF-7). Usadha Journal of Pharmacy. 2022; 1(1): 1-12. doi:doi.org/10.1234/ujp.v1i1.1

9.      Yuniarto A, Selifiana N. Aktivitas Inhibisi Enzim Alfa-glukosidase dari Ekstrak Rimpang Bangle (Zingiber cassumunar Roxb.) secara In vitro. MPI (Media Pharmaceutica Indonesiana). 2018; 2(1): 22-25. doi:10.24123/mpi.v2i1.1299

10.   Venkata Lakshmi J, Chitturi CMK, Rao CA. In Vitro Evaluation of Alpha Amylase and Alpha Glucosidase Inhibitory Activity of Kamadhenu Ark extracts of Climate Smart Crop Millets. Research Journal of Pharmacy and Technology. 2024; 17(1): 81-86. doi:10.52711/0974-360X.2024.00013

11.   Kusumawati N, Haryoto H, Indrayudha P. Penghambatan Enzim Alpha-Glukosidase oleh Daun Mimba (Azadirachta indica) dan Rimpang Temu Mangga (Curcuma mangga) | Jurnal Kefarmasian Indonesia. Jurnal Kefarmasian Indonesia. 2021; 11(1): 56-64.

12.   Riwanti P, Izazih F, Amaliyah A. Pengaruh Perbedaan Konsentrasi Etanol pada Kadar Flavonoid Total Ekstrak Etanol 50,70 dan 96% Sargassum polycystum dari Madura. Journal of Pharmaceutical-care Anwar Medika. 2018; 2(2): 35-48. doi:10.36932/jpcam.v2i2.1

13.   Tari M, Alta U, Indriani O. Penetapan Kadar Flavonoid Secara Spektrofotometri Visibel Pada Daun Jambu BIJI (Psidium guajava L) Dengan Perbedaan Suhu Pengeringan Simplisia. Jurnal’ Aisyiyah Medika. 2022; 7(1). doi:10.36729/jam.v7i1.776

14.   Purwitasari A, Hendrawan Y, Yulianingsih R. Pengaruh Suhu dan Waktu Ekstraksi Terhadap Sifat Fisik Kimia dalam Pembuatan Konsentrat Protein Kacang Komak (Lablab purpureus (L.) sweet) Influence of Temperature and Extraction Time to the Physical Chemistry Characreristic in Preparation Protein Con. Bioproses Komoditas Tropis. 2014; 2(1): 42-53.

15.   Panche AN, Diwan AD, Chandra SR. Flavonoids: an overview. J Nutr Sci. 2016;5:e47. doi:10.1017/jns.2016.41

16.   Sathish M, Nandhini M, Suresh R. In vitro Alpha amylase and Alpha glucosidase enzyme inhibition of leaf extracts of Jatropha glandulifera Roxb. Research Journal of Pharmacy and Technology. 2022; 15(6): 2493-2497. doi:10.52711/0974-360X.2022.00416

17.   Anjitha R, Subbaraya U, Narasimhan G. Integrated Invitro-Insilico screening strategy of Ethanolic extract of leaves of Passiflora edulis Sims var. flavicarpa against Alpha Glucosidase. Research Journal of Pharmacy and Technology. 2020; 13(3): 1309-1313. doi:10.5958/0974-360X.2020.00241.3

18.   Modh PG, Patel LJ. In vitro screening on Alpha amylase and Alpha glucosidase inhibitory activities of some novel Quinazolinone derivatives. Research Journal of Pharmacy and Technology. 2022; 15(9): 4226-4229. doi:10.52711/0974-360X.2022.00710

 

 

 

Received on 16.03.2024      Revised on 13.06.2024

Accepted on 03.09.2024      Published on 27.03.2025

Available online from March 27, 2025

Research J. Pharmacy and Technology. 2025;18(3):1108-1112.

DOI: 10.52711/0974-360X.2025.00159

© RJPT All right reserved

 

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