Available online on 15.06.2022 at http://jddtonline.info

Journal of Drug Delivery and Therapeutics

Open Access to Pharmaceutical and Medical Research

Copyright  © 2011-2022 The  Author(s): This is an open-access article distributed under the terms of the CC BY-NC 4.0 which permits unrestricted use, distribution, and reproduction in any medium for non-commercial use provided the original author and source are credited

Open Access   Full Text Article                                                                                                                                                                Research Article 

Antiradical and inhibitory activities of hydroethanolic extracts of Phyllanthus amarus and Striga hermonthica on α-amylase

ZANTE Abdoul-Aziz * ^ac , SOMDA Martin Bienvenu ^bd , OUATTARA Lassina ^ac , ZOUNGO Daouda ^ac , PODA F. Daniel ^ac , OUEDRAOGO R. Justin ^ac , SANOU Yacouba ^ac , KABRE B. Pawendé ^ac , KONATE K. Abdourasmane ^e, OUÉDRAOGO Georges Anicet ^cd 

a Département de Sciences Biologiques, Unité de Formation et de Recherche en Sciences de la Vie et de la Terre (UFR-SVT), Université Nazi BONI, 01 BP 1091 Bobo-Dioulasso 01, Burkina Faso.

b Centre International de Recherche-Development sur l’Elevage en zone Subhumide (CIRDES)

c Laboratoire de Recherche et d’Enseignement en Santé et Biotechnologies Animales, Université Nazi BONI, 01 BP 1091 Bobo-Dioulasso 01, Burkina Faso.

d Institut du Développement Rural, Université Nazi BONI, 01 BP 1091 Bobo-Dioulasso 01, Burkina Faso.

e Institut de l’Environnement et de la Recherches Agricoles/Bobo (INERA/Bobo)

Oxidative stress is defined as an imbalance between oxidants and antioxidant defenses¹. It is involved in a large number of chronic pathologies, including diabetes ². Thus, diabetes mellitus is a chronic disease resulting from a lack of insulin production and/or poor use of this hormone by the body³. Indeed, in 2019, 463 million people were diabetic worldwide. This figure could reach 578 million by 2030 if nothing is done⁴. According to WHO estimates, it will be the seventh leading cause of death worldwide in 2030³. As a result, it constitutes a major public health problem³. Despite the efforts made by modern medicine via synthetic products such as α-amylase inhibitors (acarbose), which is a therapeutic approach aimed at reducing postprandial hyperglycaemia, mortality has continued to increase worldwide. In addition, the high cost, the unavailability of drugs and their adverse effects are also factors contributing to this increase in mortality⁵. Faced with this situation, the World Health Organization (WHO) encourages researchers to explore new avenues of control such as herbal medicine⁶. To this end, the effectiveness of this discipline is proven and its undeniable benefits for health⁷ because of the active principles contained in the plants. Among these active ingredients, polyphenols are known for their antioxidant and antidiabetic properties^8,9. Thus, Phyllanthus (P.) amarus Schum & Thonn. and Striga (S.) hermonthica are plants used in traditional medicine throughout the world, particularly by the herbalists of Bobo-Dioulasso in the management of diabetes ^10,11,12. This study has been initiated in order to investigate the free DPPH radicals scavenging and α-amylase inhibitory activities of these plants.

2.1. Plant material

The whole plant of P. amarus and S. hermonthica were collected in Dinderesso (11°13’367” N 4°25’750” W), a village located about 15km from Bobo-Dioulasso, in September 2020. Two varieties of sorghum (Sorghum bicolor) namely IMS127 and KMTC 274 from the Institute of the Environment and Agricultural Research were used as a source of α-amylase.

2.2. Preparation and hydroethanolic extraction

The samples were dried in the dark, pulverized and stored in suitable bags before use. After measuring the moisture using by KERN (MLS 50-3C, Germany), 10 g of each powder were macerated 100 mL ethanol-water 70% (v/v) for 48 hours at room temperature with stirring according to the method described by Souhila et al.¹³ with some modifications. Then, the crude extracts were obtained following filtration using filter paper N°4 (Belle France) followed by evaporation of the solvent. Finally, these collected extracts were weighed and stored in the refrigerator in tightly closed sterile bottles to determine the antiradical and α-amylase inhibitory activity in vitro.

2.3. α-amylase extract preparation

The enzyme extracts were obtained using the protocol described by Adewale et al.^14,15 with some modifications. The protein concentration of these enzymatic extracts was determined using the bicinchoninic acid (BCA) kit (Pierce, Rockford, IL) with Bovine Serum Albumin (BSA) as standard (Sigma-Aldrich, LLC, USA). Finally, the specific activities of these extracts were also determined according to the method described by Xiao et al.¹⁶. The results were expressed in μg of starch consumed/minutes/mg of protein using the following regression equation ( 

2.4. Determination of total polyphenols

Polyphenol content of the extracts was evaluated by the Folin-Ciocalteu method¹⁷. Thus, 125 μL of each extract (100 μg/mL) was added to 625 μL of 0.2 N Folin-Ciocalteu reagent (MP Biomedicals, LLC, USA). After 5 min incubation, 500 μL of 7.5% sodium carbonate (CARLO ERBA Reagents S.A.S, France) were added to the first mixture. The whole was incubated at room temperature for 2 hours in the dark. The optical densities (OD) were read against a blank using a spectrophotometer (Spectrumlab 23A) at 760 nm. For this purpose, a calibration curve of gallic acid (Sigma- Aldrich Chemie, Steinheim, China) solution (0 to 200 mg/ L) of equation ( ) was established and the results were expressed in mg of gallic acid equivalent per 100 mg of dry extract (GAE /100 mg) using the calibration curve.

2.5. Assessment of antiradical activity

DPPH free radical scaveger activity was carried out by the DPPH method (1, 1-diphenyl-2-picryl-hydrazil)^18,19. In fact, 375 µL of each extract at different concentrations were mixed with 750 µL of DPPH (0.02 mg/mL) (Acros Organics, Spain) following by 15 minutes incubation at room temperature. Then OD was read against a blank at 517 nm. Quercetin (Sigma- Aldrich Chemie, Steinheim, China) was used as reference scavenger. The IC₅₀ was determined and the results were expressed in µg/mL.

2.6. Evaluation of the anti α-amylase activity

Plant extracts capacity to inhib alpha-amylase was evaluated by the 3,5-dinitrosalicylic (DNS) method^20,21. A mixture of phosphate buffer (pH=6.9; 0.02M) (Carlo Erba Reagents, France), plant extract and enzyme extract were made with the same volume (100 μL). This mixture was incubated at 37°C for 10 minutes. Then, 100 μL of 1% starch (Carlo Erba Reagents S.A, France) were added and the whole was incubated at 37°C for 15 minutes. After that, 200 µL of 1% DNS was added to stop the enzymatic reaction. The reaction mixture was incubated at 100°C for 8 minutes followed by cooling in an ice bath. Following this, was read against a blank at 540 nm. The inhibition percentage (%I) was calculated according to the formula:  .

2.7. Statistical analysis

The Microsoft Excel (2016) table was used to calculate the means, standard deviations of the data and the graphs. The R version 4.0.1 software was used to compare the means by a Student's T test at the 5% threshold.

3.1. Polyphenol content

The average yield of the hydroethanolic extracts was respectively 12.15 ± 0.07% (w/w) and 7.77 ± 0.11% (w/w) for the extract of P. amarus and S. hermonthica. Table 1 showed that the polyphenol content of the extract of P. amarus is significantly higher than that of S. hermonthica (p = 0.02).

Table 1: Polyphenol content

There is significant difference between a and b; DE: Dry Extract; GAE: Gallic Acid Equivalent

3.2. Anti-radical activity

The DPPH radical scavenging activity was carried out by determining the IC₅₀ values of the hydroethanolic extracts. The results were obtained using the regression curve for each sample (Figure 1).

Figure 1: Regression curve of P. amarus (PA), S. hermonthica (SH) and Quercetin(Q)

Thus, the extract of P. amarus exhibited an IC₅₀ of 77.74 ± 2.73 µg/mL versus 339.06 ± 1.43 µg/mL for S. hermonthica. The IC₅₀ of quercetin was 9.66 ± 0.10 µg/mL (Figure 2).

Figure 2: IC₅₀ of P. amarus, S. hermonthica  and Quercetin

3.3. Anti α-amylase activity

The inhibitory activities of the hydroethanolic extracts at 10 mg/mL on IMS 127 and KMTC 274 were evaluated. Thus, the extract of P. amarus presented the strongest inhibition whatever the malt extract used against a weak inhibition for the extract of S. hermonthica (Table 2).

Table 2: Inhibition of P. amarus extract and S. hermonthica on both malt extracts

Pc: Protein concentration; As: Specific activity U* in μg of starch consumed/minutes

The present study reported high polyphenol content for P. amarus extract unlike S. hermonthica extract which showed low content (p = 0.02). Other studies have shown the presence of polyphenols in these plants^22,23. Thus, the use of hydroethanolic solvent could explain this presence of these compounds because it has the advantage of solubilizing these compounds²⁴. The antiradical activity was evaluated using the DPPH test, most often used for the screening of molecules endowed with antioxidant activities present in plant extracts because of its rapidity²⁵. This activity made it possible to determine the IC₅₀ which is the concentration required to inhibit the DPPH radicals by half. In fact, the lower this concentration (IC₅₀), the higher the antioxidant activity²⁶. From the study, the extract of P. amarus exhibited a lower IC₅₀ than that of S. hermonthica. Therefore, the P. amarus extract was significantly more antiradical than the S. hermonthica extract (p = 2.2e-14). These results could be justified by their respective polyphenol content. Other studies have confirmed that these compounds are endowed with such activity^8,9. In short, the two extracts having this ability to trap DPPH free radicals are then potential antioxidants. Beyond these antiradical properties, polyphenols are also involved in the inhibition of α-amylase²⁷. During this study, both extracts exhibited an anti α-amylase effect on both varieties of sorghum malt. P. amarus extract showed higher inhibition compared to S. hermonthica extract which showed weak inhibition. This difference would be due to their respective content of polyphenolic compounds. Other studies have reported this inhibition of α-amylase par P. amarus ^28,29. In addition, this plant appears to be involved in lowering blood sugar levels in rats rendered diabetic by Alloxan ³⁰. All these results could support the use of these plants by the herbalists of Bobo-Dioulasso in the management of diabetes.

The aim of this study was to evaluate the antiradical and α-amylase inhibitory potential of extracts of S. hermonthica and P. amarus. The results of this study have highlighted the presence of polyphenols in the hydroethanolic extracts. The highest polyphenol content was obtained with P. amarus extract. In addition, all extracts showed both antiradical properties and inhibitory effects on α-amylase. These results partly explain the use of these plants in traditional medicine to treat or relieve people suffering from diabetes. For future investigation, it will be interesting to explore not only other complementary methods of antioxidant evaluation such as 2,2-Azinobis 3-ethyl-BenzoThyazoline 6-Sulphonate (ABTS) and Ferric Reducing Antioxidant Power (FRAP) but also to use a pure enzyme to determine the kinetic parameters in order to better appreciate the antidiabetic activity.

Acknowledgements

We are thankful to the herbalists of the city of Bobo-Dioulasso for sharing their precious recipes.

Conflicts of interest

The authors declare that they have no competing interests.

REFERENCES

[1] Pincemail J, Haleng J, Le Goff C, Charlier C, Chapelle JP, Defraigne JO, Oxidative stress: How to assess patient status, Nutrition info, 2007; 47-50.

[2] Favier A, Oxidative stress conceptual and experimental interest in understanding disease mechanisms and therapeutic potential, Chemical news, 2003; 108-115.

[3] WHO World Diabetes Report: Executive Summary, Geneva, 2016.

[4] FID. Report: IDF Diabetes Atlas, 9th _ editing, 2019; 1-176.

[5] Kreatsoulas C, Anand SS, The impact of social determinants on cardiovascular disease. Journal of Cardiology, 2010; 8-13. https://doi.org/10.1016/S0828-282X(10)71075-8

[6] WHO Traditional Medicine Strategy 2002-2005, Geneva, 2002.

[7] Béné K, Camara D, Fofie NBY, Kanga Y, Yapi AB, Yapo YC, Ambe SA, Zirihi G N, Ethnobotanical study of medicinal plants used in the Department of Transua, Zanzan District (Côte d' Ivory), Journal of Animal & Plant Sciences, 2016; 4230-4250. http://www.m.elewa.org/JAPS ;

[8] Patel JR, Tripathi P, Sharma V, Chauhan NS, Dixit VK, Phyllanthus amarus: Ethnomedicinal uses, phytochemistry and pharmacology: A review, Journal of Ethnopharmacology, 2011; 286-313. https://doi.org/10.1016/j.jep.2011.09.040

[9] Guha G, Rajkumar V, Ashok Kumar R, Mathew L, Aqueous extract of Phyllanthus amarus inhibits chromium (VI) -induced toxicity in MDA-MB-435S cells, Food and Chemical Toxicology, 2010; 396-401. https://doi.org/10.1016/j.fct.2009.10.028

[10] Boussim IJ, The Phanerogam parasites of Burkina Faso: Inventory, Taxonomy, Ecology and some aspects of their Biology, Special case of Loranthaceae parasites of Shea, thesis of State Doctorate of Natural Sciences, University of Ouagadougou, 2002; 1-298.

[11] Matou M, Bercion S, Merciris P, Meyssonier N, Fernand D, Marianne-Pepin T, Study of the chemical composition and associated pharmacological potential of Phyllanthus amarus Schum and Thonn. (1827) "Grenn anba fèy", International Symposium on Aromatic and Medicinal Plants (CIPAM) - 9th edition, Cayenne, French Guiana, 2016; 1-10.

[12] Ouédraogo RJ, Evaluation of antioxidant and α-amylase inhibitory activities of potentially antidiabetic plants: Case of Mitragyna inermis (willd) O. Kuntze and Tamarindus indica Linn.. Master's thesis in Applied Biological Sciences, Nazi BONI University, Burkina Faso, 2020; 1-50.

[13] Souhila M, Mustapha K, Nacéra M, Study of the extraction of phenolic compounds from different parts of the artichoke flower (Cynara scolymus L.), Nature & Technology, 2013; 35-40.

[14] Adewale IO, Agumanu NE, Otih-Okoronkwo FI, Comparative studies on α-amylases from malted maize (Zea mays), millet (Euleusina coracana) and Sorghum (Sorghum bicolor), Carbohydrate Polymers, 2006; 71-74. https://doi.org/10.1016/j.carbpol.2006.02.022

[15] Adewale IO, Oladejo A, Properties of the isoforms of α-amylase from kilned and unkilned malted sorghum (Sorghum bicolor), Carbohydrate Polymers, 2009; 105-109. https://doi.org/10.1016/j.carbpol.2008.12.011

[16] Xiao Z, Storms R, Tsang A, A quantitative starch -iodine method for measuring αamylase and glucoamylase activities, Analytical Biochemistry, 2006; 146-148. https://doi.org/10.1016/j.ab.2006.01.036

https://10.1016/j.ab.2006.01.036

[17] Singleton VL, Orthofer R, Lamuela-Raventós RM Analysis of total phenols and other oxidation substrates and antioxidants by means of folin-ciocalteu reagent, In Methods in enzymology, 1999; 152-178. https://doi.org/10.1016/S0076-6879(99)99017-1

[18] Brand-Williams W, Cuvelier ME, Berset C, Use of a Free Radical Method to Evaluate Antioxidant Activity, LWT-Food Science and Technology, 1995; 25-30. https://doi.org/10.1016/S0023-6438(95)80008-5

[19] Meda A, Lamien CE, Romito M, Millogo J, Nacoulma OG, 2005. Determination of the total phenolic, flavonoid and proline contents in Burkina Faso honey, as well as their radical scavenging activity, Food Chemistry, 2005; 571-577. https://doi.org/10.1016/j.foodchem.2004.10.006

 [20] Thalapaneni NR, Chidambaram KA, Ellappan T, Sabapati ML, Mandal SC, Journal of Complementary and Integrative Medicine, 2008; 1-10.

[21] Jaiswal P, Kumar P, α-amylase inhibitory activity of different extract of bark of Albizia lebbeck (L.) Benth, International Journal of Pharmacy and Pharmaceutical Sciences, 2017; 119-122. https://doi.org/10.22159/ijpps.2017v9i8.19411

[22] N'Guessan K, Kadja B, Zirihi G, Traoré D, Aké-Assi L, Phytochemical screening of some Ivorian medicinal plants used in Krobou country (Agboville, Ivory Coast), Natural Sciences, 2009;1-15.

[23] Kiendrebeogo M, Dijoux-Franca MG, Lamien CE, Meda A, Wouessidjewe D, Nacoulma OG, Acute toxicity and antioxidant property of Striga hermonthica (Del.) Benth (Schrophulariaceae), African Journal of Biotechnology, 2005; 919-922.

[24] Velavan S, Phytochemical techniques - a review, World Journal of Science and Research, 2015; 80-91. https://doi.org/10.1002/rwm3.20270

[25] Yi Z, Yan Y, Liang Y, Zeng B, In vitro antioxidant and antimicrobial activities of Pericarpium citric Reticulatae of a new Citrus Cultivar and its main flavonoid, LWT-Food Science and Technology, 2008; 597-603. https://doi.org/10.1016/j.lwt.2007.04.008

[26] Khoudali S, Benmenssaoud left D, Essaqui A, Zertoubi M, Azzi M, Benaissa M, Study of the antioxidant activity and anti-corrosion action of the methanolic extract of saw palmetto leaves (Chamaerops humilis L.) from Morocco, Journal of Materials and Environmental Science, 2014; 887-898.

[27] Sales PM, Souza PM, Simeoni LA, Magalhães PO, Silveira D, α-Amylase inhibitors: a review of raw material and isolated compound from plant source, Journal of Pharmacy & Pharmaceutical Sciences, 2012; 141-183. https://doi.org/10.18433/J35S3K

[28] Ali H, Houghton PJ, Soumyanath A, α-Amylase inhibitory activity of some Malaysian plants used to treat diabetes, with particular reference to Phyllanthus amarus, Journal of Ethnopharmacology, 2006; 449-455. https://doi.org/10.1016/j.jep.2006.04.004

[29] Tamil IG, Dineshkumar B, Nandhakumar M, Senthilkumar M, Mitra A, In vitro study on α-amylase inhibitory activity of an Indian medicinal plant, Phyllanthus amarus, Indian Journal of Pharmacology, 2010; 280-282. https://doi.org/10.4103/0253-7613.70107

[30] Mbagwu HOC, Jackson C, Jackson I, Ekpe G, Eyaekop U, Essien G, Evaluation of the hypoglycemic effect of aqueous extract of Phyllanthus amarus in alloxan-induced diabetic albino rats, International Journal of Pharmaceutical and Biomedical Research, 2011; 158-160.