Biology, Medicine, & Natural Product Chemistry ISSN 2089-6514 (paper) Volume 12, Number 2, October 2023 | Pages: 681-685 | DOI: 10.14421/biomedich.2023.122.681-685 ISSN 2540-9328 (online) In vivo Alpha-amylase and Alpha-glucosidase Inhibitory Potentials of Panicum maximum Jacq. (Guinea grass) Leaf Extract on Wister Rats Godwin Ndarake Enin1,*, Jude Efiom Okokon2, Enobong Mfon David1, Saviour Elisha Emmanuel1, Esther Michael Ekanem1, Bassey Sunday Antia1 1Department of Chemistry, Faculty of Science; 2Department of Pharmacology and Toxicology, Faculty of Pharmacy, University of Uyo, Uyo, Nigeria. Corresponding author* enin.godwin@gmail.com Manuscript received: 21 September, 2023. Revision accepted: 13 March, 2024. Published: 14 March, 2024. Abstract Panicum maximum Jacq. (Guinea grass) a medicinal plant used traditionally in the treatment of diseases including diabetes was evaluated for its effect on alpha amylase and alpha glucosidase enzymes in vivo. The crude ethanol extracts (150, 300 and 450 mg/kg) of P. maximum were investigated using starch, sucrose, glucose and maltose as substrates and acarbose as reference drug. The leaf extract caused significant (p<0.05) reduction in blood glucose levels of the treated rats with the four substrates used. The findings show that the leaf extract of Panicum maximum has the potentials to inhibit alpha amylase and alpha glucosidase in rats. Keywords: Panicum maximum; alpha amylase; alpha glucosidase; phytochemicals. INTRODUCTION Plants have therapeutic substances because of their potency and huge benefits, and have been widely researched and adopted in our society for the treatment and management of some diseases. In the last few decades, bioactive compounds have been trapped, purified and characterized into active drugs and, administered in rural and urban communities of the world (Coulibaly et al., 2023). Recent findings demonstrate that the world sales of medicinal plant-based products have escalated to 100.9 billion dollars representing 7.2% annual turn-over and that global population of up to 80% are utilizing medicinal plants to treat various diseases (Yusupova et al., 2023; Karahan et al., 2020). With the increasing rate of spread of diseases and infections, there is need to explore more plant-based materials for possible discoveries and publications of new and efficient remedies for these challenges. One of such herbs which could find possibility in combating current health challenges such as diabetes is Panicum maximum commonly called “Guinea grass”. Panicum maximum Jacq. is a perennial grass of the Poacace family distributed widely in Africa and other tropical regions of the world (Van Oudtshoorn, 1999). The ethnopharmacology of the plant indicates that, the plant has been employed for the treatment of malaria, microbial infections, rheumatic pain, inflammation and diabetes (Antia et al., 2010). In Nigeria, particularly in Ibibio ethnomedicine, the leaf is used to treat malaria, microbial infections and rheumatism. Other biological activities of the leaves and roots include antidiabetic (Antia et al., 2010), antimalarial and analgesic (Okokon et al., 2012), antibacterial (Gothandam et al., 2010; Doss et al., 2011a; Doss et al., 2011b), anti-inflammatory and antipyretic (Okokon et al., 2011), antifungal (Kanife, 2012), anticancer, antioxidative and antileishmanial (Okokon et al., 2014). Phytochemical studies of the root have shown the presence of alkaloid, flavonoid, tannins, terpenes, saponin, and cardiac glycosides (Okokon et al., 2016). In this study, we investigated the inhibitory activities of Panicum maximum Jacq. ethanol leaf extract on alpha-amylase and alpha-glucosidase in Wister rats. MATERIALS AND METHODS Materials The materials used include Panicum maxima powdered leaf extract, oral gastric gavage, weighing balance, gloves, scissors, glucometer and strips (fine test), distilled water, acarbose (Aldrich sigma, USA; standard drug), stirrer, beakers, 1mL syringe, starch, sucrose, maltose (Aldrich sigma, USA). Plant Collection Fresh leaves of Panicum maxima were harvested from a farmland in Use offot, Uyo, Akwa Ibom State, Nigeria, in May, 2023. The plant was identified and authenticated by Dr. Margaret Bassey, a taxonomist in the Department https://doi.org/10.14421/biomedich.2023.122.681-685 682 Biology, Medicine, & Natural Product Chemistry 12 (2), 2023: 681-685 of Botany and Ecological Studies, University of Uyo, Uyo, Nigeria. Herbarium specimen was deposited at the Department of Botany and Ecological Study, University of Uyo. Extraction The fresh leaves (2 kg) of the plant were dried on a laboratory table for 2 weeks and reduced to powder. Powdered sample (500 g) was macerated in 95% ethanol (5000 mL) for 72 hours. The liquid filtrate obtained was concentrated in vacuo at 40˚C to completely remove the ethanol. The yield was calculated, and the extract was stored in a refrigerator at 4˚C until used. Phytochemical Screening Phytochemical screening for the presence of saponins, tannins, flavonoids, alkaloids, cardiac glycosides and anthraquinones was conducted following previously reported standard procedures (Enin et al., 2023). Animals Albino Wistar rats (120 -135 g) of either sex maintained at animal house of the Department of Pharmacology and Toxicology, Faculty of Pharmacy, University of Uyo, Nigeria were used for the study. The animals were housed in standard cages and were maintained on a standard pelleted feed (Guinea feed) and water ad libitum. Alpha-Amylase inhibitory study Thirty Wistar rats were divided into 6 groups of 5 rats each. The rats in all groups were fasted for 18 h and fasting blood glucose concentration was first taken at 0 min before administration. Group I, as the normal control, received distilled water (10 mL/kg). Group II rats were orally administered starch at 2 g/kg body weight (orally with distilled water as vehicle) and distilled water (10 mL/kg) simultaneously. Rats in group III were administered starch (2 g/kg) and the standard drug (acarbose) at 100 mg/kg simultaneously. Groups IV, V and VI were administered simultaneously, starch (2 g/kg) and Panicum maximum leaf extract at 150, 300 and 450 mg/kg respectively. All administrations were done orally and blood glucose concentration was monitored at 30, 60, 120 and 180 min (Gidado et al., 2019). The blood glucose level was used to assess the effect of extract on the enzyme activity. Alpha Glucosidase inhibitory study. The procedure as described above was used for this study but with sucrose and maltose used as substrates (Gidado et al., 2019). Blood Glucose Determination Drops of blood from tip of rats tails were dropped on stripes and glucose concentration was measured using a glucometer according to manufacturer’s specifications (fine test). The glucometer works with the following principle; the blood sample is exposed to a membrane covering the reagent pad (strip), which is coated with an enzyme (glucose oxidase, glucose dehydrogenase). The reaction causes a colour change and the intensity of this change is directly proportional to the amount of glucose in the blood sample. Light from an LED strikes the pad surface and is reflected to a photodiode, which measures the light intensity and converts it to electrical signals. An electrode sensor measures the current produced when the enzyme converts glucose to gluconic acid. The resulting current is directly proportional to the amount of glucose in the sample (WHO, 2011). Statistical Analysis Data obtained were analyzed statistically using one –way ANOVA followed by Tukey-Kramer multiple comparison test using InstatR Graphpad software, (San Diego, USA). Differences between means were considered significant at p< 0.05 and very significant at p < 0.001. RESULTS AND DISCUSSION Extraction The extraction yield was 6.56% w/w. Phytochemical screening The results of the phytochemical screening revealed the presence of saponins, tannins, flavonoids, alkaloids, cardiac glycosides. The presence of anthraquinone was not observed in the study. In vivo alpha amylase and glucosidase inhibition assay Administration of starch (2 g/kg) to fasted rats caused varying percentages of increase in blood glucose levels of the treated animals after 30 mins. The percentages were starch (66.56%), P maximum leaf extract-treated groups (37.17 - 47.26%), and acarbose-treated group (17.97%). These increases were reduced after 60 min with only the groups treated with higher doses of the extract (300 and 450 mg/kg) having percentage increases of 3.82 and 18.44 % respectively. All the extract-treated groups had their BGL reduced to a normal level at 120 min and this was sustained throughout the study. Also, co-administration of the starch with acarbose prominently inhibited the rise in the blood glucose concentrations (Table 1). Enin et al. – In vivo Alpha-amylase and Alpha-glucosidase Inhibitory … 683 Table 1. Effect of ethanol leaf extract of Panicum maximum on Blood Glucose Level of rat after oral administration of starch load. TREATMENT DOSE BLOOD GLUCOSE LEVEL mg/dL IN MIN mg/kg 0 min 30 min 60 min 120 min 180 min Control normal saline - 86.00±11.53 87.66±7.12(1.93) 87.66±7.62(1.93) 91.0±7.50(5.81) 80.00±6.02 Starch 80.0±4.54 133.25±6.86a(66.56) 112.25±4.73(40.31) 92.50±1.70(15.62) 87.25±6.52(9.06) Acarbose 100 72.33±2.69 85.33±12.97(17.97) 80.33±7.21(11.06) 74.0±1.00(2.30) 72.33±8.68(0) Extract 150 95.75±5.20 133.50±7.83(39.42) 92.0±9.71a() 82.50±3.22a() 67.25±3.42a() 300 91.50±5.56 134.75±8.49(47.26) 95.0±9.44(3.82) 88.50±5.23b() 72.75±8.94a() 450 86.75±12.41 119.0±2.12(37.17) 102.75±1.48(18.44) 85.50±6.89a() 85.25±5.88() Data is expressed as MEAN ± SEM, Significant at ap<0.05, bp< 0.01, when compared to control (n=6). Values in parenthesis are percentage increases in blood glucose concentrations compared to 0 min in the same group. Administration of sucrose (2 g/kg) produced a 44.14% increase in blood glucose concentration 30 minutes post-administration of the sucrose in the control group. BGL increments of 12.07-34.60 % were also recorded in groups treated with 150, 300 and 450 mg/kg of P maximum leaf extract and 3.37% for acarbose treated group. At 60 min, percentage increases in BGL of groups treated with 150 and 450 mg/kg of extract were 19.94 and 9.22 % respectively, while the BGL of the group treated with 300 mg/kg was reduced to normal. Similarly, trend was also recorded at 120 min. There was no increment in BGL of all the extract-treated groups at 180 min (Table 2). Table 2. Effect of ethanol leaf extract of Panicum maximum on Blood Glucose Level of rat after oral administration of sucrose load. TREATMENT DOSE BLOOD GLUCOSE LEVEL mg/dL IN MIN mg/kg 0 min 30 min 60 min 120 min 180 min Control normal saline - 100.00±4.25 88.33±1.85 92.33±4.25 89.0±4.35 87.33±3.84 Sucrose 2000 81.0±4.50 116.75±6.57b(44.14) 112.66±1.45a(39.08) 97.33±1.63(20.16) 94.15±4.81(16.23) Acarbose 100 90.33±2.48 86.66±2.90 82.0±6.00 71.66±3.75 78.0±3.78 Extract 150 85.25±3.27 114.75±11.22c(34.60) 102.25±4.82(19.94) 90.25±3.27(5.86) 83.50±4.48 300 95.25±2.98 106.75±2.98b(12.07) 89.50±4.94() 85.0±2.48() 72.75±5.93() 450 84.0±5.87 101.25± 2.28(20.53) 91.75±4.55(9.22) 89.0±2.67(5.95) 76.50±3.52() Data is expressed as MEAN ± SEM. Significant at ap<0.05, bp< 0.01, when compared to control (n=6). Values in parenthesis are percentage increases in blood glucose concentrations compared to 0 min in the same group. Administration of glucose (2 g/kg) to fasted rats caused varying percentages of increase in blood glucose levels of the treated animals after 30 mins. The percentages were glucose (64.98%), P maximum leaf extract-treated groups (57.10 - 61.90%), and acarbose- treated group had no increment. All the extract-treated groups had their BGL reduced to a normal level at 120 min and this was sustained throughout the study (Table 3). Table 3. Effect of ethanol leaf extract of Panicum maximum on Blood Glucose Level of rat after oral administration of glucose load. TREATMENT DOSE BLOOD GLUCOSE LEVEL mg/dL IN MIN mg/kg 0 min 30 min 60 min 120 min 180 min Normal Control - 100.00±4.25 88.33±1.85 92.33±4.25(1.80) 89.0±4.35(1.55) 87.33±3.84(3.98) Glucose 2000 84.25±1.49 139.0±1.78b(64.98) 126.75±0.47b(50.44) 106.0±1.87b(25.81) 96.75±2.78a(14.83) Acarbose 100 85.34±1.36 84.21±0.90 84.0±1.20 82.16±2.14a 80.00±1.10 Extract 150 84.0±2.16 136.0±2.16a(61.90) 89.50±8.56a(6.54) 84.0±7.70a() 76.50±6.38a() 300 84.75±1.43 136.25±1.03b(60.76) 92.50±4.17a(9.14) 81.50±2.72a() 74.25±4.49() 450 86.25±1.10 135.50±1.44b(57.10) 88.0±2.04b(2.02) 78.75±2.52b() 68.25±3.86a() Data is expressed as MEAN ± SEM, Significant at ap<0.05, bp< 0.01, when compared to control. (n=6). Values in parenthesis are percentage increases in blood glucose concentrations compared to 0 min in the same group. Administration of maltose (2 g/kg) to fasted rats caused varying percentages of increase in blood glucose levels of the treated animals after 30 mins. The percentages were maltose (65.86%), P. maximum leaf extract-treated groups (48.17-67.87%), and acarbose- treated group (3.37%). These increases were reduced 684 Biology, Medicine, & Natural Product Chemistry 12 (2), 2023: 681-685 after 60 min with only the low dose (150 mg/kg) and middle dose (300 mg/kg) treated group having BGL increment of 14.28 and 53.93% respectively. All the extract-treated groups had their BGL reduced to a normal level at 180 min. Also, co-administration of the maltose with acarbose prominently inhibited the rise in the blood glucose concentrations (Table 4). Table 4. Effect of ethanol leaf extract of Panicum maximum on Blood Glucose Level of rat after oral administration of maltose load. TREATMENT DOSE BLOOD GLUCOSE LEVEL mg/dL IN MIN mg/kg 0 min 30 min 60 min 120 min 180 min Control normal saline - 86.00±11.53 87.66±7.12(1.93) 87.66±7.62(1.93) 91.0±7.50(5.81) 80.00±6.02 Maltose 83.50±1.19 138.50±8.10a(65.86) 120.25±2.95(44.01) 97.25±2.05(16.46) 87.25±2.13(4.49) Acarbose 100 85.34±1.36 88.22±1.10(3.37) 86.0±2.20(0.77) 84.26±1.14a() 82.28±2.26() Extract 150 75.25±5.13 111.50±9.64(48.17) 86.0±7.62(14.28) 72.25±3.98() 68.0±2.98() 300 82.5±4.02 138.50±13.17(67.87) 127.0±18.35(53.93) 90.25±20.67(9.39) 68.75±6.83() 450 83.50±1.19 138.50±8.10(65.86) 80.25±2.95() 67.25±2.05() 66.25±2.13() Data is expressed as MEAN ± SEM, Significant at ap<0.05, bp< 0.01, when compared to control (n=6). Values in parenthesis are percentage increases in blood glucose concentrations compared to 0 min in the same group. Discussion The leaf ethanol extract was found to inhibit increases in blood glucose concentration significantly following starch administration though non-dose-dependently. It has been reported that complete digestion of dietary polysaccharides like starch is achieved by the combined action of α-amylases and α-glucosidase enzymes. The α- amylase enzyme digests α-bonds of the α-linked polysaccharides yielding disaccharides, like maltose, which are further reduced to monosaccharides by membrane bound α-glucosidase enzymes (Kalra, 2014; Alongi and Anese, 2018). Inhibitions of these enzymes delay the digestion of ingested carbohydrates thereby resulting in a small rise in blood glucose concentrations following carbohydrate meals as was observed in this study. As a target for managing Type 2 diabetes mellitus, many medicinal plants have been reported to possess α- amylase and α- glucosidase inhibitory potential (Ibrahim et al., 2014; Esimone et al., 2001). Similarly, the leaf extract significantly inhibited blood glucose rises when co-administered with maltose, glucose and sucrose. Acarbose, the standard drug used in this study significantly inhibited blood glucose rise when co- administered with starch, maltose and sucrose. The results of this study support the antidiabetic activity earlier reported on the root extract (Antia et al., 2010) and further suggest the involvement of inhibitory effects on alpha glucosidase and amylase as one of the modes of antidiabetic activity of the root extract. The inhibitory activities of plant extracts are linked to their phytochemical constituents. The leaf extract of P. maximum has been reported to be rich in flavonoids, terpenes, tannins amongst others (Okokon et al., 2011). Our results of the phytochemical screening also revealed the presence of: saponins, tannins, alkaloids, flavonoids and cardiac glycosides. These compounds have been variously reported to inhibit alpha glucosidase and alpha amylase activities (Proença et al., 2017; Su and Tang, 2019). Moreso, Phenols have been reported to inhibit alpha amylase and alpha glucosidase (Oboh et al., 2017). Also, polyphenolic compounds from plants are known to cause several effects on the biological systems which include enzymes inhibitions (Kalita et al., 2018; Funke and Melzig, 2005). The phenolic compounds are known to be strong metal ion chelators and protein precipitation agents forming insoluble complexes with proteins as well as acting as biological oxidants (Ishnava and Metisariya, 2018). The presence of the polyphenolic compounds and terpenes in the root extract may suggest that their inhibitory potential on α-amylase and the membrane- bound intestinal α-glucosidase enzymes. The presence of these compounds in the extract may have contributed to the observed activity of this study and therefore explains the antidiabetic mechanism of the leaf of P. maximum. CONCLUSION The results of this study suggest that inhibition of alpha amylase and alpha glucosidase enzymes maybe one of the modes of antidiabetic activity of the leaf extract of Panicum maximum which may be attributed to the activities of its phytochemical constituents. Acknowledgements: Mr. Nsikan Malachy of Pharmacology and Toxicology Department, University of Uyo is gratefully acknowledged for providing technical assistance. Authors’ Contributions: GNE conceived the study, GNE, JEO & ABS designed the study. EME & SEE carried out the laboratory work. JEO analyzed the data. GNE & JEO wrote the manuscript, EMD edit the manuscript. All authors read and approved the final version of the manuscript. Enin et al. – In vivo Alpha-amylase and Alpha-glucosidase Inhibitory … 685 Competing Interests: The authors declare that there are no competing interests. 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In vitro study on α-amylase inhibitory activity of selected ethnobotanical plant extra its and its herbal formulations. International Journal of Pharmacognosy and Chinese Medicine 2(3): 01-10. https://dx.doi.org/10.4314/ijbcs.v17i2.1 https://doi.org/10.1016/j.foodres.2018.05.061 http://dx.doi.org/10.4314/tjpr.v18i3.20 THIS PAGE INTENTIONALLY LEFT BLANK