Carbohydrates Test and In Vitro Inhibitory Activities of Alpha- Glucosidase and Alpha-Amylase of Stem Bark Extracts of Musanga Cecropioides

Introduction

Musanga cecropioides is known in Cameroon and Equatorial Guinea as “Asseng” in Fang, in the Yoruba language as “Aga”, in Linguala (the Democratic Republic of Congo) as “Mombambo”; in English as West African corkwood, corkwood African umbrella tree. Musanga cecropioides is used in Cameroon, Gabon, Southwest of Nigeria and the Democratic Republic of Congo in the treatment of many diseases, amongst which, cough, constipation, schizophrenia, chest infection, rheumatism, leprosy, trypanosomiasis, hypertension, toothache, malaria, wounds, and jaundice [1, 2, 3]. Several parts of the plant are used in traditional medicine. A bark decoction is drunk in case of cough or high blood pressure [4, 5]. The juice of the stems is administered in the event of dysmenorrhea and as a galactagogue, the juice of the roots for stomach spasm, diarrhea, gonorrhea, pulmonary affection, trypanosomiasis, skin disease, otitis, rheumatism, edema and epilepsy, and to facilitate childbirth [4]. In Cameroon, the leaves are used in the treatment of hypertension. A decoction of inflorescences is prescribed to facilitate childbirth. The plant is employed as dehydrant, anthelminthic, antidysenteric and analgesic, for treating frailty in infants, for the restoration of appetency. The barks macerate for treating ache and as boiling for respiratory organ troubles. It’s conjointly helpful as a cough medication, for dressing wounds and bruises. Bark scrapings may be used as an aphrodisiac [6, 7, 8]. The aerial stilt-roots and conjointly the younger branches are known for their capability of yielding an outsized quantity of consumable sap, “a large portion of a bucketful” is affirmed to be available from one tree overnight [7].

Several scientific studies have demonstrated the hypotensive, hypoglycemic, antidiabetic, antidiarrheal and bactericide properties of M. cecropioides extracts [3, 9, 10, 11]. Previous studies established uterotonic effects of leaf in rats [8], and the hypotensive effects of the water extracts of the leaf and stem bark [12,13 ]. The sap is absorbed as blood-purifier, to wash abdomen, for gonorrhea, cough and chest infections, as a galactogogue, and ordinarily as a wash for persons with cephalitis, Hansen’s disease and fevers to alleviate aches and rheumatism; the wood is additionally used for construction works [8, 14]. Between doses of 1-4 g/ kg, the water extract of M. cecropioides was ascertained to be tolerated in mice as no obvious signs of toxicity were found on the animals [15]. Additionally, Medou, et al. [16] found no acute nor subacute toxicity in rats with the water extract of M. cecropioides stem barks.

Lontsi, et al. [17] isolated many compounds from the stem bark of M. cecropioides: a novel triterpenic A-ring seco derivative has been obtained, methyl 16α, 19α-dihydroxy-2, 3-secours-12-ene-2, 3. 28-trioate 3, oleanolic and ursolic acids, 2α-hydroxy oleanolic and ursolic acids, benthamic acid, 3-rhamnosyl benthamic acid, and tormentic acid [18]. Lontsi, et al. [19] identified from the trunk wood of M. cecropioides, cecropic acid methyl ester, euscaphic and tormentic acid methyl esters, methyl cecropiaceate, and methyl pomolate, methyl arjunolate and ß-D-glucosyl tormentate. Ojinnaka and Okogun [20] reported the isolation of tormentic acid, 2-acetyl tormentic acid, 3-acetyl tormentic acid, and euscaphic acid, from the rootwood of M. cecropioides. From the same part, three seco-triacidic triterpenes, cecropiacic acid, musangic acids A (2, R1 = H) and B (3, R1= H) were isolated [21], and two A-ring contracted triterpenes, musancropic acids A and B, from the rootwood of Musanga cecropioides [22]. Methyl musangicate and methyl euscaphate are two pentacyclic triterpenes isolated from the methylated rootwood extract of Musanga cecropioides [23]. Methyl kalaate, methyl ursolate, methyl oleanolate, methyl 2α-hydroxyursolate, methyl 2α-hydroxyoleanolate, and methyl pomolate have been identified from the methylated stem bark polar extracts of Musanga cecropioides [14]. Methyl tormentate, methyl 2-acetyltormentate, methyl 28-glucosyltormentate, methyl pomolate, methyl euscaphate, and methyl cecropioate are other triterpenes separated from rootwood extracts of Musanga cecropioides [24]. In furtherance of studies on Musanga cecropioides, protocatechuic acid (3, 4-dihydroxybenzoic acid) and protocatechualdehyde (3, 4-dihydroxybenzaldehyde were obtained from the stem bark [25]. Protocatechuic acid has been found also in the leaves of M. cecropioides [14], and Hibiscus sabdariffa in which it was reported to exhibit remarkable inhibitory effects against lipopolysaccharide-induced hepatic damage [26]. The plant sap contains female hormones. Isovitexin, vitexin, chlorogenic acid, catechin, and procyanidins are isolated from the leaves of M. cecropioides. Alternative phytochemical studies have additionally also suggested the presence of kalaic acid within the stem bark and a few different triterpenoid acids within the leaves, stem bark and also the root [14]. Additionally, Lacaille Dubois, et al. [27] reported the isolation of isovitexin, vitexin, orientin, isoorientin, chlorogenic acid, flavane-3-ols (+)-catechin and (–)-epicatechin, the flavone isovitexin and the flavonol isoquercitrin, procyanidins B2 and C1 from the leaves of this plant.

Following the previous study done by Nyunaï, et al. [28] which demonstrated the anti-hyperglycemic potential of the water-ethanol extract of the stem bark of Musanga cecropiodes, This work aims to evaluate the inhibitions of α-glucosidase and α-amylase activities by Musanga cecropioides fractions, and to determine the effect of the aqueous extract in the maltose tolerance test, and the starch tolerance test.

Statistical Analysis

difference in Postprandial Blood Glucose (PBG) and AUC between control and treatment groups was determined using GraphPad Prism software version 5.03. One-way analysis of variance (ANOVA) followed by Dunnet test for post hoc analysis. Estimated median inhibitory concentration (IC50) values were determined using the nonlinear regression analysis functions within the software. For all the comparisons in the study, P< 0.05 was considered significant.

Discussion

With the increasing number of diabetics worldwide, and given the limitations of existing drugs, more and more research has intensified to find new molecules that are effective and accessible. Plants are an inexhaustible source of these new molecules. Musanga cecropioides is an herb traditionally used in the treatment of diabetes [9]. Nyunaï, et al. [28] highlighted the anti-hyperglycemic potential of the water-ethanol extract of Musanga cecropioides stem bark effect. It is, therefore, seemed useful to us from the perspective of determining the active compounds of this plant to determine the α-glucosidase and α-amylase activity of the fractions derived from this plant and evaluate the effect of aqueous extract in maltose tolerance test, and starch tolerance test.

The carbohydrate test revealed that the aqueous extract of M. cecropioides used at doses of 250 mg/kg and 500 mg/ kg for the hyperglycemia induced by maltose or starch carried out in normal male rats does not reduce the Area under the Curve, unlike glibenclamide. This would suggest that the aqueous extract of M. cecropioides does not act in the same way as glibenclamide. Previous work has shown that the water-ethanol extract of stem bark of Musanga cecropioides extracts reduced blood glucose levels in diabetic rats and normal rats during the glucose tolerance test [28]. The ineffectiveness of the aqueous extract in this study could be explained by the fact that maltose and starch are polysaccharides and release more glucose molecules.

Results revealed a dose-dependent response because increasing the concentration of fractions extract increased the inhibitory activity. Likewise, in terms of half-maximal inhibitory concentration (IC50) which is the concentration needed for the sample to inhibit α-glucosidase by 50%, Table 2 shows that fraction B has a highest IC50 value of 83.10 µg/ mL compared to fraction D with IC50 value of 0.7 µg/mL and the highest inhibitory activity of 87,097% with IC50 at 0.7 µg/ mL. This suggests the presence of α-glucosidase inhibitors separated from other compounds in the extract. The results of the present study indicate that out of acarbose, fraction A and fraction D showed the maximum α-glucosidase inhibitory activity. Fraction D is the most active extract in this test because it has the lowest IC50 value. This result is related to phytochemical compounds in the fraction extract that can inhibit α-glucosidase, in contrast to acarbose which is a pure compound, besides, to determine the bioactive compounds of these fractions, it is necessary to characterize them. The expected bioactive compounds could be flavonols or phenolic acids as the literature indicates a big link among polyphenols and anti-diabetic action of plant extracts [32]. As friedelan-3-one (triterpenoid) is the major compound in Fraction A (result not shown), suggest that this compound has α-amylase inhibition properties and might be responsible for a major part of that activity of the total extract of M. cecropioides.

Fractions B and C showed a negative value, which indicated that no inhibition occurred at 6.25 µg/mL and 12 µg/mL. Some of the negative values were quite large, e.g. especially fraction C at 6.25 µg/mL gave a negative value of -190.86% and this could indicate that the α-glucosidase is activated rather than inhibited. If this were to occur in vivo, it would aggravate, rather than alleviate, the diabetic condition since the rate of glucose production would be increased and thereby the serum levels rise more rapidly. However, it might be that an increase in the reaction product levels may be due to a conformational change derived from the binding of compounds to the enzyme [33]. This could also indicate that these fractions no longer contained α-glucosidase inhibitors. Since these fractions were collected in the latter part of elution, they may already contain wash residues, pigments and other flow-through components which are not α-glucosidase inhibitors but larger molecules that might mask the activity of the α-glucosidase inhibitor activity.

The treatment goal of diabetes patients is to maintain near-normal levels of glycemic control, in both the fasting and postprandial states. One of the strategies to monitor blood glucose for type II diabetes is to either inhibit or reduce the release of glucose from the small intestine. Many natural resources have been investigated for the suppression of glucose production from carbohydrates in the gut or glucose absorption from the intestine [34]. Thus, natural products of great structural diversity are still a good source for finding such inhibitors, thereby motivating to explore biologically active compounds from the extremely various plants. α-amylase catalyzes the hydrolysis of α-(1,4)-glucosidic linkages of starch, glycogen, and various oligosaccharides and α-glucosidase further breaks down the disaccharides into simpler sugars, readily available for the intestinal absorption. The inhibition of their activity in the digestive tract of humans is considered to be effective to control diabetes by diminishing the absorption of glucose decomposed from starch by these enzymes [35]. Therefore, effective and non- toxic inhibitors of α-amylase and α-glucosidase have long been sought. Several α-glucosidase inhibitors have been remoted from medicinal plants for the development of the latest drugs with increased efficiency and decrease adverse effects than the present medication [36].

Conclusion

The results show for the first time, the potential role of fractions from M. cecropioides to inhibit α-glucosidase and α-amylase inhibition activities. Due to it having the best inhibitory α-glucosidase effect, the fraction D of M.

cecropioides will be selected for further investigation, involving bioassay-guided fractionation, to isolate the constituents responsible for the inhibitory effect of the Fraction. Given the results obtained, the mechanism of action of this plant would mainly be through the inhibition of α-glucosidase, but also secondarily through the presence of the inhibiting α-amylase compounds.

Acknowledgment

The authors are thankful to The World Academy of Sciences (TWAS), for the research grant N° TWAS Research Grant Award_17-432 RG_BIO_AF awarded to Dr. NYUNAÏ NYEMB, Senior Researcher at Medical Research Centre, Institute of Medical Research and Medicinal Plants Studies (IMPM), Yaoundé, Cameroon.

Availability of Data and Materials

The datasets generated during and/or analyzed during the study are available from the corresponding author on request.

Conflict of Interest

The authors declare no conflict of interest.