Phytochemical analysis, in vitro and in silico effects from Alstonia boonei De Wild stem bark on selected digestive enzymes and adipogenesis in 3T3-L1 preadipocytes

Abstract

Background: Obesity is a global health issue arising from the unhealthy accumulation of fat. Medicinal plants such as Alstonia boonei stem bark has been reported to possess body weight reducing effect in obese rats. Thus, this study sought to investigate the in vitro and in silico effects of fractions from Alstonia boonei stem bark on selected obesity-related digestive enzymes and adipogenesis in 3T3-L1 preadipocytes.

Method: Two fractions were prepared from A. boonei: crude alkaloid fraction (CAF) and crude saponin fraction (CSF), and their phytochemical compounds were profiled using Liquid chromatography with tandem mass spectrometry (LCMS/MS). The fractions were assayed for inhibitory activity against lipase, α-amylase and α-glucosidase, likewise their antiadipogenic effect in 3T3-L1 adipocytes. The binding properties with the 3 enzymes were also assessed using in silico tools.

Results: Eleven alkaloids and six saponin phytochemical compounds were identified in the CAF and CSF using LCMS/MS. The CAF and CSF revealed good inhibitory activity against pancreatic lipase enzyme, but weak and good activity against amylase respectively while only CSF had inhibitory activity against α-glucosidase. Both fractions showed antiadipogenic effect in the clearance of adipocytes and reduction of lipid content in 3T3-L1 adipocytes. The LCMS/MS identified compounds (41) from both fractions demonstrated good binding properties with the 3 enzymes, with at least the top ten compounds having higher binding energies than the reference inhibitors (acarbose and orlistat). The best two docked compounds to the three enzymes were firmly anchored in the substrate binding pockets of the enzymes. In a similar binding pattern as the reference acarbose, Estradiol-17-phenylpropionate (-11.0 kcal/mol) and 3α-O-trans-Feruloyl-2 α -hydroxy-12-ursen-28-oic acid (-10.0 kcal/mol) interacted with Asp197 a catalytic nucleophile of pancreatic amylase. Estradiol-17-phenylpropionate (-10.8 kcal/mol) and 10-Hydroxyyohimbine (-10.4 kcal/mol) interacted with the catalytic triad (Ser152-Asp176-His263) of pancreatic lipase while Estradiol-17-phenylpropionate (-10.1 kcal/mol) and 10-Hydroxyyohimbine (-9.9 kcal/mol) interacted with Asp616 and Asp518 the acid/base and nucleophilic residues of modelled α-glucosidase.

Conclusion: The antiobesity effect of A. boonei was displayed by both the alkaloid and saponin fractions of the plant via inhibition of pancreatic lipase and adipogenesis.

Keywords: Alkaloid; Alstonia boonei; Amylase; Lipase; Obesity; Saponin.

Fig. 1

Total Compound Chromatogram of fraction of A. boonei via LCMS/MS analysis. A TCC of CAF of A. boonei via LCMS/MS analysis in positive polarity; B TCC of CSF of A. boonei via LCMS/MS analysis in negative polarity. The details of the numbers on each peak corresponding to the identified compounds from A. boonei are presented in Tables 1 and 2

Fig. 2

Inhibitory enzyme activities of A. boonei fractions. A & B are lipase activities, C is α-amylase and D is α-glucosidase activity. CAF – crude alkaloid fraction, CSF – crude saponin fraction, ORL- Orlistat and ACA—Acarbose. Values are mean ± SEM

Fig. 3

Effect of the fractions of A. boonei on differentiated 3T3-L1 adipocytes. A cell viability; B relative lipid content; C glycerol release; D adipocytes after staining with Oil Red O. Control is at 0 mg/dL of plant fractions; n=3

Fig. 4

Top docked compounds of A. boonei fractions. Compounds from CAF are estradiol-17-phenylpropionate and 10-hydroxyyohimbine, and from CSF is 3α-O-trans-Feruloyl-2α -hydroxy-12-ursen-28-oic acid

Fig. 5

Details of binding mode: (s) solvent-accessible surface view (i-iii) Interaction view of ligands in binding pocket of human pancreatic alpha-amylase (a), human pancreatic lipase (b) and human lysosomal α-glucosidase (c). Colours indicate the stick representations of ligands for (a) as (i) green: acarbose (reference inhibitors) (ii) gold: 3-O-cis-coumaroylmaslinic_acid (iii) red: Furfural diethyl acetal. Colours indicate the stick representations of ligands for (b) as (i) green: orlistat (referencer inhibitors) (ii) gold: estradiol-17-phenylpropionate (iii) red: 10-hydroxyyohimbine (iv) cyan: methoxyundecyl phosphonic acid (MUP) (interaction not shown). Colours indicate the stick representations of ligands for (c) as (i) green: acarbose (referencer inhibitors) (ii) gold: 3α-O-trans-feruloyl-2α-hydroxy-12-ursen-28-oic acid (iii) red: estradiol-17-phenylpropionate