Biochemical investigation of the upstream anti-sickling mechanisms of soursop (Annona muricata): 15-acetyl guanacone as an inhibitor of deoxyhaemoglobin polymerisation

Abstract

Current sickle cell disease (SCD) therapies are limited and inefficient. The ethnomedicinal values of Annona muricata in the treatment of SCD, leading to this present research. Leaves and fruits of Annona muricata were processed using solvent extraction and partitioning; aqueous, chloroform and ethyl acetate fractions. In vitro (anti-oxidant and anti-sickling), in silico, quantitative (amino acids) and kinetic simulation experiments were done. 15-acetyl guanacone, was used, in silico against 2,3-bisphosphoglycerate (2, 3-BPG) mutase and deoxyhaemoglobin. The ethyl acetate and chloroform fractions better NO^● scavengers, iron-chelators and ferric reducing. In vitro unsickling (UT₅₀) had ethyl acetate = 5 h and methanol = 7 h. Chloroform fraction had EC₅₀ 1.00 mg/mL (EC₅₀ = 546 mg/mL) to 10.00 mg/mL (EC₅₀ = 99 mg/mL). EC₅₀ and IC₅₀ of ethyl acetate fraction had steady-decrease. At higher concentration, chloroform fraction had higher Bmax (1.48 × 10²¹ U/mL) and higher Kd (3.66 × 10¹⁹ mg/mL), whereas, at a lower concentration, the ethyl acetate fraction demonstrated higher Bmax (7.23 × 10¹² U/mL) and lower Kd (2.12 × 10¹¹ mg/mL); The relative affinity (BP) of chloroform fraction increased progressively with concentration. The amino acid profile revealed rich concentrations glycine, valine, leucine, lysine, phenylalanine, histidine, arginine, and tryptophan. From the in silico experiments, 15-acetyl guanacone specifically targeted the A and B chains, with greater affinity for the beta subunit. This suggested that 15-acetyl guanacone might be able to prevent the polymerisation of deoxyHbSS, induce an allosteric conformational change that increases the oxygen affinity, and decrease the cellular 2, 3-BPG concentration.

Communicated by Ramaswamy H. Sarma

Keywords:

• Sickle cell disease
• deoxyhaemoglobin
• Annona muricata
• acetogenins
• amino acids

Acknowledgements

We hereby wish to acknowledge the immense supports of Mrs. Anne Oghenerukevwe Obayiuwana (for assisting with Figure 1) and Dr. Kennedy Modugu for assisting with the grammatical revision. Mr. Christopher Ugbodaga, Mr. Reuben Ofeimun and Ms. Ngozi Rose-Mary Agageme are also appreciated for assisting to obtain and process the plant materials used for this research.

Authors’ contribution

KCA and PNO designed the project design, protocols, as well as, supervision of the research; CU and RO assisted with the processing of the plant material; KCA, MA, RI, MCT, JJO, SKO and ORE carried out the in vitro anti-sickling experiments; KCA carried out the enzyme kinetic simulation experiments; JO and AK carried out the amino acid analysis; KCA, LNE and OA carried out the in vitro anti-oxidant experiments; KCA and NRA did the statistical analysis of the research; KCA and OE carried out the in silico experiments; KCA, MA, RI, and LNE wrote the manuscript.

Additional information

Funding

This research was funded by Tertiary Fund (TETFUND) 2018/2019 research projects intervention, batch 13, with reference: TETFUND/DESS/UNI/BENIN/2018/RP/VOL.1.