Development of isolation and purification method for extraction of acetogennin from the seeds of custard apple

Introduction

Acetogenins are a class of natural compounds primarily found in the Annonaceae family, notably in species like Annona squamosa (sugar apple or sweetsop), Annona muricata (soursop), and Annona cherimola (cherimoya). These polyketide-derived molecules, characterized by a long aliphatic chain with a terminal lactone ring, are predominantly located in the seeds, leaves, and bark of these plants. Acetogenins exhibit a range of biological activities, including anticancer, antiparasitic, insect-repellent, and neurotoxic properties, making them a subject of significant pharmacological and agricultural interest. Their primary mode of action involves inhibiting mitochondrial complex I (NADH: ubiquinone oxidoreductase), disrupting ATP production, which selectively targets energydemanding cells like cancer cells or parasites (Alali et al., 1999) [1] . Additionally, acetogenins inhibit multidrug resistance proteins, enhancing their potential in overcoming chemotherapy resistance (Oberlies et al., 1997) [7] . The chemical structure of acetogenins typically includes a 35-37 carbon backbone with oxygenated functional groups, such as hydroxyls, ketones, or epoxides, and one to three tetrahydrofuran (THF) rings, contributing to their bioactivity. Over 400 acetogenins have been identified, with notable examples like annonacin, squamocin, and bullatacin, each varying in potency and specificity. For instance, annonacin, abundant in Annona muricata, has shown cytotoxicity against various cancer cell lines, including breast, lung, and colon cancers, with IC50 values often in the nanomolar range (Liaw et al., 2010) [6] . However, their neurotoxicity, particularly linked to annonacin, raises concerns, as chronic consumption of annonaceous fruits or teas has been epidemiologically associated with atypical parkinsonism in regions like Guadeloupe, likely due to mitochondrial dysfunction in neurons (Höllerhage et al., 2009) [3] . In agriculture, acetogenins serve as natural insecticides and antifeedants. Powdered seeds of Annona squamosa, rich in squamocin, are traditionally used to control lice and pests, leveraging their toxicity to disrupt insect mitochondrial metabolism (Leatemia & Isman, 2004) [5] . Despite their therapeutic potential, clinical applications are limited by challenges in solubility, bioavailability, and toxicity. Acetogenins are poorly water-soluble, requiring advanced delivery systems like liposomes or nanoparticles to enhance efficacy. Moreover, their non-selective toxicity necessitates careful dose optimization to avoid adverse effects on healthy cells. Current research focuses on semi-synthetic derivatives to improve specificity and reduce neurotoxic risks, alongside exploring their antimalarial and antiviral properties (Bermejo et al., 2005) [2] . The extraction of acetogenins from plants in the Annonaceae family, such as Annona squamosa (sugar apple) and Annona muricata (soursop), is a critical process for studying their potent bioactivities, including anticancer, antiparasitic, and insect-repellent properties. Acetogenins, polyketide-derived compounds with a long aliphatic chain and a terminal lactone ring, are primarily concentrated in seeds, leaves, and bark, requiring careful extraction to preserve their structural integrity and bioactivity. Further purification is achieved through column chromatography, often employing silica gel with a gradient of hexane and ethyl acetate, which allows for the isolation of individual acetogenins based on their differential interactions with the stationary phase (Bermejo et al., 2005) [2] . High-performance liquid chromatography (HPLC) is frequently used for final purification, with reversed-phase C18 columns and methanol-water or acetonitrile-water mobile phases to achieve high-purity acetogenin fractions. Detection is typically performed using UV-Vis spectroscopy at 210-220 nm, as acetogenins exhibit weak UV absorption due to their lactone moiety (Liaw et al., 2010) [6] . For acetogenins, which are lipophilic with varying degrees of polarity due to hydroxyl and tetrahydrofuran groups, silica gel column chromatography effectively separates them from less polar lipids and more polar phenolics. Fractions are collected in small volumes, monitored by thin-layer chromatography (TLC) with visualization under UV light or staining with reagents like vanillin-sulfuric acid, which detects acetogenins' lactone moieties (Alali et al., 1999) [1] .

The elution gradient is tailored to the target compounds; for example, a hexane:ethyl acetate ratio starting at 9:1 and gradually increasing to 1:1 is common for isolating acetogennins like squamocin or annonacin from Annona squamosa seed extracts. The technique's versatility allows for scale-up in preparative chromatography, where larger columns and automated fraction collectors enhance throughput for industrial applications (Snyder et al., 2012) [8] .

TLC for Isolation of Actogennins

The choice of mobile phase is critical, with typical ratios like hexane:ethyl acetate (7:3 or 8:2) effectively separating acetogennins due to their intermediate polarity, influenced by hydroxyl and tetrahydrofuran moieties. After development, acetogennins are visualized under UV light at 254 nm, where their lactone rings exhibit weak absorption, or by spraying with reagents like vanillin-sulphuric acid, which produces colored spots (e.g., pink or purple) specific to acetogennins, facilitating their identification (Bermejo et al., 2005) [2] . Retention factor (Rf) values, calculated as the ratio of the distance traveled by the compound to that of the solvent front, are used to characterize acetogennins, with Rf values typically ranging from 0.3 to 0.6 in hexane:ethyl acetate systems for compounds like annonacin (Liaw et al., 2010) [6] . Preparative TLC, using thicker silica gel plates (e.g., 1-2 mm), allows for larger sample loads to isolate milligram quantities of acetogenins, where bands are scraped off, eluted with methanol or chloroform, and further purified, often by high-performance liquid chromatography (HPLC) for higher resolution (Hostettmann et al., 1998) [4] . TLC's advantages include its rapid setup, low cost, and ability to process multiple samples simultaneously, making it ideal for screening crude extracts and monitoring column chromatography fractions. However, limitations include lower resolution compared to HPLC, potential overlap of structurally similar acetogenins, and sensitivity to environmental factors like humidity, which can affect Rf values. To enhance separation, two-dimensional TLC or high-performance TLC (HPTLC) with finer particle size stationary phases can be employed, offering improved resolution and reproducibility (Snyder et al., 2012) [8] . For acetogenin isolation, TLC is often a complementary step, guiding the purification process by identifying fractions rich in target compounds before final purification. Its role in acetogenin research underscores its importance in natural product chemistry, balancing accessibility with sufficient sensitivity for preliminary isolation tasks. 30 cm column was used to fractionate the sample. F4 bit was transferred to an open column chromatography with silica gel 60 as the stationary phase. Detergents used as eluents were hexane, hexane-chloroform (82), hexanechloroform (11) and ethyl acetate. Pink colour spot on TLC distance after dipping in kedde's reagent confirm the presence of acetogenin in CC bit.

Materials and Methods

Acetogennins are bioactive polyketides found in plants of the Annonaceae family, known for their cytotoxic, antitumor, and pesticidal properties. The extraction, isolation, and identification of acetogennins involve a series of phytochemical techniques aimed at obtaining pure compounds for further analysis or application.

Sample Extraction

The process begins with grinding seeds to increase surface area, followed by sequential extraction using solvents of increasing polarity (n-hexane, chloroform, methanol) to isolate acetogennins, which are polar compounds. Non-polar solvents remove lipids, while methanol extracts polar acetogennins. The methanol extract is partitioned with ethyl acetate to concentrate acetogennins in the organic phase. This method leverages the principle of differential solubility to separate compounds based on polarity (Harborne, 1998) [4] .

Column Chromatography for Isolation

Column chromatography uses silica gel (SiO₂) as a stationary phase and a gradient of n-hexane: chloroform as the mobile phase to separate acetogennins based on their affinity for the stationary phase. The gradient elution ensures that compounds with varying polarities are eluted sequentially, allowing for the isolation of acetogennins in specific fractions (Liaw et al., 2010) [6] .

3.3

Thin-Layer Chromatography (TLC) for Identification TLC confirms the presence of acetogennins using silica gel 60 as the stationary phase and chloroform: methanol (9:1) as the mobile phase. Visualization with Kedde's reagent, which reacts with the lactone ring of acetogennins to produce a pinkish color, confirms their presence. The Rf value aids in identification by comparing migration distances (Stahl, 1969) [9] .

Conclusion

Annona squamosa seeds contain powerful bioactive compounds, especially acetogenins, which exhibit strong cytotoxic and anti-proliferative effects against a wide variety of cancer cell lines. These effects are achieved through mechanisms such as mitochondrial inhibition, apoptosis induction, ROS generation, and cell cycle arrest.

The cytotoxic activity has been proven in both in vitro and in vivo models, showing potential for clinical applications. However, care must be taken due to potential toxicity, and future work should focus on purification, formulation, and human testing. Thus, with proper scientific validation and formulation, Annona squamosa seed extract may serve as a promising natural source for anticancer therapies.

References

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