Evaluation of In Vitro Anti-sickling Potential of Syzygium aromaticum

Mukesh Kumar Kurre^1*, Suneeta Patra², P.K. Patra³

^1&2 Government N. P. G. College of Science, Raipur, Chhattisgarh, India

3 Pt. Deendayal Upadhyay Memorial Health Sciences and Ayush University of Chhattisgarh

ABSTRACT

Sickle cell disease (SCD) is a hereditary genetic blood disorder affects millions of people across the globe. The SCD is due to point mutation in globin gene of heamolobin, which causes normal red blood cells to deform into a sickle shape. These investigations aimed to screening of the phytochemicals and assess the anti-sickling potential of different solvent fractions of S. aromaticum (Clove) using in vitro models. Analysis of S. aromaticum extract through phytochemical screening revealed it contains flavonoids, alkaloids, saponins, phenols, glycosides, and tannins. The anti-sickling activity of fractions (n-hexane, ethyl acetate, butanol, and aqueous) was assessed using sodium metabisulphite-induced sickling of HbSS erythrocytes. Based on results demonstrated that among the fractions, the ethyl acetate fraction showed the highest sickling reversal activity at (78%), followed by butanol (68%), aqueous (52%), and n-hexane (38%) fractions.  Significantly sickled cells returned to their typical biconcave shape following treatment according to microscopic observation. Based on these results S. aromaticum may be a promising phytotherapeutic agent for the treatment of sickle cell disease (SCD) and contains bioactive compounds that can reverse erythrocyte sickling.

Keywords: Anti-sickling, Phytochemical, S. aromaticum.

1.     Introduction

Sickle Cell Disease (SCD) also known as Sickle Cell Anemia is a hereditary genetic disorder that affects millions of people worldwide, especially those of African, Mediterranean, Middle Eastern, and Indian descent [1]. In India, central and western states like Madhya Pradesh, Maharashtra, Odisha, Gujarat, Tamil Nadu, Kerala and Chhattisgarh have remarkably significant rates of sickle cell anemia [2]. In individuals with SCD, a point mutation in the β-globin gene results the replacement of a single amino acid (valine to glutamic acid), which are leading to the production of abnormal hemoglobin known as hemoglobin S (HbS). This genetic mutation affects the β-globin chain, a subunit of hemoglobin that carries oxygen through the bloodstream [3]. Under certain conditions, such as inappropriate oxygen supply or dehydration, HbS molecules are polymerized so that forcing red blood cells to form a sickle shape. These sickled cells are rigid and sticks to the walls of blood vessel to obstructing blood flow and cause a series of complications [4]. However, despite the fact that there is no specific drug for the treatment of these genetic diseases, researchers are exploring innovative therapies that target the underlying genetic mutations. The first line of clinical treatment for sickle cell disease includes the use of hydroxyurea, antibiotics, antimalarial prophylaxis, folic acid, blood transfusions, and bone marrow transplantation. The first-line clinical treatments are expensive and come with risks [5].

Therefore there is a need for continuous research on alternative therapies for SCD. Over the past few decades, there has been an increasing interest in investigating natural substances for potential therapeutic benefits in a variety of disorders, including SCD. The concept of anti-sickling activity revolves around the ability of certain compounds to might prevent the polymerization of hemoglobin S which stops normal red blood cells from converting sickle shaped. [6] These compounds mitigate the risk of vaso-occlusive crises and improve blood flow by maintaining the normal biconcave shape of blood cell, therefore reducing the symptoms of sickle cell disease. [7] Across various cultures, traditional therapists have long utilized various medicinal plants with purported anti-sickling properties as part of their therapeutic arsenal. Some of plants they have reported Antisickling activities are; Carica papaya linn, Psidum guajava linn. and Terminalia catappa [8]

Syzygium aromaticum is a spice of the family Myrtaceae, native to Indonesia with the aromatic flower buds widely recognized as clove. S. aromaticum has a rich phytochemical composition and proven pharmacological characteristics, it has become one of the more appealing options. In World traditional medicine, S. aromaticum has been used for the treatment of several diseases such as analgesic, Oxidative Stress, Cancer, Antidiabitic, Inflammation and Antithrombotic activities have been validated in recent pharmacological studies [9-13]. However, a recent ethno botanical study found that S. aromaticum are traditionally utilized in the treatment of SCD [14]. This provides some validity for the plant’s ethnomedicinal applications in the prevention and management of SCD. This study aims to evaluate the phytochemical constituents of S. aromaticum and investigate the anti-sickling activity of its various solvent fractions using in vitro models of sodium metabisulphite-induced sickling of HbSS erythrocytes.

Syzygium aromaticum represent one of the major vegetal sources of phenolic compounds such as phenolic acids (Gallic acid, caffeic acid, ferulic acid, and ellagic acid), flavonoids (Kaempferol and quercetin), carotenoids and Phytosterols (oleanolic acid and stigmasterol), and tannins which produce various pharmacological effects [15-18].

Figure 1: Clove buds and Clove buds powder

2.     Materials and methods

2.1 Plant sample collection and preparation of plant extract

 The flower buds of S. aromaticum were procured from a local market, shade-dried, and pulverized into a fine powder. A 500 g portion of the powdered material was subjected to cold maceration in 80% ethanol for seven days in large amber bottles with intermittent shaking. The mixture was filtered by a Whatman filter paper (no. 1), and the resulting filtrate was concentrated under reduced pressure using a vacuum evaporator to obtain the crude extract, which was used for preliminary analysis.

2.2 Fractionation of crude extract

The crude extract was further fractionated by successive liquid–liquid partitioning with solvents of increasing polarity, namely n-hexane, ethyl acetate, and butanol. Crude extract was suspended in 250 ml of distilled water in a separatory funnel. The aqueous portion was partitioned thrice with 250 ml of n-hexane to n-hexane fraction. Then, aqueous residue was further fractionated thrice with 250 ml of ethyl acetate and same as for butanol to obtain the different fraction. Finally, the aqueous solution was collected as the forth fraction. Each solvent fraction was then concentrated using the same vacuum evaporation technique [19–20]. The obtained fractions were reconstituted in normal saline (0.9% NaCl) to a final concentration of 250 μg/mL for use in the anti-sickling assay.

2.3 Phytochemical Evaluation

Preliminary phytochemical screening was performed on the crude extracts to investigate the presence of alkaloids, saponins, flavonoids, tannins, terpenes and steroids according to standard protocol described by [21-22].

2.4 Blood Collection and Sample Preparation

The homozygous HbS/HbS (SS) blood sample was obtained in sterile tubes containing EDTA as an anticoagulant from Pt. Jawaharlal Nehru Memorial Medical College, Raipur. All anti-sickling assays were carried out using of SS blood sample. To confirm the homozygous sickle cell (SS) status, the samples were subjected to hemoglobin electrophoresis on cellulose acetate gel at pH 8.5. The electrophoretic analysis confirmed the SS genotype, after which the samples were stored at 4°C until further use [23].

2.5 Anti-sickling activity assay (In vitro induction of sickling)

Blood samples (5 mL) collected from patients were centrifuged at 5,000 rpm for 10 minutes and washed three times with saline to isolate red blood cells (RBCs). The recovered RBCs were resuspended in normal saline and used for further analysis following the method described by [24]. For the sickling assay, 100 μL of SS RBC suspension were combined with 100 μL of 2% sodium metabisulfite (Na₂S₂O₅) solution and incubated at 37°C for 30 minutes to induce sickling. The extent of erythrocyte sickling was assessed microscopically, and the percentage of sickled cells was calculated using the formula:
% Sickling = (Number of sickled cells / Total number of cells) × 100 [25].

The in vitro anti-sickling activity of various S. aromaticum extracts was evaluated using this method. In the assay, 100 μL of SS RBCs pre-treated with 2% sodium metabisulfite were incubated with 100 μL of each extract solution at a final concentration of 250 μg/mL. The mixtures were incubated at 37°C for 2 hours, the time required to achieve maximum sickling. After incubation, 10 μL of each mixture was diluted 100-fold, and a drop of the diluted sample was placed on a microscope slide for analysis. Sickled and total erythrocytes were counted in five randomly selected microscopic fields. For the negative control, the extract solution was replaced with normal saline. The percentage of sickled cells was then determined.

 3. Result and Discussion

3.1 Phytochemical screening

When we performed qualitative tests for phytochemicals in clove bud extract a number of phytochemicals shows positive results in their specific tests. Presence of alkaloids, saponins, tannins, flavonoids, glycosides, and flavonoids were recorded in the present study [table 1].  These findings are in accordance to a previous study by Yadav and Agarwala, [26] the results show extracts of Syzygium aromaticum are rich in a diverse array of phytochemicals, several of which are known for their medicinal and pharmacological properties. The high content of phenolic compounds highlights the potential of these extracts for further exploration in antioxidant and antisickling therapies, as supported by existing literature. [27-29]

Table 1: Results of phytochemical screening of crude extract of S. aromaticum buds

The symbol (–) represents absence, while symbol (+) represents presence

3.2 Anti-sickling activity of different fractions from S. aromaticum

The image below shows different micrographs of SS blood alone and SS blood in the presence of various extracts.

Figure 2: sickle-red blood cells alone in a NaCl 0.9% solution (control)

Figures: 3a to 3d display the morphological changes in sickle cell erythrocytes treated with different solvent fractions of S. aromaticum: n-hexane (Fig. 3a), ethyl acetate (Fig. 3b), butanol (Fig. 3c), and aqueous (Fig. 3d) fractions.

The current work evaluated in vitro assays to assess the phytochemical content and anti-sickling effectiveness of different solvent fractions of S. aromaticum. The selection of solvent was based on their increasing polarity, which facilitates the separation of phytochemicals ranging from non-polar to highly polar compounds. [30] Figure 2 illustrates that, under hypoxic conditions, the majority of red blood cells (RBCs) exhibit a sickle-shaped morphology, confirming the SS genotype of the blood samples used as control. In contrast, Figures 3a to 3d show that when sickled erythrocytes are treated with n-hexane, ethyl acetate, butanol and aqueous fractions of S. aromaticum under the same experimental conditions. Microscopic observation confirmed that a significant proportion of the treated HbSS erythrocytes regained their typical biconcave shape from the sickled form of erythrocytes, the treated SS RBCs closely resembled with normal cells in morphology. Among the fractions tested, the ethyl acetate and butanol extracts exhibited the most potent anti-sickling activity, while the n-hexane and aqueous fractions demonstrated comparatively less effects. Quantitatively, the normalization rates were 38.82% for the n-hexane fraction, 78.42% for ethyl acetate, 68.32% for butanol, and 52.42% for the aqueous extract, as presented in Table 2. . The superior anti-sickling activity of the ethyl acetate fraction in this study is consistent with findings from other recent investigations [31–33], which have also reported enhanced efficacy for ethyl acetate-soluble phytoconstituents. Some bioactive compounds phenolics, flavonoids, and terpenoids in these extracts would interact with HbS and interfere with the polymerization process. This would prevent the red blood cells from sickling and also it may restrict the cellular processes that cause erythrocyte sickling, which helps sickled cells return to their normal morphology.

Table 2: Normalization rate (%) of examined fractions with their effectiveness

Figure 4: Percentage reversal of sickling by normal saline and partitioned fraction of S. aromaticum at 250 μg/ml

Multiple investigations into various ethnomedicinal plants species such as Jatropha gossypiifolia, Jatropha secunda, and Phyllanthus nigrescens exhibit notable anti-sickling activity, largely attributed to their high polyphenolic content [34]. The normalization rates observed in those studies were comparable to the values obtained in the present investigation, underscoring the crucial role of polyphenols and other secondary metabolites in preventing erythrocyte sickling. These results support the ethnopharmacological use of S. aromaticum in traditional medicine for the management of sickle cell disorder and also support the hypothesis that solvent polarity plays a significant role in extracting bioactive compounds with anti-sickling potential.

4. Conclusion

According to this study, S. aromaticum has a wide range of phytochemicals that support its anti-sickling properties. Among the different solvent fractions tested, the ethyl acetate fraction showed the highest ability to reverse sickled erythrocytes, followed by the aqueous, butanol, and n-hexane fractions. These results suggest that moderately polar compounds in S. aromaticum extract play an important role in its antisickling activity. Overall, S. aromaticum offers a promising natural source for the development of affordable and accessible treatments for sickle cell disease, especially in regions where the disease is prevalent and access to conventional therapies is limited.

Acknowledgement

I wish to thank Council of Scientific and Industrial Research-Human Resource Development Group (CSIR-HRDG), New Delhi, for providing junior Research Fellowship and they are also acknowledging Pt. Jawaharlal Nehru Memorial Medical College in Raipur for help in obtaining the blood samples used in this study.

 Conflict of interest

The authors declare that they have no any conflict of interest in this study.