Introduction

Elephant grass (Pennisetum purpureum Schumach), commonly called “achara” by the Igbo-speaking people of Southeastern Nigeria belongs to the Poaceae family and is native to tropical Africa[14]. It is a tall perennial grass with bamboo-like stems of about 4-7m and 2-5cm in diameter. It has a rapid growth rate, with high productivity, regeneration capacity, tolerance to adverse conditions and high adaptability, among others. Elephant grass is generally used as forage for grazing animals[3,26]. It is used as an ornamental plant, improves soil fertility and protects lands from erosion. It is also used for fencing, firebreaks, wind breaks, in paper pulp production and most recently used to produce bio-oil, biogas and charcoal[18]. Elephant grass is the most important fodder crop for dairy farmers in East Africa [5,28]. Its high productivity makes it particularly suited to feeding cattle and buffalo. However, young shoot culms are edible and enjoyable as food in some parts of Igbo land in Southeastern Nigeria, where they serve as a traditional relish and as a vegetable for soup and stew from prehistoric times. The young shoots and leaves contain dietary fiber, protein, cellulose, hemicellulose, lignin, moisture and dry matter. It contains a variety of vitamins and minerals, including vitamin C, calcium, potassium and magnesium, as well as antioxidants and phytochemicals which include alkaloids[8, 22]

Elephant grass helps in digestion, boosts energy level and detoxifies the body[9]. The anti-inflammatory properties of the components, chlorogenic acid, caffeoylquinic acids and sesquiterpene lactones make Elephant grass effective in [8] the relief of pain and stiffness. Also, it plays a role in the prevention and management of inflammatory conditions, such as cardiovascular diseases, arthritis and cancer. The plant may help to protect against colds and flues because of its anti-bacteria and antiviral properties, which are conferred on it by antioxidants and phytochemicals that help to boost the immune system by scavenging harmful toxins and fighting off infections[6, 23]. Using Elephant grass as a culinary may help to maintain good health and reduce the risk of developing serious illnesses.

Very little research has been carried out to study the anti-sickling properties of this very important crop. Sickle cell disease is a genetic condition that occurs due to a mutation at the sixth residue of the ß–ß-ß-globin chain of hemoglobin and leads to the production of sickle hemoglobin (HbS), causing red blood cells to become rigid and sickle-shaped. It reduces the ability of hemoglobin in red blood cells to transport oxygen. Sickle-cells tend to stick together, blocking small blood vessels, causing painful and damaging complications[20]. This devastating blood disorder causes severe clinical complications such as vaso-occlusive crisis, acute

Chest syndrome, hemolytic anemia, pulmonary hypertension, nephritic syndromes, and may also manifest into entire organ damage [16]. Sickle cell disease has a profound impact on individuals, families, and communities. It causes significant morbidity and mortality [15], chronic pain and disability[1], increased risk of infections, stroke, organ damage, reduced quality of life, and life expectancy ([21]. It places a substantial economic burden on healthcare systems and families. Most people with sickle disease are from sub-Saharan Africa and their descendants. In Africa, access to medical care and public health strategies to decrease mortality and morbidity are not uniformly available. The conventional medicines and techniques are costly and unaffordable to many poor people in Africa [24]. Consequently, a search for alternative therapeutics using indigenous crops that are readily available to the people becomes a priority. Hence, the study of anti-sickling potentials of Elephant grass was carried out to determine the ability of its molecular constituents to affect the hydrophobic bonds in sickle cells and reverse them to normal cells, thereby reducing the severity and crises of the disease in its numerous patients.

Materials and Methods Plant Material

Fresh P. purpureum was obtained from the University of Nigeria community in Nsukka, Enugu State, Nigeria. The plant sample was chopped into small chunks at room temperature and blended using an electric blender before use.

Instruments and Equipment

Sephadex G25 (Merck, Germany), Centrifuge (Jiangsu Jinyi, China), Spectrumlab 23A Spectrophotometer (Zhejiang Nade, China), Electronic Scale (G&G, China), Light Microscope (Motic, China), pH meter (Polycase, USA), HH-S4 Water Bath (Searchtech, UK), BUCK M910 Gas Chromatography, Pasteur Pipette, Micropipette, Electrophoresis Paper, Microscope Slides, Microscope Coverslips, Test Tubes, EDTA Bottles, Filter Paper, Hand Gloves, Syringes, Plain Vacutainers, and Glass Chromatography Column.

Reagents

Potassium Phosphate Buffer (10 mM/0.01 M), Ethanol, Distilled Water, Sodium Metabisulfite, and Sodium Chloride

Methodology

Preparation of Aqueous Plant Extract

The aqueous plant extract was prepared following the methods outlined by previously [4] The fresh plant sample of Pennisetum purpureum, weighing 273.67g was subjected to extraction by chopping it into small chunks and then homogenizing it with 500ml of distilled water using an electronic blender. Subsequently, the extract underwent filtration and was stored in a refrigerated environment for the assays. Various concentrations, including 1000, 500, and 250μg/ml, were meticulously prepared and utilized for the anti-sickling study.

Extraction of Phytochemicals

The extraction of phytochemicals was carried out following [2]. A sample of 1g was weighed into a test tube, and 15 ml of ethanol, 10 ml 50 % w/v KOH was added and allowed to sit in a water bath at 60 °C for 1 hr. After, the content was transferred to a separatory funnel. The tube was washed successively with 20 ml of ethanol, 10 ml of cold water, 10 ml of hot water, and 3ml of hexane, all transferred to the funnel. This extract was combined and washed thrice with 10ml of 10 % v/v ethanol aqueous solution. The solution was dried with anhydrous sodium sulfate, evaporating the solvent. The sample was solubilized in 1000 μl (1ml) of pyridine, of which 200μl (0.2ml) was transferred to a vial for analysis.

Quantification of Phytochemicals by Gas Chromatography-Flame Ionization Detector

The analysis of phytochemicals was performed on a BUCK M910 Gas chromatography equipped with a flame ionization detector. A syringe was used to draw 0.1 ml of the extract and injected into the gas chromatography GC machine equipped with FID. In principle, an FID uses a flame to ionize organic compounds containing carbon. Following the separation of the sample in the GC column, each analyte passes through a flame, fueled by hydrogen and zero air, which ionizes the carbon atoms.

Blood Collection

The blood collection was done as performed previously [4]. Venous blood (AA and SS) was collected from willing human participants using sterile techniques. Both were immediately placed in anticoagulant tubes containing Ethylenediaminetetraacetic acid (EDTA) to prevent clotting and preserve the blood samples’ integrity. They were then stored in a cooling flask with ice.

Preparation of 10mM Potassium Phosphate Buffer pH 7.8

The potassium phosphate buffer was prepared according to [19]. The anhydrous dipotassium hydrogen phosphate (K2HPO4) has a molecular weight (Mw) of 174.18 g. To prepare a 10 mM (0.01 M) potassium phosphate buffer, a precise amount of 0.870s9 g K2HPO4 was weighed and dissolved in 500 ml of distilled water. The pH level of the solution was then.

Meticulously adjusted to 7.8 using sodium hydroxide (NaOH) while continuously monitoring it with a pH meter.

Preparation of Sephadex G25

Preparation of Sephadex G25 was done according to the method previously described [17]with modifications. Sephadex G25, weighing ten grams, was hydrated by soaking it in 1000 mL of distilled water. The mixture was then gently boiled at 50 °C for 30 minutes, with regular stirring every 3 minutes to ensure complete hydration. After boiling, the mixture was left to cool, and the distilled water was decanted. Next, 500 ml of potassium phosphate buffer was added to the mixture and left to stand for 24 hours. Finally, the mixture was carefully transferred to a column for gel exclusion chromatography.

Equilibration of the Chromatography Column

Equilibration of the Chromatography Column was done according to [11]with modifications. To assemble a 50 ml column, the entry point was blocked using glass wool and secured with a glass rod to pin down the glass wool. Sephadex G25 was then added meticulously, ensuring no air bubbles were present. The glass rod was gradually removed after pouring the Sephadex G25. The column was equilibrated by slowly adding potassium phosphate buffer (10 mM, pH 7.8) for 30 minutes until the pH reached 7.8. Throughout the experiment, the flow rate of the column was kept constant at 15 drops per minute. Using a slower flow rate is advisable to achieve a uniform flow rate. As a preventive measure, a clip was carefully positioned at the column outlet to ensure the potassium buffer solution did not escape the column.

Washing Blood Cells with 1% Normal Saline

To maintain the proper function and purity of blood cells, they were meticulously washed three times using a 1% normal saline solution, which is an isotonic solution. This isotonic solution was created by dissolving 5 g of NaCl in 500 ml of distilled water to prevent potential hemolysis. The solution was then thoroughly mixed by shaking, and two 5 ml AA blood samples were washed with 5 ml saline each in 10 ml centrifuge tubes. Additionally, 2 ml of SS blood was washed with 2 ml of saline in 10ml centrifuge tubes. During each wash, a laboratory centrifuge was used to spin the samples at 4000 rpm for 10 minutes.

Removal of Serum

After centrifugation, the serum (supernatant) was extracted precisely using a Pasteur pipette and a micropipette. This washing procedure was repeated three times to guarantee the complete elimination of plasma components. The serum contains all proteins, excluding clotting factors responsible for blood clotting, and comprises electrolytes, antibodies, antigens, hormones, and any

External substances like drugs or microorganisms. Once the serum was extracted, the red blood cell volume decreased, and the solution was doubled in volume with 1% normal saline. Also, washing was carried out until the third times.

Isolation of Hemoglobin

Following the third wash, the two centrifuge tubes contained 2ml of washed red blood cells, which were then lysed using distilled water. The resulting solution from the third wash was doubled in volume with 2 ml of distilled water, and the reagents were gently swirled before being left to stand for an hour at room temperature. The mixture was spun at 4000rpm for 30 minutes using a laboratory centrifuge. Unfortunately, no distinct layers were observed in the tubes after centrifugation, so the process was repeated twice, each time adding 2 ml of distilled water. After the third centrifugation, no distinct layers were visible, so 1 ml of distilled water was added to the 8 ml mixture, and the hemolysis process was repeated. Finally, after centrifugation, two distinct layers could be seen. The supernatant, the hemolysate, was transferred to the EDTA tube, while the pellet containing the plasma membrane, glucose 6-phosphate dehydrogenase, and other enzymes used for metabolism was discarded.

Gel Exclusion Chromatography

The clip at the column outlet was removed to allow the potassium phosphate buffer solution to settle at the top of the gel. Then, using a Pasteur pipette, the hemolysate solution was gently and uniformly introduced into the column while rotating around the circumference to ensure a smooth flow. As the hemolysate progressed, the colors shifted from dark to light red, and the column eluate was collected at 2.5ml per Vacutainer labelled from 0 to 8. This collected elute contains the hemoglobin (oxyhemoglobin), with tube 0 reserved for UV-visible spectroscopy. Gel exclusion chromatography’s principle showed that larger molecules moved faster than smaller ones, and since hemoglobin has a molecular weight of 64,000Daltons, it moves quickly out of the gel beads. Finally, the collected eluate was stored with ice in a cooling flask to preserve its quality.

UV-Visible Spectroscopy of Pennisetum purpureum on AA and SS Blood Hemoglobin

The UV-visible spectroscopy was done according to the method previously used [10]with modifications. A baseline was established using 300μl of potassium phosphate buffer to obtain accurate results. The hemoglobin spectra were obtained through UV-visible spectroscopy by dissolving 100μl of the blood hemoglobin (AA and SS) in 300μl of the same buffer. Then, 100μl of the hemoglobin mixture was combined with 100μl of three different doses of plant extract (1000μg/ml, 500μg/ml, and 250μg/ml), and the resulting mixture was observed for 0, 10, and 20 minutes. UV-visible spectroscopy was conducted using a 1cm cuvette, and absorbance was

Scanned between 400 and 700nm. Differences in absorbance at the α-peak (540 or 541 nm) and β-peak (576 or 577nm) were recorded and compared. The purity of hemoglobin was calculated using the formula below:

The concentration of hemoglobin was calculated with Beer-Lambert’s Law using the maximum peak between the two bands and extinction coefficient of 15.54 cm^-1mM^-1 and 16.61cm^-1mM^-1 for the α-peak and β-peak, respectively, as reported by [12]. Hence, the formula for hemoglobin concentration below:

Microscopy of Pennisetum purpureum on SS Red Blood Cells

The microscopy procedures were conducted based on methods described previously [4,13]. After centrifugation, the SS red blood cells were washed to prepare the blood sample slides. A drop of 2% sodium metabisulfite and SS red blood cells were added to a microscope slide and covered with a cover slip to serve as the control. To examine the anti-sickling potential of Zapoteca portoricencis stem bark, a drop of 2% sodium metabisulfite and SS red blood cells were added to a drop of the different doses of the plant extract (1000μg/ml, 500μg/ml, and 250μg/ml) on the slide and covered with a cover slip. The red blood cells were counted using a bright field microscope at x40 lens and x10 eyepiece magnification.

The percentage of sickled red blood cells was calculated as follows:

Finally, the percentage of reverse sickling was calculated as follows:

Hemolysis Study of Pennisetum purpureum on SS Red Blood Cells

In the hemolysis studies of SS red blood cells, the plant extract was tested at its highest concentration of 1000μg/ml. The experiment consisted of 11 test tubes with different volumes of saline and distilled water. Saline quantities varied from 0ml to 10ml, while distilled water made up the remaining volume from 10ml to 0ml. For this experiment, 50μl of saline was added to each test tube, followed by 10ml of solvent (varying amounts of saline and distilled water), and finally,

50μl of SS red blood cells were added to serve as the control. For the plant extract tests, 50μl of 1000μg/ml plant extract was added to each test tube, followed by 10 ml of solvent (varying amounts of saline and distilled water), and then 50μl of SS red blood cells.

RESULTS

Phytochemical Content of the Aqueous Extract of Pennisetum purpureum

The result presented in Table 1 below shows the chemical components of the P. purpureum extract as detected using the GC-FID. Twenty-four compounds were detected in varying concentrations, including quercetin, catechin, gallocatechin, resveratrol, vanillic, and ellagic acid.

Effect of the Aqueous Extract of Pennisetum purpureum on AA Blood Hemoglobin Oxidation

Figures 1-10 show the effect of P. purpureum extract on AA blood hemoglobin oxidation at 0, 10, and 20 minutes for 1000µg/ml, 500µg/ml, and 250µg/ml of the plant extract. The results showed a decrease in the hemoglobin’s absorbance upon adding the plant extract compared to the control.

Effect of the Aqueous Extract of Pennisetum purpureumon SS Blood Hemoglobin Oxidation

Figures 11 – 20 show the effect of P. purpureum extract on SS blood hemoglobin oxidation at 0, 10, and 20 minutes for 1000µg/ml, 500µg/ml, and 250µg/ml of the plant extract. The results showed a decrease in the hemoglobin’s absorbance upon adding the plant extract compared to the control.

Purity and Concentration of AA Blood Hemoglobin

The result presented in Table 2 shows the purity and concentration of the AA Blood hemoglobin at 0, 10, and 20 minutes for the 1000µg/ml, 500µg/ml, and 250µg/ml of the plant extract. The result showed that the purity and concentration of hemoglobin increased over time upon adding plant extract compared to the control.

Purity and Concentration of SS Blood Hemoglobin

The result presented in Table 2 shows the purity and concentration of the SS blood hemoglobin at 0, 10, and 20 minutes for the 1000µg/ml, 500µg/ml, and 250µg/ml of the plant extract. The result showed that the purity of hemoglobin increased over time upon adding plant extract compared to the control, whereas the hemoglobin concentration decreased over time.

Anti-sickling Potential of Aqueous Extract of Pennisetum purpureum (Achara)

The results in Figures 21-23 below show the anti-sickling potential of P. purpureum extract on SS red blood cells for 1000, 500, and 250µg/ml concentrations. The percentage of sickling was calculated for the control (2% sodium metabisulphite + SS blood). In comparison, the percentage of reverse sickling was calculated upon adding the different concentrations of P. purpureum (2% sodium metabisulphite + SS blood + plant extract) at other times. The results showed that P. purpureum (achara) could reverse and decrease sickling.

Effect of Pennisetum purpureum (Achara) on Hemolysis of SS Red Blood Cells

The result presented in Figure 11 shows the hemolysis effect of the ethanol extract of P. purpureum stem bark on SS red blood cells for the 1000µg/ml concentration. The hemolysis curve was plotted for the control and the addition of the plant extract, where the plant extract showed a stabilizing hemolytic effect compared to the control. Eleven test tubes were used for the hemolysis study, and the absorbance was plotted against each test tube number. Test Tube 0 to 10 comprised 0ml to 10ml saline and 10ml to 0ml distilled water, respectively.

DISCUSSION AND CONCLUSION

Identifying bioactive molecules in plant extracts is crucial in searching for new drugs or alternative therapeutic approaches using natural sources. In the current study, phytochemical analysis of Pennisetum purpureum (Achara) extract revealed the presence of several bioactive compounds, including flavonoids, catechins, polyphenols, and a cyanogenic glycoside. The extract’s effect on sickling cells was evaluated using 2% sodium metabisulfite to induce sickling in SS red blood cells in vitro. The results showed that the extract reduced sickling in red blood cells in a dose-dependent manner, with the 1000µg/ml concentration providing the best outcome. Further analysis of the plant’s bioactive compounds revealed the presence of aromatic molecules with antioxidant properties, such as resveratrol, kaempferol, quercetin, naringenin, genistein,

epicatechin, vanillic and ellagic acid. Research has shown that these compounds inhibit sickling in red blood cells. The results from UV-visible spectroscopy on AA cells revealed that the effect of P. purpureum on hemoglobin oxidation is minimal. The concentration of hemoglobin (0.0486 mM) in the control was reduced by 54.93% to 0.0222mM upon adding the plant extract, showing a minimal effect on hemoglobin concentration. This could result from possible interactions between the components of the plant extract and the hemoglobin. The microscopic results showed that P. purpureum has a significant anti-sickling potential. A maximum percentage of reverse sickling (87.50%) was observed in the 250μg/ml ethanol extract of P. purpureum at 55 minutes, showing a significant anti-sickling effect compared to the control with 100.00% of sickled cells. Other concentrations of the plant extract (1000 and 500μg/ml) still showed a significant anti-sickling effect. However, the anti-sickling activity appeared dose-dependent, with the lowest concentration (250μg/ml) showing the best outcome. Interestingly, the anti-sickling properties of

P. purpureum increases over time, indicating that P. purpureum (achara) can convert the sickled cells to normal round cells. While several anti-sickling agents target hemoglobin, this study indicates that some anti-sickling compounds can interact with the plasma membrane, preventing hemoglobin oxidation and consequent sickling. The hemolysis result showed that the absorbance of the red blood cells was stabilized upon adding the plant extract compared to the control, indicating that P. purpureum is anti-hemolytic. Research has shown that 12 out of the 15 detected compounds in the phytochemical result are anti-hemolytic, including kaempferol, resveratrol, genistein, daidzein, epicatechin, ellagic acid, catechin, quercetin, luteolin, naringenin, apigenin, and myricetin [2].

This study has revealed that Pennisetum purpureum (Achara) exhibits both anti-sickling and anti-hemolytic properties, along with other known pharmacological benefits. Our observations suggest that individuals with sickle cell disease would benefit from incorporating Achara in their diet or into their nutritional regimen as a natural therapeutic alternative [27]. Additionally, the study indicates that certain anti-sickling compounds can interact with the red blood cell membrane, preventing hemoglobin oxidation and subsequent sickling, unlike other agents that target hemoglobin exclusively.

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