Investigation of the antianemic activity of phyllanthus reticulatus poir. In 2,4-dinitrophenylhydrazine-induced anemic ratsAbstract
Growing concerns regarding the side effects of conventional drugs have heightened interest in medicinal plants as alternative therapeutic agents. Phyllanthus reticulatus, widely used in traditional medicine across Asia and Africa, is recognized for its diverse pharmacological properties. This study aimed to evaluate the potential antianemic effects of P. reticulatus in anemic rats. Fresh leaves of P. reticulatus were collected, air-dried, powdered, and extracted using methanol. The extract was subjected to phytochemical analysis and assessed for antianemic activity in 2,4-dinitrophenylhydrazine-induced anemic rats at doses of 250, 500, and 1,000 mg/kg body weight, with folic acid serving as the positive control. Hematological parameters, including white blood cell (WBC) count, red blood cell (RBC) count, hemoglobin (Hb) levels, packed cell volume (PCV), mean corpuscular hemoglobin concentration (MCHC), and mean corpuscular volume (MCV), were evaluated. Phytochemical analysis revealed the presence of alkaloids, flavonoids, tannins, saponins, phenols, and cardiac glycosides. Among the tested doses, P. reticulatus at 250 mg/kg demonstrated the highest efficacy, significantly increasing RBC count, Hb levels, and PCV, with effects comparable to those of the positive control. However, its influence on WBC count, MCHC, and MCV was comparatively lower. In conclusion, P. reticulatus exhibited promising antianemic potential by increasing RBC count, Hb levels, and PCV, suggesting its potential as a natural therapeutic agent for anemia.
Keywords:
Phyllanthus reticulatus; Phytochemicals; Anemic; 2; 4 dinitrophenyl hydrazine; Hematological parameters.
INTRODUCTION
Anemia is a medical condition characterized bya deficiency of red blood cells (RBC) or insufficient hemoglobin (Hb) levels, leading to a reduced ability to carry oxygen to tissues and organs (WHO, 2023).It can arise from various underlying factors, including nutrient deficiencies-such as iron, vitamin B12, and folate deficiencies-chronic illnesses, inherited disorders, bone marrow dysfunction, and toxic exposures. Common symptoms of anemia include generalized weakness, fatigue, pallor, shortness of breath, dizziness, and an increased heart rate (WHO, 2023). Due to its impact on oxygen transport, anemia affects both physical and mental health. Several types of anemia have been identified, with iron-deficiency anemia being the most prevalent. Other forms include hemolytic anemia, megaloblastic anemia, and vitamindeficiency anemia, some of which have a genetic basis (Chaparro, Suchdev, 2019).
Treatment strategies for anemia vary depending on its underlying cause and may include iron or vitamin supplementation, blood transfusions, and other therapeutic interventions. However, the safety, long-term efficacy, and potential side effects of these treatments require careful consideration (Chaparro, Suchdev, 2019). Medicinal plants have been widely used in traditional medicine across different cultures and continue to play a significant role in global health care. Their therapeutic potential is primarily attributed to the presence of bioactive phytochemicals, which have been linked to various medicinal benefits (Rabizadeh et al., 2022).
Phyllanthus reticulatus is a small tree belonging to the Euphorbiaceae family, characterized by a cylindrical trunk and capable of reaching up to 10 meters in height. It is widely distributed across subtropical regions of Africa, Asia, Australia, and the Pacific Islands (Orwa et al. 2009). This species thrives in diverse environments, including forests, savannas, and disturbed areas such as roadsides and agricultural fields. The leaves of P. reticulatus are simple, alternate, and typically ovate or elliptical, measuring 4-10 cm in length and 2-6 cm in width, with a smooth surface and entire margins (Orwa et al., 2009). The tree produces small, greenish-yellow unisexual flowers that grow in axillary clusters and measure approximately 3-5 mm. Its fruit is a small, three-lobed capsule, initially green and turning brown upon ripening, with a diameter of 6-7 mm (Orwa et al., 2009). As the tree matures, its bark becomes fissured and scaly, while its roots develop into thick, woody structures that extend deep into the soil. P. reticulatus is typically propagated through seeds or stem cuttings and thrives in warm, humid climates (Orwa et al., 2009).
P. reticulatus is widely recognized as a medicinal plant traditionally used across various cultures, including those in China, India, Brazil, Nigeria, and Southeast Asian countries, for the treatment of numerous ailments. It has been employed in managing conditions such as hepatitis, malaria, diabetes, renal calculi, jaundice, and digestive disorders (Mao et al., 2016). Several bioactive compounds have been isolated from P. reticulatus, including corilagin, kaempferol 3-rutinoside, ellagic acid, astragalin, and isoquercitrin (Lan et al., 2010). Other compounds include (5R*,6R*)- 4,6-dimethoxycarbonyl-5-[2′,3′,4′-trihydroxy-6′- (methoxycarbonyl) phenyl]-5,6-dihydro-2H-pyran- 2-one, 3,4,3′-tri-O-methylellagic acid, and methyl gallate (Pojchaijongdee et al., 2010). Phyllanthusmin C, isotachioside, hovetrichoside A, mananthoside I, and reticulateside A have also been extracted from P. reticulatus (Ma et al., 2012).
P. reticulatus has been reported to have a range of pharmacological activities, including antifungal and antioxidant properties (Chellappandian et al., 2018) as well as anti-inflammatory and analgesic effects (Saha et al., 2007). Additionally, it has demonstrated anti-ulcer (Izhar et al., 2021), anti-diabetic (Sathasivampillai et al., 2017), antimicrobial, anticancer, and hepatoprotective properties (Stavropoulou et al., 2021). Despite its diverse therapeutic potential, little to no research has explored the possible antianemic effects of P. reticulatus. Therefore, this study aimed to evaluate the antianemic activity of P. reticulatus leaves, a plant of significant relevance in Nigerian ethnomedicine.
MATERIAL AND METHODS
Ethics statement
This study was conducted in accordance with established guidelines for animal care and welfare. All experimental procedures were reviewed and approved by the Animal Research Ethics Committee of the Department of Pharmacology, University of Jos, Nigeria.
Material/equipment
Materials and equipment used in this study included Wistar rats as test animals. Laboratory equipment comprised an analytical weighing balance, a weighing scale, a needle and syringe, an electric blender (Flourish FL-1600), a maceration jar, a rotary evaporator (Shanghai RE-2000B, China), an auto-analyzer (Genesis™ HA6000), and a dissection kit. Consumables used in the study included adsorbent cotton wool, hand gloves, EDTA bottles, and distilled water. Chemicals and reagents consisted of methanol (analytical grade), folic acid, and 2.5 g of 2,4-dinitrophenylhydrazine.
Plant collection and preparation
Fresh leaves of P. reticulatus (Figure 1) were collected in April 2023 from Area 1, Abuja, Nigeria. The plant was identified and authenticated by the curator (Mr. Vincent Jackson) and the botanist (Dr. Tayo Famojuro) at the Department of Pharmacognosy Herbarium, Bingham University, Karu, Nigeria. A voucher specimen was deposited under the reference code DPHBHU 0042.
Contaminants and extraneous materials were removed from the plant, which was then thoroughly washed under running water, cut into small pieces, and air-dried at room temperature (27ºC) for four weeks. The dried plant material was then ground into a coarse powder using an electric blender and soaked in methanol for 72 hours, with occasional stirring every 3 hours. The mixture was subsequently filtered using adsorbent cotton wool, and the filtrate was concentrated to dryness using a rotary evaporator at 40ºC. The resulting extract was stored in an airtight container and refrigerated until further use.
Phytochemical screening of P. reticulatus leaves
The extract of P. reticulatus leaves was analyzed for the presence of various phytochemicals, including alkaloids, saponins, flavonoids, cardiac glycosides, anthraquinones, and tannins, using standard phytochemicalscreeningtechniques(Famojuro et al., 2023).
Preparation of stock solutions and dose calculation
2,4-dinitrophenylhydrazine
A stock solution of 2,4-dinitrophenylhydrazine was prepared by dissolving 1 g of the compound in 100 mL of distilled water. The final volume of the administered dose (in mL) was calculated using the following formula:
Folic acid tablet
A 2.5 mg folic acid (vitamin B9) tablet was obtained from a pharmacy and dissolved in 100 mL of distilled water to prepare the stock solution. The final administered dose (in mL) was calculated using the same formula:
Plant extract
A stock solution of the plant extract was prepared by dissolving 2 g of the extract in 100 mL of distilled water. The final administered dose (in mL) was calculated using the following formula:
Experimental animals and study design
A total of 25 male Wistar rats weighing between 130 and 200 g were obtained from the Bingham University Animal Care Unit for this study. Prior to the experiment, the rats were housed under standard environmental conditions, maintained on a 12-hour light/dark cycle at an ambient temperature. They were kept in cages with unrestricted access to food and water.
The study followed a modified method proposed by Berger (2007) and was conducted over three weeks. Rats were randomly assigned to five groups, each consisting of five rats (n = 5).
All 25 rats received 2,4-dinitrophenylhydrazine at a dose of 40 mg/kg body weight, administered orally once daily for five days using a feeding cannula to induce anemia. On the sixth day, blood samples were collected by making a small incision at the tip of the tail, allowing blood to flow into capillary tubes for hematological analysis. Rats that exhibited a packed cell volume (PCV) reduction of more than 30% were classified as anemic and included in the study.
Antianemic treatment
Treatment with the plant extract commenced immediately after anemia was confirmed in the rats. The treatment groups received different doses of the extract (250, 500, and 1,000 mg/kg). The positive control group was administered folic acid (2.5 mg/kg), while the negative control group remained untreated, receiving only distilled water (10 mg/kg). All treatments were administered orally once daily for 14 consecutive days using an oral cannula.
Rats were divided into five groups in the following manner: Group 1 (negative control) consisted of anemic rats that received only distilled water (10 mg/kg). Group 2 included anemic rats treated with 250 mg/kg of P. reticulatus extract, while Group 3 received 500 mg/kg of the same extract. Group 4 was composed of anemic rats treated with 1,000 mg/kg of P. reticulatus extract. Finally, Group 5 (positive control) comprised anemic rats treated with 2.5 mg/kg of folic acid.
Hematological assay
The hematological effects of P. reticulatus extract on blood samples were assessed using the method described by Delwatta et al. (2018), with some minor modifications. Hematological analysis was conducted using an automated analyzer (Genesis™ HA6000).
After two weeks of treatment with the methanol extract of P. reticulatus, blood samples were collected from the rats and stored in EDTA bottles to prevent clotting. Such samples were then analyzed using an automated analyzer, which quantified the number and types of blood cells. Parameters measured included white blood cell (WBC) count, RBC count, PCV, Hb levels, mean corpuscular Hb concentration (MCHC), and mean corpuscular volume (MCV) (Sarma, 1990).
Statistical analysis
All experiments were conducted in triplicate, and the results are expressed as the mean ± standard error of the mean (SEM). Each experimental group consisted of five rats. Data were analyzed using one-way Analysis of Variance (ANOVA), followed by Fisher’s least significant difference (LSD) multiple comparisons. The significance level was set at p < 0.05. No significant differences were observed when compared to the control groups (both positive and negative). Statistical analyses were performed using SPSS version 22.0.
RESULTS
Phytochemical screening
Phytochemical analysis revealed the presence of key bioactive compounds, except for anthraquinones, terpenoids, and phytosterols (Table I).
WBC analysis
Table II presents the effects of different treatments on WBC counts in rats. Group 1 had no significant changes in WBC count compared to baseline, with a mean value of 2.96 × 106 cells/μL. Group 2 had a mean WBC count of 2.56 × 106 cells/μL, which did not significantly differ from the control. However, Group 3 had a notable reduction in WBC count to 2.22 × 106 cells/μL. Group 4 had a mean WBC count of 2.80 × 106 cells/μL, which remained comparable to Group 1. Group 5 had a significant decrease in mean WBC count to 2.32 × 106 cells/μL compared to Group 1.
Values are presented as mean ± SEM. Five rats were assigned per group. Data were analyzed using one-way ANOVA followed by Fisher’s least significant difference (LSD) multiple comparisons. The significance level was set at 0.05. “*” denotes values that differ significantly from the negative control (distilled water) group. “a” denotes values that differ significantly from the positive control (folic acid) group.
RBC analysis
Table III presents the effects of different treatments on RBC counts in rats. Group 1 maintained a mean RBC count of 2.72 million/μL. Group 2 had a significant increase in RBC count (4.52 million/μL) comparable to Group 5. Such a result suggests P. reticulatus at 250 mg/kg increases RBC production. Group 3 had a mean RBC count of 3.62 million/μL, which did not significantly differ from Group 1. Similarly, Group 4 had a mean RBC count of 3.12 million/μL, with no statistically significant difference from Group 1. Group 5 had a mean RBC count of 3.68 million/μL, indicating a statistically significant increase compared to Group 1.
In summary, P. reticulatus at 250 mg/kg significantly increased RBC counts comparable to folic acid at 2.5 mg/kg, suggesting its potential role in mitigating anemia.
Values are presented as mean ± SEM. Five rats were assigned per group. Data were analyzed using one-way ANOVA followed by Fisher’s least significant difference (LSD) multiple comparisons. The significance level was set at 0.05. “*” denotes values that differ significantly from the negative control (distilled water) group. “a” denotes values that differ significantly from the positive control (folic acid) group.
HB count
Table IV presents the effects of different treatments on Hb levels in rats. Group 1 maintained a mean Hb level of 3.54 g/dL. Group 2 a significant increase in mean Hb levels (5.92 g/dL). Similarly, Groups 3 and 4 had significant increases in Hb levels when compared to Group 1. Group 5 had a mean Hb level of 4.68 g/dL, a value significantly different from Group 1.
In summary, P. reticulatus at 250 mg/kg significantly increased Hb levels comparable to folic acid at 2.5 mg/kg. Such findings highlight the potential role of P. reticulatus in improving anemia by increasing Hb levels.
PCV analysis
Table V presents the effects of different treatments on PCV in rats. Group 1 maintained a mean PCV of 14.2%. Group 2 had a mean PCV of 14.4%, showing no statistically significant difference from Group 1. Group 3 had a mean PCV of 15%, which also did not significantly differ from the control. In contrast, Group 4 had a substantial increase in mean PCV (17.6%), indicating a statistically significant difference from Group 1. Group 5 had a mean PCV of 18.2%, a value significantly different from Group 1.
In summary, P. reticulatus at 1,000 mg/kg significantly increased PCV comparable to folic acid at 2.5 mg/kg. Such findings highlight the potential role of P. reticulatus in improving anemia by increasing PCV.
MCHC analysis
Table VI presents the effects of different treatments on MCHC after treatment. No group had significative changes in MCHC compared to Group 1, which maintained a mean MCHC of 33.4 g/dL with no substantial deviation from baseline. Groups 2, 3, 4, and 5 had MCHC levels that did not significantly differ from the negative control. Such findings suggest MCHC remained stable across all treatment groups.
MCV analysis
Table VII presents the effects of different treatments on MCV after treatment. No group had significative changes in MCV compared to Group 1, which maintained a mean MCV of 46.8 fL with no substantial deviation from baseline. Similarly, MCV values for Groups 2, 3, 4, and 5 showed no statistically significant differences from the negative control group. Such findings suggest MCV remained stable across all treatment groups.
DISCUSSION
Phytochemical analysis confirmed the presence of several bioactive compounds, including alkaloids, tannins, flavonoids, saponins, carbohydrates, proteins, phenols, cardiac glycosides, and reducing sugars. However, anthraquinones, terpenoids, and phytosterols were not detected. These findings align with those reported by Mythili (2020), who documented the presence of similar phytochemicals. The absence of these three specific compounds in our study may be attributed to differences in geographical location, environmental conditions, or variations in the solvent and extraction methods used. The presence of these bioactive compounds in P. reticulatus likely contributes to its diverse therapeutic properties, reinforcing its traditional use in managing various health conditions.
These findings provide valuable insights into the potential antianemic properties of P. reticulatus in a rat model of 2,4-dinitrophenylhydrazine-induced anemia. Hematological parameters, including WBC count, RBC count, Hb levels, PCV, MCHC, and MCV, were assessed following treatment with varying doses of P. reticulatus and positive control, folic acid.
The administration of P. reticulatus extract did not lead to a significant increase in WBC counts. This finding is consistent with the study by Ofem, Ani, and Eno (2012), which reported a similar outcome when evaluating the effects of Ocimum gratissimum extract on hematological parameters in rats. Such results suggest P. reticulatus may have limited therapeutic value in conditions characterized by decreased WBC counts.
P. reticulatus extract at 250 mg/kg significantly increased both RBC count and Hb levels, demonstrating an effect comparable to that observed in the positive control group treated with folic acid. Such findings suggest P. reticulatus may enhance erythropoiesis, thereby promoting RBC production and increasing Hb levels. This aligns with previous studies reported by Silitonga, Restuati and Silitonga (2018), Ighodaro et al. (2020), and Kondo et al. (2023).
Comparable to folic acid at 2.5 mg/kg, P. reticulatus at 1,000 mg/kg significantly increased PCV levels. Since PCV reflects the proportion of blood volume occupied by RBCs, this increase suggests an improvement in RBC mass and oxygen-carrying capacity (Ighodaro et al., 2020).
Interestingly, no significant changes in MCHC were observed across all treatment groups, indicating Hb concentration within RBCs remained stable. This suggests the treatments did not affect Hb distribution within RBCs. Similarly, MCV remained consistent across all groups, with no statistically significant differences detected, aligning with findings reported by Pluncevic et al. (2019). Such results imply the treatments had no notable effect on average volumeof RBCs.
The findings of this study suggest P. reticulatus, particularly at a dose of 250 mg/kg, exhibits potential antianemic effects by significantly increasing RBC count, Hb levels, and PCV. These results are aligned with those observed in the positive control group treated with folic acid, a well-established therapy for certain types of anemia (Nagao, Hirokawa, 2017; Palika et al., 2022). The significant increase in RBC count, Hb levels, and PCV at 250 mg/kg may indicate an optimal therapeutic dose, as higher doses (500 and 1,000 mg/kg) did not further enhance these hematological parameters. However, P. reticulatus had little to no effect on WBC count, MCHC, or MCV. Such results highlight the potential of P. reticulatus as a natural therapeutic agent for anemia, likely by promoting erythropoiesis and increasing RBC production (Ofem, Ani, Eno, 2012; Dasofunjo et al., 2020).
The antianemic effects of P. reticulatus observed in this study may be attributed to the presence of key phytochemicals, such as flavonoids and alkaloids, which are known to protect RBCs (Sheethal, Krishnakumar, Mathew, 2023), potentially reversing anemia in the treated rats. However, further research is necessary to elucidate the underlying mechanisms of action, assess the safety profile, and determine the optimal therapeutic dose of P. reticulatus. Additionally, clinical studies involving human participants are needed to validate these findings and explore the potential of P. reticulatus as a complementary treatment for anemia in clinical settings.
CONCLUSION
This study provides novel insights into the potential antianemic properties of P. reticulatus in a rat model of 2,4-dinitrophenylhydrazine-induced anemia. Findings indicate P. reticulatus, particularly at a dose of 250 mg/ kg, significantly increased RBC count, Hb levels, and PCV, demonstrating effects comparable to those of the positive control, folic acid. Thus, P. reticulatus may serve as an antianemic agent by potentially enhancing erythropoiesis and improving RBC parameters. However, its effects on WBC count, MCHC, and MCV were less pronounced.
While these findings are promising, further research is essential to elucidate the underlying mechanisms of action, assess the safety profile, and determine the optimal doses of P. reticulatus for clinical applications. Ultimately, this study enhances our understanding of the therapeutic potential of P. reticulatus in managing anemia and highlights the need for future investigations and clinical trials to validate its efficacy in humans.
DATA AVAILABILITY STATEMENT
Not Informed.
ACKNOWLEDGMENTS
The authors express their gratitude to Mr. Solomon Gamde and Mr. Audu Jonah of the Department of Medical Laboratory Science, Bingham University, Nigeria, for their invaluable contributions to this study. Professional English language editing for this manuscript was provided by Aptos Comunicação Editorial (contato@aptoseditorial.com) upon recommendation from the Brazilian Journal of Pharmaceutical Sciences.
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Associate Editor:
Severino Matias de Alencar
