Evaluation of Platelet Augmentation Potential of Setplet in Cyclophosphamide-Induced Thrombocytopenia in Wistar Rats

Thrombocytopenia is a clinical condition characterized by a decrease in platelet counts, defined as having 150,000 or fewer platelets per mm³ of blood, along with a shorter platelet survival time. Platelets, or thrombocytes, are colourless blood cells that are crucial for the blood clotting process. A reduction in platelet count can impair the clotting mechanism and extend bleeding time. Thrombocytopenia may arise from various factors, including viral infections, dengue fever, hepatitis C, cancer, immune thrombocytopenic purpura, or during pregnancy. The standard first-line treatments typically involve corticosteroids, immunoglobulins, and splenectomy. However, these treatments may not be effective for 25 to 30% of patients with chronic idiopathic thrombocytopenic purpura (ITP). In this study, we utilized Cyclophosphamide, an antineoplastic and immunosuppressive agent, as an inducer of stable thrombocytopenia. Administering intraperitoneal injections of Cyclophosphamide (25 mg/kg/day) for three consecutive days has proven to be a straightforward and reliable model for inducing thrombocytopenia in rats. Plants and plant-based herbal products have historically served as key sources for drug discovery and development, largely due to their high availability and relatively mild side effects. The successful application of herbal products in treating thrombocytopenia has opened new research avenues in herbal product screening. Setplet syrup is a unique platelet booster with hematinic properties, specifically designed for dogs and cats affected by vector-borne blood parasitic diseases. Its primary objective is to enhance platelet counts. Carica papaya leaves contain a range of complex biochemical components, including flavonoids, glycosides, alkaloids, glutathione, and glucosinolates, which contribute to boosting platelet counts by stimulating bone marrow. Additionally, the elemental iron in Carica papaya, along with folic acid, cobalt, copper, and vitamin B12, plays a significant role in hematopoiesis. Setplet syrup combines Carica papaya leaf extract with elemental iron, folic acid, cobalt, copper, vitamin B12, and vitamin C. It serves as a supportive therapy for thrombocytopenia and anemia, as well as an adjunct in postoperative care and in treating vector-borne parasitic diseases. This study aims to investigate the effects of Setplet on platelet recovery in an animal model of thrombocytopenia. Therefore, the in vivo efficacy of Setplet will be assessed in rats to confirm its role in managing thrombocytopenia and anemia.

MATERIAL AND METHODS

Drug and Chemicals

This is a sample of Setplet used for research purposes from Vetrina Healthcare Pvt. Ltd. located in Pune, India, accompanied by a no-objection certificate from the manufacturer. Additionally, Cyclophosphamide injection was purchased from a local pharmacy (Fresenius Kabi Oncology Ltd.) and administered at a dosage of 25 mg per kg of body weight for three consecutive days. Setplet syrup contains an extract of Carica papaya, as well as elemental iron, folic acid, cobalt, copper, and vitamin B12, all of which contribute to hematopoiesis. The formulation of Setplet syrup combines Carica papaya leaf extract with elemental iron, folic acid, cobalt, copper, vitamin B12, and vitamin C. The diagnostic kits used in this study were procured from Transasia Biomedicals Ltd. in Mumbai. All other chemicals and reagents used in the study were of analytical grade.

Experimental animal

The study received approval from the Institutional Animal Ethics Committee (IAEC) at Mumbai Veterinary College. All animal experiments and protocols were conducted in accordance with the guidelines set forth by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA). The experiment involved healthy adult albino Sprague-Dawley (SD) rats of both sexes, each weighing between 200 and 250 grams. The rats were housed in polypropylene cages under standardized conditions, including a 12-hour light/dark cycle, a temperature of 24°C, and humidity levels between 35% and 60%. They were given unrestricted access to a diet and purified drinking water.

Induction of Thrombocytopenia:

Thrombocytopenia was induced in experimental Sprague Dawley rats through a intraperitoneal injection of Cyclophosphamide @ 25 mg/kg/ day body weight for three consecutive days (Fresenius Kabi Oncology Ltd) regardless of sex. The study lasted for 21 days.

Experimental design:

Albino SD rats (weighting between 200–250 g) of either sex were divided into five groups, each consisting of ten rats. Group I received normal saline from day 1 to day 21 and served as the normal control group. Group II received cyclophosphamide (25 mg/kg, intraperitoneally) from day 1 to day 3, followed by normal saline (1 ml/kg, orally) from day 4 to day 21, serving as the toxic control group. Groups III and IV also received cyclophosphamide (50 mg/kg, intraperitoneally) from day 1 to day 3, followed by Setplet (0.2 ml, 0.4 ml, and 1 ml, orally, respectively) for 21 days, serving as the treatment groups. Blood samples were collected 1 hour before the start of the experiment, and again on days 7 and 21 after administration of Setplet. The blood was drawn from the retro-orbital plexus of the rats under light anaesthesia, using glass capillaries, and was stored with disodium ethylenediaminetetraacetate for biochemical parameter estimation. Total platelet count (PC), Total leucocyte count (TLC), and Differential leucocyte count (DLC) including Neutrophils, Lymphocytes, Monocytes, Eosinophils, and Basophils were measured. The rats were closely monitored for general activity, fur condition, food and water intake, changes in weight, and mortality rate. The animals were weighed daily for 21 days.

Determination of clotting time

At 21 days, a glass capillary (inner diameter=0.9~1.1 mm, length=10 cm) was inserted into one eye of rat. Then the capillary was removed and placed on the desk horizontally until the blood full filled it. Every 30 seconds the capillary was broken and stretched gently to see if there were fibrin threads of blood at the breakage point. The time from bleeding to the fibrin threads was recorded.

Determination of bleeding time

Bleeding time was determined by a modified Duke method. The animal was kept in a rat restrainer with the tail exposed out. The animal tail was cleaned using hot water, rectified spirit, and its tip was punctured using a sterile needle and blotted on Whatman® filter paper until bleeding stopped. Bleeding time was recorded in seconds.

Statistical analysis

Values were expressed as mean ± SEM and data were analysed using one-way ANOVA followed by the Dunnett’s Multiple Comparison Test at 95% confidence interval using GraphPad Prism (version 8; Graph- Pad Software Inc., San Diego, CA, USA). The significance level was set at p<0.05.

RESULT AND DISCUSSION:

Effect of Setplet on Platelet counts

The present study aimed to evaluate the platelet augmentation potential of the commercially available Setplet in a rat model of Cyclophosphamide-induced thrombocytopenia. Prior to the induction of thrombocytopenia, there were no significant differences in mean platelet counts among the groups (see Table 1). However, by day 7, the platelet count in the disease control group significantly decreased (p<0.01) compared to the normal control group, confirming the successful induction of thrombocytopenia by cyclophosphamide. Animal pretreatment with Setplet at doses of 0.2 ml, 0.4 ml, and 1 ml effectively mitigated the drastic reduction in platelet counts observed in the toxic control group. Furthermore, as treatment with Setplet continued, we observed a statistically significant increase in platelet count (p<0.001) compared to the toxic control group (see Fig. 1). All treatment groups demonstrated a significant change in total platelet count relative to the disease control group, indicating that Setplet had a positive effect. The data suggest that at the end of the study, the platelet counts in all treatment groups were higher than they were on Day 1. The highest drop in platelet count occurred on day 7 in the disease control group, which recorded a count of (2.33 ± 0.74) × 10^5 /μL, compared to (6.21 ± 0.68) × 10^5 /μL on Day 1. After reaching this low, the platelet count began to increase gradually, with readings of (5.62 ± 0.68) × 10^5 /μL on Day 7 and (5.62 ± 0.48) × 10^5 /μL on Day 21, indicating significant decreases of 49.33%, 44.84%, and 32.75%, respectively.

Table 1: Effect of Setplet on Platelet counts (105/uL) in Cyclophosphamide induced thrombocytopenia

Effects of Setplet on total WBC count

Total WBC significantly (p<0.001, p<0.05) decreased in the toxic control group as compared to normal controls on days 7 and 21. Treatment with Setplet (0.2ml, 0.4ml or 1ml) caused no significant differences in the total WBC on day 7, but significantly increased (p<0.001) the total WBC on day 21 as compared to CP treated rats (Fig. 2).

Bleeding time and clotting time

The bleeding time and clotting time at day 14 showed no significant differences between the groups. This may be due to improvement in the platelet count of the treated as well as non-treated groups (Figs 3, 4)

Table 3: Effect of Setplet on bleeding time in Cyclophosphamide induced thrombocytopenia

Table 4: Effect of Setplet on clotting time in Cyclophosphamide induced thrombocytopenia:

DISCUSSION