Anaemia is a common blood disorder which affects people of all ages and poses a significant threat to global healthcare. Globally, anaemia affects 1.62 billion people, which corresponds to 24.8% of the population of nine out of ten anaemia sufferers living in developing countries
Tephrosia purpurea is an herbaceous perennial and branched plant found in tropical regions. This plant possesses various pharmacological as well as medicinal properties. Tephrosia purpurea is commonly used in Ayurveda traditional medicine in India to treat ailments such as heart, liver, spleen, and asthma
The whole Tephrosia purpurea was purchased from Mr. Chelladurai, a scientist in Botany Chennai, India. The material procured was in the pulverized form. The plant material was identified and authenticated by Dr. P.E. Rajasekharan, Principal Scientist, Division of Plant Genetic Resources
Pulverised plants (45 g) were placed in a Soxhlet extractor in a round-bottom flask. Methanol (80%) was used as the solvent for extraction. Methanol (400 mL) and distilled water (100 mL) were mixed and poured into a condenser to initiate the extraction process. The setup was placed in a heating mantle at 40–60°C for 6 h. After the extraction, the solvent was recovered using the same process. Subsequently, the solvent was evaporated using a Rota evaporator. After extraction, the practical yield was calculated.
The prepared extracts were subjected to phytochemical analysis and antioxidant capacity determination. The remaining extract was stored in a refrigerator until further use.
The aqueous alcoholic extract was evaporated, diluted with HCl, added to the residue, and properly mixed and filtered. The filtrate was tested for the presence of alkaloids.
2 mL of water, 0.1 g of sodium bicarbonate, and 20 mg of ferrous sulphate were added to 2 mL of 2% w/v extract and allowed to stand, and a deep violet colour was formed. Subsequently, 5 mL of 1M H2SO4, was added and the violet colour disappeared, indicating the presence of vitamin C.
The antioxidant activity of METP was measured using the 2,2-diphenyl-2-picrylhydrazyl (DPPH) method. One millilitre of DPPH (0.01mM) was added to 3 mL of METP at various concentrations. The reaction mixture was then incubated in the dark at room temperature for 30 min. The absorbance was measured at 517 nm against a blank. The free radical-scavenging activity of the plant extract was determined by comparison with that of the methanol control. A lower absorbance of the reaction mixture indicated a higher free radical scavenging activity.
Female Sprague Dawley rats weighing 200± 50 g were housed at 25 ± 5°C in a well-ventilated animal house under a 12:12 h light/dark cycle. The experimental protocol was approved by the Institutional Animal Ethics Committee. The animals were maintained under standard conditions in an animal house approved by the Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA). All rats were provided a normal pellet diet and water ad libitum prior to dietary manipulation. The Institutional Ethical Committee approved the experimental protocol No.2016/PCOL/02/KCP/IAEC.
A phenyl hydrazine hydrochloride solution was prepared in 0.1M potassium phosphate buffer (pH 7.4) and sterilized by filtration prior to use. Anaemia was induced by intraperitoneal administration of phenylhydrazine (60 mg/kg body weight) for 2 days.
Group 1: Negative control, treated with vehicle 5 mg/kg p. o. for 14 days.
Group 2: Positive control, treated with PHZ (60 mg/kg, i. p.) for 2 days.
Group 3: Low-dose METP 200 mg/kg p. o. for 14 days after the induction of anaemia.
Group 4: Medium dose of METP 300 mg/kg p. o. for 14 days after anaemia induction.
Group 5: High dose of METP 400 mg/kg p. o. for 14 days after induction of anaemia.
Group 6: Standard group; standard iron (Fefol capsule) 0.012 mg/kg p. o. for 14 days after induction of anaemia
Blood was collected from the rats into EDTA tubes/heparinized tubes using the tail vein method
N/10 HCl was placed in a diluting tube to mark 20. Then, blood was pipetted up to the 20-cubic-mm-mark and blown into the diluting tube, and rinsed the pipette, after 10 minutes distilled water was added drop by drop until the colour matched exactly to the standard and noted the reading that indicated the haemoglobin percentage.
Mean corpuscular haemoglobin (MCH) is the average amount of haemoglobin per red blood cell in a blood sample. MCH is used to diagnose the type, cause, and severity of anaemia. The normal range for MCH is 27-31 picograms/cell in adults.
WBCs counts were estimated using Neubauer’s chamber.
A subcutaneous injection of low molecular weight heparin, at a dose of 2000 IU/kg, was injected into the rats daily for 10 days.
Group 1: Positive control (vehicle, 5 mg/kg) p. o. for 14 days.
Group 2: Negative control Heparin 2000 IU/kg) s.c for 10 Days
Group 3: Low dose of METP 200mg/kg p.o. For 14 days
Group 4: Medium dose of METP 300mg/kg p.o for 14 days
Group 5: High dose of METP 400mg/kg p.o for 14 days
Group 6: Standard drug Prednisone 7mg/kg/day p.o for 10 days
Platelet count was determined by hemocytometry following the method described by Rees and Ecker (1923).
Blood was drawn into capillary tubes. The time of appearance of the blood drop in the cut tail was recorded. The capillary glass tube was then placed between the palms of both hands for 30 s to maintain it at body temperature. After 30 s, the tube was removed and a small portion of the capillary tube was broken at regular intervals of 30 s until a thread of clotted blood appeared between the two pieces of the capillary glass tube. The time interval between the appearance of the drop of blood and the thread of the blood clot was considered as the clotting time of rats expressed in minutes.
Determination of bleeding time: The bleeding time was determined using the modified Duke’s method. The animals were kept in restrainers, and the tail was exposed. The animal tail was cleaned using hot water, and the rectified spirit and its tip were punctured using a sterile needle and blotted on Whatman filter paper until the bleeding stopped. The bleeding time was recorded in seconds.
All results are presented as mean ± SEM. The significance of the differences compared to the positive control groups was determined using one-way analysis of variance (ANOVA), followed by Dunnett’s test using GraphPad Prism version 5. Statistical significance was set at p < 0.05.
The pulverized form of the whole plant was subjected to extraction using a Soxhlet apparatus with 80% methanol, and the yield was 6.52%.
Extracts subjected to preliminary phytochemical investigation showed the presence of various phytoconstituents such as carbohydrates, flavonoids, alkaloids, tannins, and vitamin C. The results are presented in
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a. |
Mayer’s test |
Brown precipitate |
++ |
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b. |
Dragendroff's test |
Precipitate |
++ |
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c. |
Hager’s test |
Yellow precipitate |
++ |
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a. |
Benedict’s test |
Green colour |
+ |
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b. |
Molish test |
Violet ring appeared |
+ |
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c. |
Fehling’s test |
Brick red precipitate |
+ |
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a. |
Libermann's test |
Green colour |
+ |
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b. |
Salkowski's test |
Reddish brown colour |
+ |
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a. |
Salkowski reaction |
Greenish yellow colour |
++ |
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b. |
Libermannburchard test |
Fluorescence green |
++ |
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a. |
Foam test |
No foam appeared |
_ |
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a. |
Shinoda test |
Pink colour |
++ |
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b. |
Alkaline reagent test |
Yellow colour |
++ |
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a. |
Biuret test |
No pink colour |
- |
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b. |
Millon’s test |
No red colour |
- |
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a. |
5%FeCl3 solution |
Deep blue colour |
++ |
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b. |
Lead acetate |
Yellow precipitate |
++ |
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Yellow precipitate |
++ |
‘+’ represents presence of compound, ‘++’ shows more prominence in color formation and '-' represent absence of compound
METP demonstrated a dose-dependent increase in free radical scavenging activity. The results are shown in
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25 |
0.948 |
21.46 |
5 |
0.559 |
16 |
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50 |
0.661 |
46.72 |
10 |
0.626 |
30 |
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75 |
0.50 |
59.67 |
15 |
0.727 |
51 |
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100 |
0.302 |
75.63 |
20 |
0.801 |
66 |
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125 |
0.106 |
91.42 |
25 |
0.886 |
84 |
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150 |
0.022 |
98.21 |
30 |
0.956 |
98.4 |
The anti-anaemic activity of METP against phenylhydrazine-induced anaemia in Sprague-Dawley rats was evaluated. Haemoglobin, red blood cell (RBCs, MCH, and western blot WBCs counts were measured. After treatment with METP for 14 days, the study observed an increase in RBC and Hb counts, body weight, and WBC, and a decrease in MCH when compared to the negative control group. At all three doses at a low dose, mid dose and high dose there was an increase in RBCs, Hb, and WBCs in a dose-dependent manner, demonstrating the effectiveness of the anti-anemic property of Tephrosia purpurea (
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1 |
Negative Control |
221.7 |
13.10 |
7.328 |
17.98 |
9.68±0.33 |
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2 |
Positive control (PHZ 60mg/kg i.p 2 days) |
186.7 |
8.433 |
2.670 |
36.23 |
5.01±90.18 |
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3 |
Low dose of METP (200mg/kg p.o 14 days) |
205.0 |
9.633 |
4.855 |
21.50 |
4.48±0.23 |
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4 |
Medium dose of METP (300mg/kg p.o 14 days) |
203.3 |
11.07 |
5.992 |
19.44 |
5.89±0.16 |
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5 |
High dose of METP (400mg/kg p.o 14 days) |
200.0 |
12.13 |
7.330 |
16.74 |
8.69±0.323 |
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6 |
Standard (Fefol capsule 0.012mg/kg p.o for 14) |
203.3 |
12.53 |
6.175 |
21.42 |
8.36±0.16 |
All Values are given as mean ± SEM, (n=6)
Note: a, b and c represent ***, **, * respectively.
All the results were calculated using ANOVA followed by Dunnett’s' test by graph pad prism version 5.
The anti-thrombocytopenic activity of METP in heparin-induced anaemia in Sprague Dawley rats was evaluated. The platelet count, bleeding time, and clotting time were recorded. After treatment with METP for 14 days at a low dose (200 mg/kg), mid-dose (300 mg/kg), and high dose (400 mg/kg), there was an increase in body weight and platelet count and a decrease in bleeding time and clotting time compared to the negative control. There was a dose-dependent increase in body weight and platelet count, and a dose-dependent decrease in bleeding and clotting times. Tephrosia purpurea showed anti-thrombocytopenic activity against heparin-induced thrombocytopenia in Sprague Dawley rats (
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1 |
Negative Control |
208.3±3.073 |
756.0±40.16 |
75.83 ±3.005 |
123.3± 4.944 |
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2 |
Positive control (Heparin 2000 IU/kg s.c for 10 Days) |
225.0±4.282 |
412.5±40.41 |
116.7± 4.944 |
175.0± 7.638 |
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3 |
Low dose of METP (200mg/kg p.o 14 days) |
238.3±6.009 |
503.5±33.75 |
101.7± 4.773 |
151.7± 7.491 |
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4 |
Medium dose of METP (300mg/kg p.o 14 days |
248.3±6.009 |
542.2±43.65 |
90.00± 3.651 |
135.0± 4.282 |
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5 |
High dose of METP (400mg/kg p.o 14 days) |
255.0±6.191 |
641.0±25.40 |
70.00 ±3.651 |
131.7± 4.773 |
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6 |
Standard ((prednisone 7mg/kg p.o 10 days) |
255.0±6.708 |
670.3±14.22a |
69.17 ±3.745 |
130.0± 3.651 |
All Values are given as mean ± SEM, (n=6)
Note: a, b and c represent ***, **, * respectively.
All the results were calculated using ANOVA method followed by Dunnett’s test by using graph pad prism version 5.
The present study aimed to investigate the potential benefits of Tephrosia purpurea on anaemia and thrombocytopenia in Sprague-Dawley rats, followed by a phenylhydrazine-induced anaemia and heparin-induced thrombocytopenia animal model. The study was initiated with the authentication of Tephrosia purpurea, which belongs to the family Fabaceae. The pulverized plants were subjected to Soxhlet extraction to facilitate the extraction process. The solvent that was used for the extraction was methanol. Based on a literature review, methanol is an ideal solvent for extracting flavonoid and polyphenol compounds, the main phytoconstituent of interest for the expected pharmacological activity in future work
A preliminary study of anti-anaemic activity was carried out using Tephrosia purpurea extract, followed by an in vivo phenylhydrazine model. Phenylhydrazine was administered and subjected to develop anaemia in Sprague Dawley rats. The use of phenylhydrazine resulted in decreased Hb and HCT levels, which lasted for 8–12 days
RBCs are cellular components of the blood. The primary role of Red Blood Cells (RBCs) transport oxygen to body tissues. However, in certain pathological conditions, such as anaemia, the function of RBCs may be altered, which can negatively impact normal functioning of the body. In the current investigation, administration of phenylhydrazine resulted in a decrease in the red blood cell count of the blood during the experimental phase, a finding that aligns with previous findings
Haemoglobin (Hb) is a critical factor in evaluating the effectiveness of anti-anaemic drugs, as a decrease in Hb levels can result in a reduction in the oxygen-carrying capacity of blood. In this study, the administration of phenylhydrazine led to a decrease in Hb content during the experimental period, which was consistent with previous findings
HCT, also known as packed cell volume, is the ratio of the volume of packed RBCs to the total blood volume. A decrease in HCT is a common indication for anaemia. In this study, a significant decrease in HCT levels was observed in anaemic control rats as a result of phenylhydrazine-induced haemolysis. This finding is consistent with those of earlier reports
Phenylhydrazine is known to stimulate the generation of reactive oxygen species, which can cause oxidative damage to red blood cells (RBCs). In contrast, flavonoids possess potent antioxidant properties that enable them to prevent or repair damage to RBCs
Heparin-induced thrombocytopenia (HIT) is a common drug-induced autoimmune disorder characterized by arterial and venous thromboembolism. Antibodies generated for immune complexes consist of platelet factor 4 (PF4), heparin, and immunoglobulin G (IgG)
The binding of high-affinity heparin to antithrombin is accompanied by a conformational change; this conformational change follows an initial, weak binding of heparin to antithrombin and causes a tight interaction between the polysaccharide and inhibitor, which is a prerequisite for heparin anticoagulant activity. According to some reports, conformational changes can result in a more favourable environment for the activity of proteases. The binding of the reactive site of antithrombin, which prompts a swift interaction with heparin, also encompasses coagulation proteases. Recent research suggests that this binding is not as strong or specific to antithrombin as previously thought. However, for certain enzymes, such as thrombin, Factor IXa and Factor Xia, it appears that an interaction between heparin and the protease, in addition to the polysaccharide and antithrombin, is necessary for the accelerated inhibition of these enzymes.
Heparin functions as a catalyst in the antithrombin-protease reaction, which accelerates the activity of enzymes, such as thrombin, Factor IXa, and Factor XIa. It facilitates the interaction between heparin and non-stoichiometric amounts of these enzymes without being consumed during the reaction
Tephrosia purpurea is used to treat rheumatism, an immune-mediated disease; therefore, it was assumed that METP which decreases the antibody produced for the heparin-PT4 complex, destroys platelets
Tephrosia purpurea improved phenylhydrazine-induced decreases in RBCs, Hb, and HCT levels. Hence, Tephrosia purpurea has significant anti-anaemic activity against phenylhydrazine-induced anaemia and anti-thrombocytopenic activity against heparin-induced thrombocytopenia in rats. Further investigation into the quantitative determination of phytoconstituents, coupled with in vivo evaluations, is necessary to shed light on the precise mechanism underlying the observed anti-anemic and anti-thrombocytopenic effects.