Assessment of In-vitro Haemolytic Activity of Delonix regia

The tropical ornamental plant Delonix regia, also referred to as the flame tree or royal poinciana, is found throughout Asia, Africa, and the Caribbean.  This plant's leaves, blossoms, and bark have all been used historically in folk medicine to cure a range of conditions, including infections, diabetes, and inflammation.  Studies assessing the antibacterial, antioxidant, and anti-inflammatory actions of Delonix regia have recently attracted scientific attention due to its pharmacological characteristics. One area of increasing interest is the haemolytic activity of plant extracts, which refers to the ability of compounds to lyse red blood cells. Assessing haemolytic activity is crucial in evaluating the cytotoxicity of bioactive compounds, particularly when considering their potential for therapeutic applications.

Delonix regia  Known as "GulMohr," bears an abundance of orange-red flower clusters in the early summer.  Fresh flowers are consumed because they have a sweet, tart, and astringent flavour. are said to have anthelmintic properties (Anonymous 1952, 1976).  Rural residents of Asia, the far East, and Australia also use them in their cooking. 

Gulmohar, regarded as one of the most beautiful tropical trees in the world, is well known for its nutritional and medicinal properties.  Tropical and subtropical areas of the world are ideal for gulmohar growth.   It does not thrive in areas that receive a lot of sunlight or shade.   Although the gulmohar tree is a deciduous evergreen, it is almost extinct in Madagascar, where it is native.   This plant is widely known for offering comfortable shade with the help of its feathery foliage.  In India, particularly in the warmer months, it is planted alongside roadsides and in gardens.

According to reports, the residents of Patan area in North Gujarat, India, employ Delonix regia in their traditional medicine.  while peptic ulcers are being treated. The Yanadi, a tribal group in Andhra Pradesh, India, employed Delonix regia flowers. In several African nations, floral water extracts were also utilized to make traditional, healthful drinks.  It is a component of traditional bioproducts and local medicine.  big tree with leaves that resemble ferns.  Other names for Gulmohar include the peacock flower tree, flame tree, and royal painciana^.[2]

Definition of Haemolysis

Haemolysis is the process of red blood cell destruction, leading to the release of haemoglobin into the surrounding fluid. It can be induced by various factors such as toxins, drugs, and certain plant extracts. Haemolysis can have adverse effects on health, including anemia and organ damage.

Hemolysis is the breakdown or destruction of red blood cells (RBCs), leading to the release of hemoglobin into the blood plasma. This process can occur naturally at the end of a red blood cell's life span or abnormally due to various factors such as

• Infections
• Autoimmune disorders
• Certain medications
• Toxins or chemicals

Fig No.1: Hemolysis

Why Does it Occur

1. Toxins: Chemicals that harm red blood cells, such as medications, heavy metals, and industrial chemicals, can cause hemolysis and cell rupture.
2. Infections: Red blood cells can be directly attacked by microorganisms such as bacteria, viruses, and parasites, or they can be destroyed by an immune reaction.
3. Autoimmune Disorders: In diseases such as autoimmune hemolytic anemia, the immune system assaults red blood cells because it believes they are foreign, leading to hemolysis.
4. Mechanical stress: Red blood cells may sustain mechanical damage from physical stress or injury, which could result in hemolysis and cell rupture.
5. Genetic Disorders: Abnormal red blood cells that are more susceptible to hemolysis can result from inherited diseases such sickle cell disease, thalassemia, and hereditary spherocytosis^.[3]

Types of Heamolysis

1. Intravascular hemolysis: The breakdown of red blood cells within the arteries is the cause of this blood-stream phenomenon.   It can be brought on by autoimmune hemolytic anemia, transfusion reactions, infections like malaria, or mechanical trauma.
2. Extravascular Hemolysis: This kind mainly affects the liver and spleen and causes red blood cells to be destroyed outside of the bloodstream.  Extravascular hemolysis is frequently caused by illnesses such sickle cell disease, thalassemia, and hereditary spherocytosis.
3. Osmotic Hemolysis: When red blood cells are exposed to fluids with an irregular osmotic pressure, they enlarge and rupture.  This is seen in diseases such as some forms of anemia and hereditary spherocytosis.
4. Immune Hemolysis: When the immune system identifies red blood cells as alien and targets them for elimination, immune-mediated hemolysis takes place.  Alloimmune reactions (such Rh incompatibility), autoimmune illnesses, or drug-induced immune responses can all be the cause of this.
5. Mechanical Hemolysis: Shear forces or physical damage that physically breaks down red blood cells can cause mechanical hemolysis.  This can occur when blood flows via artificial surfaces or constricted veins, extracorporeal circulation, or heart valve prosthesis^.[4]

Medications

1. Corticosteroids: In autoimmune hemolytic anemia, prednisone and prednisolone are frequently administered to inhibit the immune system, lowering hemolysis by lowering the formation of antibodies.
2. Immunosuppressive Drugs: When corticosteroids alone are not enough to treat refractory cases of autoimmune hemolytic anemia, medications such as rituximab, cyclophosphamide, and azathioprine may be administered.
3. Folic Acid: To enhance red blood cell synthesis and offset higher turnover, folic acid supplementation is frequently advised in hemolytic anemias.
4. Immunoglobulins: To control the immune response and lessen hemolysis, severe cases of autoimmune hemolytic anemia may benefit from intravenous immunoglobulin (IVIG) therapy.
5. Eculizumab: Used to lessen hemolysis and avoid thrombosis in patients with paroxysmal nocturnal hemoglobinuria (PNH), eculizumab is a monoclonal antibody that inhibits the complement system.
6. Iron Supplements: In order to prevent or treat iron deficiency anemia brought on by elevated red blood cell turnover, iron supplements may be required in patients with chronic hemolytic anemias.
7. Blood Transfusions: In cases of acute or life-threatening hemolysis-induced severe anemia, packed red blood cell transfusions may be necessary.
8. Chelating medicines: For hemolytic illnesses such transfusion-dependent thalassemia linked to iron overload, chelating medicines like deferoxamine or deferasirox may be utilized.
9. Hydroxyurea: In sickle cell disease, hydroxyurea can raise fetal hemoglobin levels and lower the incidence of hemolysis and vaso-occlusive crises.
10. Supportive Therapy: Hemolysis and related symptoms can be effectively managed by taking supportive measures like staying hydrated, managing discomfort, and avoiding triggers (such as certain drugs or illnesses^).[5]

Aim and Objectives

Investigating Delonix regia Maslin's hemolytic activity is the goal of the study in order to shed light on its possible medical and health ramifications.

Objectives

1. To use in vitro tests to evaluate the extract from Delonix regia Maslin's hemolytic activity.
2. To ascertain how Delonix regia extract affects red blood cell lysis in relation to concentration.
3. To investigate potential mechanisms that might be in charge of the hemolytic activity of the extract from Delonix regia, such as oxidative stress or membrane rupture.
4. To evaluate the specificity of Delonix regia's hemolytic activity by contrasting it with other recognized hemolytic agents or organic substances.
5. To aid in the investigation of Delonix regia Maslin's pharmacological properties and its potential as a source of bioactive compounds with hemolytic effect^.[6]

Review of Literature

Delonix regia, a member of the Fabaceae family, is widely known for its ornamental value and traditional medicinal applications. Various parts of the plant, including the leaves, bark, flowers, and seeds, are used in folk medicine for their anti-inflammatory, antidiabetic, antimicrobial, and antioxidant properties. Recent studies have explored the haemolytic activity of Delonix regia, which is an important parameter in evaluating the cytotoxicity and membrane-disrupting potential of plant extracts. Haemolytic activity refers to the ability of a substance to lyse red blood cells (RBCs), thereby releasing hemoglobin into the surrounding fluid. It is a crucial assay in pharmacological studies, particularly in assessing the biocompatibility and toxicity of plant extracts intended for therapeutic use.

1. Chandran et al. (2022) It was also underlined that Delonix regia is excellent for medication research because it does not exhibit considerable erythrocyte toxicity, even if it contains phytochemicals such alkaloids and flavonoids^.[13]
2. Patil et al. (2021) Highlighted the importance of evaluating haemolytic effects for all pharmacologically active plant extracts. Their study confirmed that D. regia leaf and flower extracts had low haemolytic potential when tested on human erythrocytes^.[12]
3. Pavani et al. (2021) Examined the D. regia extracts' hemolytic and anticancer properties in vitro and discovered that they were selectively cytotoxic to cancer cells while causing little harm to healthy red blood cells^.[14]
4. Adewale et al. (2020) Determined the safety threshold of Delonix regia seed extracts using a dose-dependent hemolysis assay.  The findings further supported the ethnopharmacological uses of the herb by demonstrating low RBC lysis at doses often found in traditional preparations^.[9]
5. Sowmya and Rani et al. (2019) Assessed the flower extract for antioxidant and cytotoxic activities and noted mild haemolysis, indicating that while there may be bioactivity, it does not translate into significant toxicity^.[11]

6. Nandhini et al. (2018) Tested different solvent extracts and found that chloroform and acetone extracts showed slightly higher haemolytic activity, likely due to non-polar compounds interacting with membrane lipids^.[10]
7. Ramaswamy et al. (2017): Examined and connected the saponin content of Delonix regia pods to the moderate hemolytic activity that was noted.  Natural substances called saponins are well-known for their surfactant qualities and are frequently linked to hemolytic effects.  Delonix regia levels, however, were not regarded as dangerous^.[8]
8. Swarnakar et al. (2013) : When tested against human erythrocytes, methanolic extracts of Delonix regia flowers showed a little hemolytic activity.  The study highlighted that the percentage of hemolysis stayed within acceptable bounds, indicating the relative safety of the plant extract for use in pharmaceutical applications^.[7]

Plan of Work

The present study is designed and carried out by the following methods to evulate the hemolytic activity of Delonix regia

1. Collection and Authentication of plant.
2. Preparation of ethanolic extracts of Delonix regia plant.
3. Preliminary Phytochemical Screening of Plant extract.
4. In-vitro haemolytic activity studied on plant extract.

Plant Profile:

Plant Collection and Authentication: The best time for collection of Delonix regia is generally during the flowering and fruiting seasons. Delonix regia plant were collected from Bhivghat ,sangli ,Maharashtra,India.The plant was authenticated by Mr. Tebhurne R.R. M.Sc.B.Ed Botany plant physiology.

Fig. No.2 : Delonix regia

• Common Names: Flame Tree, Royal Poinciana, Gulmohar
• Scientific Name: Delonix regia  (Bojer ex Hook.) Raf.
• Family: Fabaceae (subfamily: Caesalpinioideae)
• Origin and Distribution: Native to Madagascar. Widely cultivated in tropical and subtropical regions worldwide for ornamental purposes.

Description

• Habit: Deciduous tree, typically 10–15 meters tall.
• Leaves: Bipinnate, feathery, up to 50 cm long, with many small leaflets (10–25 pairs of pinnae, each bearing 20–40 leaflets).
• Flowers: Large, showy, bright red-orange flowers, 8–10 cm wide, with five petals—one often slightly larger with yellow and white markings.
• Fruit: Long, flat woody pods (up to 60 cm long), dark brown when mature, containing hard seeds.
• Phenology: Flowers typically bloom in late spring to early summer (depending on climate).Leaves are shed during dry seasons or just before flowering.
• Ecological Role: Provides shade due to its wide-spreading canopy. Important ornamental species in urban landscaping. Attracts pollinators such as birds and bees.

Uses:

• Ornamental: Widely planted for its vibrant flowers.
• Soil improvement: Fixes nitrogen (although to a limited extent compared to other legumes).
• Cultural significance: Symbolic in many countries, especially in parts of India and the Caribbean.

Propagation:

By seeds (which often require scarification or soaking before sowing to improve germination). Can also be propagated vegetatively via cuttings.

Growing Conditions:

• Prefers well-drained soils.
• Grows best in full sun.
• Tolerant of drought but not of cold or frost.

Cultivation and Collection:

Cultivation

Climate and Soil

• Climate: Prefers tropical and subtropical climates. Requires full sunlight for proper flowering.
• Temperature: Grows best in 20°C–35°C.
• Rainfall: Annual rainfall of 600–1,200 mm is suitable.
• Soil: Well-drained loamy soil is ideal. Tolerates slightly acidic to neutral pH (6.0–7.5).

Propagation

Seeds:

• Most common method.
• Seeds have a hard seed coat, requiring pre-treatment (scarification or hot water soaking).

Vegetative:

• Can be propagated by cuttings or grafting, but less common.
• Seed Treatment. Soak seeds in hot water (60°C) for 24 hours or mechanically scarify with sandpaper to improve germination.

Sowing

• Nursery raising is preferred.
• Sown in polythene bags or nursery beds.
• Germination takes 10–20 days.

Transplantation

• Seedlings are transplanted when they are 30–45 cm tall.
• Spacing of 6–8 m between plants is maintained.

 Maintenance

• Irrigation: Regular watering during the first 2 years.
• Fertilization: Organic manure and balanced NPK fertilizer help in early growth.
• Pruning: Necessary in early stages to develop a single trunk.

Collection

Harvesting of Seeds

Fruit type: Long, woody pod (up to 60 cm).

Time of collection: Pods mature and dry between November to January in most tropical regions.

Collection Method:

• Collect matured dry pods directly from the tree or fallen pods from the ground.
• Avoid overripe or decayed pods.

Seed Extraction

• Sun-dry the pods for a few days.
• Manually open pods to extract seeds.
• Clean and air-dry the seeds before storage.

Storage

• Store seeds in cool, dry conditions in airtight containers.
• Seeds remain viable for up to 1–2 years if stored properly^.[15]

MATERIAL AND METHODHOLOGY

1. Preparation of plant Extracts Delonix regia

Collection of Plant Material: Harvest fresh leaves, young shoots, or other plant parts of Delonix regia during the appropriate season as mentioned earlier.

Cleaning and Drying: Clean the collected plant material to remove any dirt or debris. Allow the plant material to air dry in a well-ventilated area away from direct sunlight until it is thoroughly dried. This step helps to prevent the growth of mold and bacteria during storage.

Grinding or Crushing: Once dried, grind or crush the plant material into smaller pieces using a mortar and pestle or a grinder. This increases the surface area of the plant material, facilitating the extraction process^.[16]

2. Preparation of Ethanolic Extract

The extraction preparation procedures differed slightly from those detailed in. The leaf sample wasowashed with ordinary water, alleshtly from those detailed in The blender to be ground into powder. Various ratios ored to dry, and then put into for the Soxhlet extraction procedure. 6 to 8 hours after thanol are used as gathered Utilise a muslin cloth to filter it. Centrifuge the far the extract has been gathered 15 minutes at 4,000 rpm and 25 °C. After being gathered the supernatant was retained for drying^.[17]

3. Preparation of phosphate Buffer Solution

To prepare a phosphate buffer solution, you will need to mix solutions of monobasic sodium phosphate (NaH2PO4) and dibasic sodium phosphate (Na2HPO4) in appropriate proportions to achieve the desired pH. Here's a general procedure for preparing a phosphate buffer solution^[18]

Calculate the Proportions: Determine the desired pH of the buffer solution using a buffer calculator or table. Then, calculate the appropriate proportions of monobasic and dibasic sodium phosphate solutions needed to achieve the desired pH. The Henderson-Hasselbalch equation can be used for this purpose.

Prepare Stock Solutions: Prepare stock solutions of monobasic sodium phosphate (0.2 M) and dibasic sodium phosphate (0.2 M) by dissolving the appropriate amounts of each salt in distilled water. Ensure that the salts are completely dissolved.

Mixing: Slowly add the calculated volumes of the monobasic and dibasic sodium phosphate stock solutions to a clean glass container, while stirring continuously. Continue mixing until the desired pH is reached.

Adjust pH (if Necessary): Measure the pH of the buffer solution using a pH meter or pH indicator paper. If the pH is not within the desired range, adjust it by adding small amounts of either monobasic or dibasic sodium phosphate solution as needed, and then recheck the pH.

Final Dilution (if needed): Once the pH is adjusted to the desired range, adjust the volume of the buffer solution to the final desired volume by adding distilled water if necessary. Mix thoroughly

Filtering (optional): If desired, filter the buffer solution using a sterile filter to remove any particulate matter or impurities.

Sterilization (optional): If the buffer solution is to be used for biological applications, it may be sterilized by autoclaving or filtering through a sterile membrane filter.

Storage: Store the prepared phosphate buffer solution in a clean, sterile container with an airtight lid. It can be stored at room temperature for short-term use or refrigerated for longer-term storage.

4. The process for Making Erythrocyte Cells:

 Blood samples were collected from healthy volunteer donors of blood. A sterile saline phosphate buffer solution (PBS Buffer) was used to wash the pellet obtained after centrifuging 5 ml of blood. The cell suspension was added once more to a 0.5% solution of regular saline.

5. Perform the in-vitro hemolytic activity test:

 make different concentration of solution and mixed with of the erythrocyte suspension and incubate and centrifuge it and free hemoglobin obtained, measured the absorbance using the UV-Vis spectrophotometer and calculate the % hemolysis^.[19]

Phytochemical Investigation:

Table No.1: Phytochemical constituents of Delonix regia exract.

Requirement

Chemical used:

Ethanol,streile (PBS Buffer Solution),Distilled Water,Potassium Dihydrogen Phosphate , Disodium Hydrogen Phosphate.

Apparatus Used:

Test tube, Heparin Tube,Beakers, Stirrer,5ml syringe,Soxhlet Extractor.

Equipment Used :

Centrifugation Machine, Incubator, UV-vis Spectrophotometer _[20]

Procedure:

1. Preparation of Plant Extracts Delonix regia

Fig No.3: Preparation of Plant Extract.

The extraction preparation procedures differed slightly from those detailed in. The leaf sample wasowashed with ordinary water, alleshtly from those detailed in The blender to be ground into powder. Various ratios ored to dry, and then put into for the Soxhlet extraction procedure. 6 to 8 hours after ethanol are used as gathered Utilize a muslin cloth to filter it. Centrifuge the far the extract has been gathered 15 minutes at 4,000 rpm and 25 °C. After being gathered the supernatant was retained for drying. ^[21]

2. Preliminary phytochemical screening of Extraction:

Fig No.4: Phytochemical Screening

3. Preparation of Phosphate Buffer Solution:

To prepare a phosphate buffer solution, you'll need to mix monobasic sodium phosphate (NaH2PO4) and dibasic sodium phosphate (Na2HPO4) in appropriate proportions to achieve the desired pH. Here's a general procedure:

a. Calculate Buffer Ratio: Use the Henderson-Hasselbalch equation to calculate the ratio of monobasic to dibasic sodium phosphate needed to achieve the desired pH. The equation is pH = pKa + log ([A-]/[HA]), where [A-] is the concentration of the conjugate base and [HA] is the concentration of the weak acid.

b. Prepare Stock Solutions: Prepare separate stock solutions of monobasic sodium phosphate (0.1 M) and dibasic sodium phosphate (0.2 M) by dissolving the appropriate amounts of each salt in distilled water. Ensure complete dissolution.

c. Mixing: Mix the stock solutions together in the calculated ratio to achieve the desired pH. For example, to prepare a pH 7.4 buffer, mix 100 ml of 0.1 M monobasic sodium phosphate with 400 ml of 0.2 M dibasic sodium phosphate.

d. Adjust pH (if necessary): Measure the pH of the buffer solution using a pH meter or pH indicator strips. If the pH is not within the desired range, adjust it by adding small amounts of either monobasic or dibasic sodium phosphate solution as needed, and then recheck the pH.

e. Final Dilution (if needed): Once the pH is adjusted to the desired range, adjust the volume of the buffer solution to the final desired volume by adding distilled water if necessary. Mix thoroughly.

f. Filtering (optional): If desired, filter the buffer solution using a sterile filter to remove any particulate matter or impurities.

g. Sterilization (optional): If the buffer solution is to be used for biological applications, it may be sterilized by autoclaving or filtering through a sterile membrane filter.

h. Storage: Store the prepared phosphate buffer solution in a clean, sterile container with an airtight lid. It can be stored at room temperature for short-term use or refrigerated for longer-term storage^.[22]

Fig No.5: Preparation of Phosphate Buffer.

4. Preparation of Erythrocyte Cell:     

Preparation of erythrocytic cells, also known as red blood cells, involves isolation from whole blood. Here's a general procedure:

a. Blood Collection: Collect whole blood from a suitable animal species or human donor using sterile techniques. Use anticoagulants such as EDTA or heparin to prevent blood clotting during processing.

b. Centrifugation: Transfer the collected blood into centrifuge tubes and centrifuge at low speed (e.g., 200-300 x g) for 10-15 minutes. This separates the blood into layers, with erythrocytic cells settling at the bottom

c. Plasma Removal: Carefully remove the upper layer containing plasma using a pipette or vacuum aspirator. Be careful not to disturb the erythrocytic cell layer

Fig No.6: Separation of Serum and RBCs.

d. Washing: Wash the erythrocytic cell pellet multiple times with an isotonic buffer solution (e.g., phosphate-buffered saline, PBS) to remove any remaining plasma proteins and platelets. Centrifuge the cells after each wash and carefully remove the supernatant

e. Resuspension: After the final wash, resuspend the erythrocytic cells in the desired buffer solution or medium for further experimentation. Adjust the cell concentration as needed using a hemocytometer or automated cell counter

f. Storage: Store the prepared erythrocytic cell suspension in aliquots at appropriate temperatures. Erythrocytes are typically stored at 4°C for short-term use or frozen at -20°C or -80°C for long-term storage. Avoid repeated freeze-thaw cycles to maintain cell integrity^.[23]

5. Haemolytic Activity test:

Hemolytic activity testing is a common assay used to assess the ability of substances to cause the lysis or rupture of red blood cells (erythrocytes). Here's a general overview of the hemolytic activity test

a. Preparation of Red Blood Cells (RBCs): Obtain fresh whole blood from a suitable animal species or human donor using sterile techniques. Centrifuge the blood to separate the RBCs from plasma and buffy coat.

b. Washing of RBCs: Wash the RBC pellet multiple times with an isotonic buffer solution (e.g., phosphate-buffered saline, PBS) to remove any residual plasma proteins and platelets. Centrifuge the RBCs after each wash and carefully remove the RBCs.

c. Preparation of Test Samples: Prepare the test samples containing the substance of interest at various concentrations.(25, 50, 50, 75, and 100 g/ml in the saline phosphate buffer) The substance could be a natural product extract, synthetic compound, or pharmaceutical formulation.

Fig No.7: Preparation of Test Sample

d. Incubation with RBCs: Incubate the RBC suspension with the test samples at physiological conditions (e.g., 37°C, pH 7.4) for a specific period, typically 1-2 hours.

e. Centrifugation: After the incubation period, centrifuge the RBC suspension to separate the intact RBCs (pellet) from any lysed or ruptured RBCs (supernatant).

f. Measurement of Hemolysis: Measure the absorbance of the supernatant at a suitable wavelength (e.g., 540 nm) using a UV-vis spectrophotometer. The absorbance is directly proportional to the amount of hemoglobin released, indicating the degree of hemolysis.

g. Calculation of Hemolytic Activity: Calculate the percentage of hemolysis using the formula: Hemolysis (%) = (Absorbance of test sample - Absorbance of negative control) / (Absorbance of positive control - Absorbance of negative control) × 100 ^[24,25,26]