INTRODUCTION:

Asthma, a chronic respiratory disease, is a global health concern with increasing prevalence, especially in industrialized nations. It is estimated that approximately 300 million individuals are currently affected worldwide, with projections indicating a further 100 million cases by 2025^1,2. The pathophysiology of asthma is characterized by airway inflammation and narrowing, resulting from the dysregulation of immune responses, infiltration of inflammatory cells (such as mast cells and lymphocytes), and the release of pro-inflammatory mediators (including cytokines). Inflammation, a fundamental immune response, plays a pivotal role in the progression of chronic diseases. Triggered by harmful stimuli, this response manifests as a complex series of events, characterized by pain, edema, redness, and heat. During inflammation, blood vessels dilate, and immune cells, including neutrophils, macrophages, and lymphocytes, accumulate at the site of injury. A key component of this process involves the release of arachidonic acid, a polyunsaturated fatty acid, from ruptured cell membranes³.

Phytomedicine, a field rooted in ancient practices, leverages the therapeutic potential of plants. Herbal remedies, derived from diverse plant parts including flowers, leaves, roots, bark, fruits, and seeds, have been employed for centuries⁴. Calotropis gigantea Linn, a member of the Asclepiadaceae family, revered in traditional medicine systems like Ayurveda, Siddha, Unani, and Chinese medicine, has garnered significant attention. This plant, commonly known as Crown Flower and Giant Indian Milkweed, is widely distributed across Africa, South Asia, and India. It is a rich source of phytochemicals, including cardiac glycosides, madrine, β-sitosterol, saponins, tannins, alkaloids, flavonoids, cardenolides, benzoylinesolone, and calotropons^5,6.

Historical and contemporary research has validated the therapeutic potential of Calotropis gigantea. Various plant parts, including seeds, leaves, and latex, have been utilized to treat a range of ailments, such as asthma, snakebites, rheumatic conditions, dental problems, skin disorders, and childbirth pain. Recent scientific investigations have further elucidated the underlying mechanisms of action. The plant's latex exhibits analgesic and wound-healing properties, its flowers possess antimicrobial and cytotoxic activities, and its leaves and root bark demonstrate anti-inflammatory, hepatoprotective, anti-fertility, and anticancer effects^7,8. This research aimed to investigate whether different solvent extracts of Calotropis flowers could protect against milk-induced leukocytosis and eosinophilia, egg albumin-induced mast cell degranulation in mice, and carrageenan-induced acute paw edema in rats.

MATERIALS AND METHODS:

Procurement, Authentication, and Extraction of Calotropis flowers

Dr. Hemantkumar A. Thakur, affiliated with the Department of Botany at GES's HPT Arts and RYK Science Institution, Nashik, Maharashtra, India, authenticated and verified the taxonomic identity of Calotropis flower specimen sourced from the indigenous Nashik region. This meticulously preserved herbarium specimen, catalogued as PS-01/2016, will serve as a valuable resource for future botanical research and reference. To facilitate subsequent phytochemical analysis, the dried flowers were coarsely powdered using a pulverizer. A Soxhlet extraction method was employed to extract the powdered flowers with different solvents such as petroleum ether, chloroform, ethanol, and water. The extracts were concentrated under reduced pressure using a rotary evaporator and subsequently freeze-dried. The yields of the freeze-dried extracts of Calotropis gigantea flowers (ECGF) were calculated. The subsequent analysis of ECGF was primarily focused on identifying the presence of bioactive components⁹.

Chemicals

Egg albumin, aluminum hydroxide, Evans blue disodium cromoglycate, dexamethasone, RPMI-1640 buffer, toluidine blue, carrageenan, and indomethacin were purchased from HiMedia Laboratories Private Limited, Mumbai, Maharashtra, India.

Animals

Adult male Wistar albino rats (150-200 g) and young adult Swiss albino mice (6-8 weeks old, 20-25 g) were used in this study. The animals were housed under standard laboratory conditions with a controlled temperature of 25°C ± 1°C, relative humidity of 45-55%, and a 12-hour light/dark cycle. Food and water were provided ad libitum. The animals were acclimatized to the laboratory environment for 7-10 days prior to the initiation of the experiment. The experimental protocol (MSG/PC/CPCSEA/II/2017/02) was approved by the Institutional Animal Ethics Committee (IAEC) of GES's Sir Dr. M. S. Gosavi College of Pharmaceutical Education and Research, Nashik, and was conducted in accordance with the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA)¹⁰.

Methodology for Experimental Investigation

Study on acute toxicity

An acute toxicity study of ECGF was conducted adhering to OECD Guideline 425¹¹. The primary aims were to determine the median lethal dose (LD50) and establish appropriate dose ranges for subsequent studies. Rats were orally administered varying doses of ECGF suspended in 1% CMC solution and monitored for 72 hours for signs of toxicity and mortality.

Investigation of anti-asthmatic potency of extracts of Calotropis gigantea flowers (ECGF)

Milk-induced leukocytosis and eosinophilia

Mice were randomly divided into ten groups of six animals each. Blood samples were collected from the retro-orbital plexus. The control group received a vehicle (1% Tween-80 solution, 5 mL/kg, i.p.). The test groups were administered ECGF at doses of 100 and 150 mg/kg, i.p., respectively. The standard group received dexamethasone (50 mg/kg, i.p.). Thirty minutes post-treatment, all groups were injected subcutaneously with 4 mL/kg of boiled and cooled milk. Total leukocyte and eosinophil counts were determined in each group both before treatment and 24 hours post-milk injection. The change in these cell counts was calculated to assess the anti-inflammatory effect of ECGF¹².

Egg albumin-induced mast cell degranulation

Mice were randomly divided into ten groups of six animals each. A three-day treatment regimen was implemented. The control group received a vehicle (1% Tween-80 solution, 5 mL/kg, i.p.). The test groups were administered ECGF at doses of 100 and 150 mg/kg, i.p., respectively. A standard anti-allergic drug, sodium cromoglycate (50 mg/kg, i.p.), was administered to animals of the standard group. On the fourth day, all mice underwent intraperitoneal injection of saline solution. Peritoneal fluid containing mast cells was collected after five minutes and subsequently centrifuged to isolate the mast cells. The isolated mast cells were washed with RPMI-1640 buffer and exposed to egg albumin (100 mg/mL) for a 10-minute incubation period at 37°C. Following incubation, the cells were stained with 1% toluidine blue and examined microscopically under 10x magnification. Degranulation, characterized by morphological changes indicative of cell rupture, was quantified by counting 100 cells per sample. The percentage of degranulated cells in each group was calculated to assess the inhibitory efficacy of the treatments¹³.

Investigation of anti-inflammatory potency of extracts of Calotropis gigantea flowers (ECGF)

Carrageenan-induced acute paw edema

Rats were randomly divided into ten groups of six animals each. A three-day treatment regimen was implemented. The control group received a vehicle (1% Tween-80 solution, 5 mL/kg, i.p.). The test groups were administered ECGF at doses of 100 and 150 mg/kg, i.p., respectively. To the animals of the standard group, indomethacin (10 mg/kg, p.o.), a standard anti-inflammatory drug, was administered. Thirty minutes post-extract administration, a 0.1 mL volume of 1% w/v carrageenan solution was subplantarly injected into the right hind paw of each rat¹⁴. The contralateral (left) paw served as an untreated control. Paw volumes of both paws were measured at 1, 2, and 3 hours post-carrageenan injection for control, standard, and extract-treated test groups. The percentage inhibition of edema for each rat and group was determined using the following formula:

% Inhibition of edema = [(Vc - Vt) / Vc] × 100

Where: Vc: increase in paw volume of the control group, and Vt: increase in paw volume of the test group.

Data collection and analysis

Data were presented as mean values ± standard error of the mean (SEM) and analyzed using a one-way analysis of variance (ANOVA) in GraphPad Prism 9.0. To further investigate group differences, a post-hoc Dunnett's multiple comparison test was conducted. Differences were considered statistically significant at a p<0.05.

RESULT:

The percentage yield of the extract and bioactive constituent’s analysis

Analysis of different extracts of Calotropis gigantea flowers (ECGF) revealed the presence of bioactive components, as shown in (table 1). The percentage yields of freeze-dried petroleum ether (PECGF), chloroform (CECGF), ethanol (EECGF), and aqueous (AECGF) extracts of Calotropis gigantea flowers were found to be 6.3, 5.2, 8.9, and 14.9% w/w, respectively.

Table 1. The percentage yield of different freeze-dried extracts of Calotropis gigantea flowers (ECGF)

Sr. No.	Name of test	Observation
Sr. No.	Name of test	PEC GF	CEC GF	EEC GF	AEC GF
1.	Test for Steroids
	Salkowski test	-	-	+	+
	Liebermann test	-	-	+	+
	Liebermann-Burchard test	-	-	+	+
2.	Test for Saponins
2.	Foam formation test	-	-	+	+
3.	Test for Alkaloids
	Dragendorff’s test	+	+	-	-
	Mayer’s test	+	+	-	-
	Wagner’s test	+	+	-	-
	Hager’s test	+	+	-	-
4.	Test for Glycoside
	Modified Borntrager’s test	-	+	+	+
	Legal’s Test	-	+	+	+
	Keller-Killiani Test	-	+	+	+
5.	Test for Flavonoids
	Shinoda Test	-	+	+	-
	Alkaline Reagent Test	-	+	+	-
6.	Test for Tannins
	Ferric Chloride test	+	-	+	+
	Lead Acetate test	+	-	+	+
	Potassium Dichromate test	+	-	+	+
7.	Test for Proteins
	Biuret test	-	-	-	+
	Xanthoproteic test	-	-	-	+
	Millon’s test	-	-	-	+
8.	Test for Amino Acids
8.	Ninhydrin test	-	+	-	-
9.	Test for Carbohydrates
	Molisch test	-	-	+	+
	Barfoed’s test	-	-	+	+
	Felhing’s test	-	-	+	+

Acute toxicity study

Administering suspensions of petroleum ether (PECGF), chloroform (CECGF), ethanol (EECGF), and aqueous (AECGF) extracts of Calotropis gigantea flowers in 1% CMC solution orally for 72 hours at a concentration of 1% (w/v) did not cause any mortality or signs of toxicity in the studied animals. While the OECD recommends a maximum tolerated dose of 2000 mg/kg for extracts of Calotropis, the chosen dosages (100 and 150 mg/kg) were considered suitable for investigating the potential effects of Calotropis in the context of asthma and inflammation.

Milk-induced leukocytosis and eosinophilia

The most pronounced increase in leucocyte (4575±114.28) and eosinophil (130.15±3.53) counts was observed in the control group 24 hours post-administration of milk (4 mL/kg, subcutaneously). EECGF, at doses of 100 and 150 mg/kg, significantly inhibited milk-induced leucocytosis and eosinophilia in a dose-dependent manner. EECGF at doses of 100 and 150 mg/kg demonstrated significant inhibitory effects comparable to dexamethasone (50 mg/kg), as illustrated in (table 2 and 3).

Table 2. Effect of different freeze-dried extracts of Calotropis gigantea flowers (ECGF) on milk-induced leucocytosis in mice.

Treatments	Dose (i.p.) mg/kg	Difference in no. of leucocytes (Before and after treatment) (Mean ± SEM) “
Control (1% Tween-80)	5 ml/kg	4575 ± 114.28
PECGF	100	3762±142.43
PECGF	150	3952±162.52
CECGF	100	3852±132.64
CECGF	150	3964±173.64
EECGF	100	2733.33±249.56**
EECGF	150	2816.67±145.87**
AECGF	100	3410±201.11
AECGF	150	3782±157.19
Dexamethasone	50	2600.00±11**

n = 6 in each group. **p<0.01 significant compared with control group.

Egg albumin-induced mast cell degranulation

The control group exhibited substantial mast cell degranulation (72.15±1.521). In contrast, pretreatment with EECGF at a dose of 100 and 150 mg/kg and sodium chromoglycate significantly attenuated mast cell degranulation, as depicted in (table 4).

Table 3. Effect of different freeze-dried extracts of Calotropis gigantea flowers (ECGF) on milk-induced eosinophilia in mice.

Treatments	Dose (i.p.) mg/kg	Difference in no. of eosinophils (Before and after treatment) (Mean ± SEM) “
Control (1% Tween-80)	5 ml/kg	130.15±3.53
PECGF	100	114±161.2
PECGF	150	143±145.42
CECGF	100	121±173.15
CECGF	150	126±212.14
EECGF	100	97±138.43**
EECGF	150	98±127.39**
AECGF	100	124±152.83
AECGF	150	125±167.43
Dexamethasone	50	95.17±2.75**

n = 6 in each group. **p<0.01 significant compared with control group.

Table 4. Effect of different freeze-dried extracts of Calotropis gigantea flowers (ECGF) on egg albumin-induced mast cell degranulation in mice.

Treatments	Dose (i.p.) mg/kg	Number of degranulated mast cells (Mean ± SEM)	% Protection of mast cells“
Control (1% Tween-80)	5 ml/kg	72.15±1.52	-
PECGF	100	56.21±21.54	31.23
PECGF	150	59.29±2.14	35.47
CECGF	100	53.45±3.24	34.62
CECGF	150	55.93±3.02	32.12
EECGF	100	30.32 ±0.13**	43.21
EECGF	150	31.41 ±1.36**	60.63
AECGF	100	52.31±2.31	49.58
AECGF	150	54.72±3.72	46.03
Sodium chromoglycate	50	27.32±1.02**	66.42

n = 6 in each group. **p<0.01 significant compared with control group.

Carrageenan-induced acute paw edema

The control group exhibited a substantial increase in paw volume at 1 (0.45±0.01), 2 (0.53±0.02), and 3 (0.60±0.03) hours post-carrageenan injection. In contrast, pretreatment with CECGF and EECGF at doses of 100 and 150 mg/kg and indomethacin showed significant reductions in paw volumes, as depicted in (table 4).

DISCUSSION:

This study evaluated the efficacy of different freeze-dried extracts of Calotropis gigantea flowers (ECGF) administered at doses of 100 and 150 mg/kg, i.p., in managing asthma-like symptoms in mice. Asthma is a chronic inflammatory disease characterized by airway inflammation, mucus hypersecretion, and airway hyperresponsiveness. Milk-induced leukocytosis and eosinophilia serve as a model for studying asthma pathogenesis^15,16. Leukocytes and eosinophils play a crucial role in the inflammatory process of asthma. Leukocytes release inflammatory mediators, including cytokines, histamine, and major basic protein, which contribute to airway inflammation. Additionally, leukocytes can generate reactive oxygen species, leading to oxidative stress and tissue damage^17,18. Eosinophils, another type of white blood cell, are involved in mucus hypersecretion and airway hyperresponsiveness¹⁹. The study demonstrated that ethanolic extract of Calotropis gigantea flowers (EECGF) treatment significantly reduced the milk-induced increase in leukocyte and eosinophil counts. This suggests that EECGF may possess anti-inflammatory properties, potentially by mitigating oxidative stress and modulating the immune response.

Anaphylactic reactions are severe, life-threatening allergic responses characterized by the rapid release of inflammatory mediators, including histamine and pro-inflammatory cytokines. These reactions can be triggered by various allergens, such as egg albumin. EECGF has demonstrated potential anti-allergic properties, particularly in inhibiting the release of inflammatory mediators and protecting mast cells from degranulation. This mechanism of action may be attributed to the presence of bioactive compounds, such as saponins and flavonoids, within its composition. Saponins have been well-documented for their mast cell-stabilizing activity^20,21, while flavonoids, including apigenin^22,23 and luteolin^24,25, have been shown to exhibit anti-inflammatory effects by inhibiting the release of histamine and other inflammatory mediators from immune cells. The current findings suggest that EECGF significantly attenuated mast cell degranulation and may be effective in mitigating type I hypersensitivity reactions, making it a promising candidate for further investigation as a potential therapeutic agent.

The carrageenan-induced rat paw edema model is a well-established experimental model for evaluating the anti-inflammatory potential of drugs. Carrageenan, a potent inflammatory agent, induces edema by stimulating the release of various inflammatory mediators, including prostaglandins, leukotrienes, histamine, bradykinin, and tumor necrosis factor-alpha (TNF-α)^26,27. Acute inflammation is biphasic²⁸. The early phase, characterized by the release of histamine, serotonin, and kinins, occurs within the first few hours post-carrageenan injection. The late phase, dominated by the release of prostaglandin-like substances, begins 2 to 3 hours later. The late phase is particularly sensitive to both steroidal and non-steroidal anti-inflammatory drugs. Pre-treatment with EECGF and chloroform extract of Calotropis gigantea flowers (CECGF) showed significant reduction in paw volumes. Both the EECGF and CECGF extracts also increased the percentage inhibition of edema.

CONCLUSION:

The presence of active bioconstituents like steroids, saponins, and flavonoids in that ethanolic extract of Calotropis gigantea flowers (EECGF) strengthened the anti-asthmatic and anti-inflammatory potencies of Calotropis in milk-induced leukocytosis and eosinophilia and egg albumin-induced mast cell degranulation in mice and the carrageenan-induced acute paw edema in rats, respectively. Furthermore, chloroform extract of Calotropis gigantea flowers (CECGF) exhibited significant anti-inflammatory activity against carrageenan-induced inflammation as evidenced by the presence of flavonoids.

CONFLICT OF INTEREST:

The authors affirm the absence of any conflicting interests.

ACKNOWLEDGMENTS:

The authors express their gratitude to the Department of Pharmacognosy and Department of Pharmacology, GES's Sir Dr. M. S. Gosavi College of Pharmaceutical Education and Research, Nashik, for providing the essential infrastructure that facilitated the conduct of this study. Additionally, the authors would like to extend their sincere thanks to Dr. Hemantkumar A. Thakur, Head of the Postgraduate Department of Botany at GES's HPT Arts and RYK Science Institution, Nashik, for his invaluable assistance in authenticating the plant materials utilized in this research.

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Received on 02.09.2024 Revised on 10.10.2024

Accepted on 02.12.2024 Published on 28.01.2025

Available online from February 27, 2025

Research J. Pharmacy and Technology. 2025;18(2):671-676.

DOI: 10.52711/0974-360X.2025.00099

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

Treatments	Dosage (i.p)	Paw volume (ml) Mean ± SEM			% Inhibition of edema
Treatments	Dosage (i.p)	1 hr	2hr	3hr	1 hr	2hr	3hr
Control (1% Tween-80)	5 ml/kg	0.45±0.01	0.53±0.02	0.60±0.03	-	-	-
PECGF	100	0.37±0.03	0.46±0.02	0.47±0.04	17.34	23.64	22.56
PECGF	150	0.38±0.01	0.47±0.01	0.49±0.02	19.84	29.54	39.84
CECGF	100	0.41±0.02*	0.42±0.01*	0.54±0.01*	20.68	25.46	26.85
CECGF	150	0.42±0.01*	0.45±0.01*	0.56±0.01*	24.93	34.45	28.62
EECGF	100	0.26±0.42**	0.38±0.03**	0.34±0.04**	29.46	27.62	28.21
EECGF	150	0.28±0.40**	0.39±0.04**	0.35±0.05**	30.48	28.43	29.72
AECGF	100	0.43±0.08	0.48±0.01	0.53±0.01	18.44	17.26	24.57
AECGF	150	0.44±0.05	0.50±0.01	0.55±0.02	21.73	15.73	28.72
Indomethacin	10 mg/kg, p.o.	0.24±0.02**	0.36±0.03**	0.33±0.02**	15.43	16.54	20.54