INTRODUCTION:

Asthma is a prevalent chronic respiratory disease that significantly impacts the quality of life for numerous individuals globally.

It is a leading cause of disability worldwide, ranking 16^th in terms of years lived with disability and 28^th in terms of overall disease burden. Currently, approximately 300 million people are affected by asthma, and this number is projected to increase by 100 million by 2025¹. Despite advancements in conventional treatments, many patients still experience persistent symptoms and adverse effects². This has led to a growing interest in alternative approaches to asthma management³.

Ayurvedic medicine, an ancient Indian system of healing, offers a holistic approach that addresses the root causes of disease, including imbalances in the body's doshas. Ayurvedic interventions often involve herbal remedies, dietary modifications, lifestyle changes, and specific breathing techniques. These interventions may offer additional benefits, such as improved quality of life, reduced reliance on conventional medications, and a more holistic approach to health and wellness^4,5. While further research is needed to fully understand the mechanisms of action and long-term efficacy of Ayurvedic therapies for asthma, they may provide a valuable adjunct to conventional treatments for many patients.

Phytomedicine, a discipline rooted in ancient practices, harnesses the therapeutic potential of plants. Herbal remedies, derived from diverse plant components, have been employed for centuries. Calotropis gigantea Linn, a species belonging to the Asclepiadaceae family, has garnered significant attention in traditional medicine systems such as Ayurveda, Siddha, Unani, and Chinese medicine⁶. This plant, commonly known as Crown Flower or Giant Indian Milkweed, is widely distributed across Africa, South Asia, and India. It is a rich source of various phytochemicals, including cardiac glycosides, alkaloids, flavonoids, and saponins^7,8. Historical and contemporary research has validated the therapeutic efficacy of C. gigantea. Different parts of the plant, including seeds, leaves, and latex, have been utilized to treat a range of conditions, such as respiratory disorders, venomous bites, rheumatic ailments, dental issues, dermatological problems, and childbirth pain. Recent scientific investigations have further elucidated the underlying mechanisms of action. The plant's latex exhibits analgesic and wound-healing properties, while its flowers possess antimicrobial and cytotoxic activities. Additionally, the leaves and root bark demonstrate anti-inflammatory, hepatoprotective, anti-fertility, and anticancer effects^9,10. The current investigation aimed to explore whether different solvent extracts from Calotropis flowers could suppress histamine-induced contraction of the goat tracheal chain, clonidine-induced catalepsy in mice, and passive cutaneous anaphylaxis in rats.

MATERIALS AND METHODS:

Collection, Validation, and Extraction of Calotropis flowers:

Dr. Hemantkumar A. Thakur, at GES's HPT Arts and RYK Science Institution in Nashik, India, validated the taxonomic identity of Calotropis flower specimens collected locally from a region native to Nashik. A preserved herbarium specimen, catalogued as PS-01/2016, was prepared to serve as a reference for future botanical research. To facilitate subsequent phytochemical analysis, the dried Calotropis flowers were pulverized into a coarse powder. Soxhlet extraction was employed to extract the powdered plant material using a sequential solvent extraction method with petroleum ether, chloroform, ethanol, and water. The resulting extracts were concentrated under reduced pressure using a rotary evaporator and subsequently freeze-dried. The yield of each freeze-dried Calotropis gigantea flowers extract (CGFE) was calculated. The subsequent analysis of CGFE was primarily focused on identifying the presence of bioactive compounds¹¹.

Chemicals:

High-quality histamine dihydrochloride (HiMedia Laboratories Private Limited, India), clonidine (Unichem Laboratories Ltd, India), chlorpheniramine maleate (Alkem Laboratories, India), and dexamethasone (HiMedia Laboratories Private Limited, India) were utilized in this study.

Animals:

Adult male Wistar albino rats (weighing 150-200g) and young adult Swiss albino mice (6-8 weeks old, weighing 20-25 g) were employed in this study. All animals were housed under standard laboratory conditions, maintained at a controlled temperature of 25°C±1°C, a relative humidity of 45-55%, and a 12-hour light/dark cycle. Food and water were provided ad libitum. Prior to the commencement of the experiment, animals were acclimatized to the laboratory environment for a period of 7-10 days. 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 strict adherence to the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA)¹².

Experimental Approach:

Acute toxicity investigation:

An acute toxicity study was conducted on test extracts in accordance with OECD guideline 425, employing the up-and-down method and a maximum dose of 2,000 mg/kg body weight¹³. Rats were fasted overnight but allowed water. An oral dose of 2,000mg/kg body weight of the test extract was given to a single rat initially. Subsequently, the rat was monitored for mortality and clinical signs, including behavioral changes such as aggression, restlessness, sedation, tremors, ataxia, paralysis, convulsions, and unusual locomotion, for 72 hours post-dosing. If the rat survived, four additional rats were dosed at 48hour intervals. The total number of rats that died within 14 days and the number of survivors were recorded.

Exploration of anti-asthmatic potency of Calotropis gigantea flowers extracts (CGFE):

Histamine induced contraction of goat tracheal chain:

Tracheal tissue was procured from a local slaughterhouse and dissected into individual rings. These rings were serially connected to form a tissue chain. The tissue chain was suspended in a Krebs' solution bath and maintained at a constant temperature of 37°C±0.5°C with continual aeration (1bubble/sec.). The isolated goat tracheal chain was equilibrated in Krebs' solution for a 45minute period under a 400mg applied load. To investigate the effects of histamine and Calotropis gigantea flowers extracts (CGFE), dose-response curves (DRCs) were generated for histamine 0.5µg/mL in both plain Krebs' solution and Krebs' solution supplemented with 2.5 to 25µg/mL of CGFE. The percentage of maximum contractile response to histamine was plotted to visualize the dose-response relationships in the absence and presence of the plant extract¹⁴.

Clonidine induced catalepsy in mice:

The study employed the bar test method to evaluate the influence of Calotropis gigantea flowers extracts (CGFE) on clonidine-induced catalepsy in mice. Mice were randomly divided into ten groups of six animals each. A control group received a vehicle (1% Tween-80 solution, 5mL/kg, i.p.). Chlorpheniramine maleate (CPM, 10mg/kg, i.p.) was administered to animals of the standard group. The test groups were administered CGFE at doses of 100 and 150mg/kg, i.p., respectively. Thirty minutes post-treatment, all groups received a subcutaneous injection of clonidine (1mg/kg) to induce catalepsy. Cataleptic behavior was evaluated by measuring the latency period for mice to remove their forepaws from a horizontal bar. Observations were recorded at 30-minute intervals for a duration of 180 minutes post-clonidine administration¹⁵.

Passive cutaneous anaphylaxis in rats:

Rats were sensitized by subcutaneous injections of egg albumin on days 1, 3, and 5. Antiserum was harvested on day 10 and stored at -20°C. The rats were randomized into ten groups of six animals each. On the day of the experiment, 0.1mL of rat homologous antiserum was injected intradermally on either side of the shaved dorsal area of rats. After 48hours, the control group received a vehicle solution (1% Tween-80, 5 mL/kg, i.p.). The test groups were administered CGFE at doses of 100 and 150mg/kg, i.p. Dexamethasone (0.5 mg/kg, i.p.) was given to standard group rats. One hour post-treatment, all groups received a mixture containing 0.5% Evan blue dye and 1% (w/v) egg albumin prepared in normal saline at a dose of 0.25mL/rat, i.v. After 30 minutes, the extent of the allergic reaction was assessed by measuring the diameter of the blue spots (mm²) formed at the injection. The percentage inhibition was thereafter calculated¹⁶.

Data collection and statistical analysis:

The experimental data was presented as mean values± standard error of the mean (SEM). Statistical analysis was performed using a one-way analysis of variance (ANOVA) followed by a Dunnett's multiple comparison test. Statistical significance was determined at a p<0.05.

RESULT:

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

An analysis of different Calotropis gigantea flowers extracts (CGFE) unveiled the presence of bioactive constituents, as shown in (table 1). The percentage yield of these extracts, specifically petroleum ether (CGFPEE), chloroform (CGFCE), ethanol (CGFEE), and aqueous (CGFAE), was quantified as 6.3, 5.2, 8.9, and 14.9% w/w, respectively.

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

Sr. No.	Name of test	Observation
Sr. No.	Name of test	CGFPEE	CGFCE	CGFEE	CGFAE
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	-	-	+	+

Table 2. Effect of different freeze-dried extracts of Calotropis gigantea flowers (ECGF) on histamine induced contraction of goat tracheal chain. **p<0.01 significant compared with control group.

Sr. No.	Conc. of Histamine (µg/ml)	Conc. of extracts (µg/ml)	Height of response (mm) Mean ± SEM”				% Relaxation of goat trachea”
Sr. No.	Conc. of Histamine (µg/ml)	Conc. of extracts (µg/ml)	CGFPEE	CGFCE	CGFEE	CGFAE	CGFPEE	CGFCE	CGFEE	CGFAE
1	0.5	-	38.34±1.34	33.54±1.65	34.56±2.65	30.62±1.75	1.57	2.75	1.54	4.24
2	0.5	2.5	32.65±1.87	29.76±0.97	30.43±0.64	28.84±0.76	2.32	5.75	4.54	3.65
3	0.5	5.0	23.76±0.75	21.97±0.84	25.74±0.53	20.65±0.64	10.73	28.82	25.92	31.75
4	0.5	10.0	21.10±0.54	20.75±0.34	23.22±0.76	22.87±0.43	29.45	31.56	11.78	18.94
5	0.5	20.0	17.74±0.43	19.68±0.64	22.97±0.64	20.86±0.32	46.72	42.87	26.48	31.75
6	0.5	25.0	16.74±0.79	18.65± 0.54	21.87±0.45**	19.65±0.65	52.73	49.659	29.65	43.65
7	0.5	-	25.65±0.44	27.97±0.77	31.86±0.88	26.54±0.22	-	-	-	-

Table 3. Effect of different freeze-dried extracts of Calotropis gigantea flowers (ECGF) on Clonidine induced catalepsy in mice. n = 6 in each group. **p<0.01 significant compared with control group.

Groups	Dose mg/kg (i.p.)	“Duration of catalepsy (sec) Mean ± SEM at”
Groups	Dose mg/kg (i.p.)	30 min	60 min	90 min	120 min	150 min	180 min
Control	-	91.42 ±4.03	99.66±5.88	109.09±7.61	94.30±3.92	98.96±2.09	92.67±2.67
CGFPEE	100	89.10 ± 0.58	95.50 ± 9.26	96.00 ± 8.045	92.07 ± 1.46	96.30 ± 1.32	90.69 ± 1.39
CGFPEE	150	90.34±1.03	97.38±3.04	102.34±0.14	93.45±0.12	97.98±2.36	91.43±3.05
CGFCE	100	88.83 ± 0.75	93.44 ± 0.85	99.36 ± 16.885	90.715 ± 2.234	95.150 ± 2.23	87.00 ± 1.231
CGFCE	150	89.62±2.1	95.34±0.34	101.45±2.04	93.45±4.05	97.56±0.53	89.49±1.56
CGFEE	100	56.85 ± 0.92**	59.37 ± 0.70**	64.01 ± 1.01**	77.53 ± 0.95**	65.57 ± 1.09**	62.33 ± 1.97**
CGFEE	150	59.52±2.26**	60.32±7.32**	67.29±0.32**	79.4±0.21**	66.54±4.32**	64.42±3.21**
CGFAE	100	87.08 ± 0.99	95.94 ± 1.27	97.20 ± 1.354	91.99 ± 0.855	94.06 ± 1.18	90.22 ± 0.57
CGFAE	150	89.67±3.62	96.62±0.32	99.42±1.54	93.73±1.03	97.53±3.02	91.54±0.63
CPM	10	54.51 ± 5.85**	58.71 ± 5.77**	61.45 ± 6.45**	76.39 ± 5.54**	62.47 ± 4.82**	60.41 ± 3.15**

Passive cutaneous anaphylaxis in rats:

In the 1% Tween-80 solution-treated control group, egg albumin exacerbated local cutaneous reactions in sensitized animals, as evidenced by increased blue coloration indicative of enhanced vascular permeability. Pretreatment with Calotropis gigantea flower ethanol extracts (CGFEE) at doses of 100 and 150mg/kg i.p. significantly reduced (p<0.01) the extent of blue coloration in a dose-dependent manner. Dexamethasone (0.5mg/kg, i.p.) demonstrated a more pronounced reduction in blue coloration (p<0.001), as depicted in (table 4).

Table 4. Effect of different freeze-dried extracts of Calotropis gigantea flowers (ECGF) on passive cutaneous anaphylaxis in rats. n = 6 in each group. **p<0.01 and ***p<0.001 significant compared with control group.

Groups	Dose mg/kg (i.p.)	Area of blue dye leakage (mm²) Mean ± SEM	% Inhibition
Control	5	43.22±1.44**	-
CGFPEE	100	37.03±1.46	38.37
CGFPEE	150	38.93±1.94	37.14
CGFCE	100	34.63±1.73	36.57
CGFCE	150	37.74±1.83	28.32
CGFEE	100	14.64±1.02**	50.27
CGFEE	150	16.74±0.4**	52.52
CGFAE	100	38.03±1.75	37.56
CGFAE	150	39.17±1.94	42.57
Dexamethasone	0.5	5.38±0.152***	90.42

DISCUSSION:

Asthma is a complex, multifaceted disorder characterized by immunological, physiological, and biochemical heterogeneity. Its etiology is multifactorial, involving a range of triggers and underlying mechanisms. This study aimed to evaluate the therapeutic potential of different Calotropis gigantea flowers extracts (CGFE) on various aspects of asthma. Specifically, the research focused on the impact of CGFE on bronchoconstriction, eosinophilia, mast cell degranulation, allergy-associated inflammation, and the lipoxygenase pathway of arachidonate metabolism. These effects were assessed using both in vitro and in vivo animal models.

Bronchial asthma is a chronic inflammatory disease characterized by airway hyperresponsiveness and inflammation. This leads to recurrent episodes of wheezing, breathlessness, chest tightness, and coughing. Multiple cell types, including mast cells, eosinophils, and T lymphocytes, play critical roles in the pathogenesis of asthma¹⁷.

Mast cells and basophils, which express IgE receptors on their surface, are key players in immediate hypersensitivity reactions. Elevated IgE levels are commonly observed in allergic asthma¹⁸. Activated mast cells release a variety of inflammatory mediators, including histamine and cysteinyl leukotrienes, which contribute to the development of asthmatic symptoms. Additionally, mast cells release pro-inflammatory cytokines, such as IL-4, IL-5, and IL-13, which promote eosinophilic inflammation and IgE synthesis¹⁹.

Histamine, a pivotal mediator of allergy, inflammation, and bronchoconstriction, was the first identified substance implicated in the pathophysiological mechanisms of asthma²⁰. It is primarily synthesized and released by mast cells within the airway wall and by circulating and infiltrating basophils.

In vitro studies of tracheal and bronchial smooth muscle contraction are frequently employed to assess the contractile and dilatory responses to agonists and antagonists. Both goat tracheal chain and strip preparations are suitable models for screening drug effects on respiratory smooth muscle function^21,22. Spasmogens such as histamine, acetylcholine, and barium chloride induce dose-dependent contractions in goat tracheal chain preparations. Goat tracheal muscle expresses H₁, M₃, and β₂ receptors. Stimulation of H₁ and M₃ receptors triggers bronchial smooth muscle contraction²³. The present study demonstrates that Calotropis gigantea flowers ethanol extract (CGFEE) significantly inhibits histamine-induced contractions in isolated goat tracheal chain preparations, indicating potential antihistaminic activity.

A α₂-adrenergic agonist, clonidine, induces dose-dependent catalepsy in mice, a phenomenon attenuated by H₁ histamine receptor antagonists but not H₂ antagonists²⁴. Similarly, intracerebroventricular administration of histamine to conscious mice elicits catalepsy, which is also inhibited by H₁ antagonists²⁵. Clonidine is known to induce histamine release from mast cells, and histamine-containing mast cells have been identified in the mouse brain; it is plausible that clonidine-induced catalepsy is mediated by histamine acting on H₁ receptors. The current study demonstrates that pretreatment with CGFEE and chlorpheniramine maleate significantly inhibits clonidine-induced catalepsy in mice. This suggests that the anti-cataleptic effect of Calotropis gigantea may be attributable to its H₁ antihistaminic activity.

The allergic reactions induced by passive cutaneous anaphylaxis (PCA) are predominantly mediated by IgE-dependent mechanisms, involving increased vascular permeability and histamine release from mast cells^27,28. It is well-established that cutaneous reactions, both immediate and delayed-type hypersensitivity, involve intricate interplay between inflammatory and immunological processes. In the present study, CGFEE and dexamethasone treatment demonstrated significant protective effects against IgE-mediated local allergic skin reactions elicited by PCA. These findings suggest that CGFEE possesses potential to modulate anti-allergic responses and may offer therapeutic benefits in the management of IgE-mediated anaphylactic reactions.

CONCLUSION:

The present investigation indicates that Calotropis gigantea flowers ethanol extract (CGFEE) exhibits substantial anti-asthmatic properties. This effect can be attributed to its potential anti-allergic, bronchodilatory, anti-histaminic, and anti-cataleptic activities. These findings corroborate traditional claims regarding the therapeutic benefits of Calotropis gigantea. The synergistic action of steroids, saponins, and flavonoids present in CGFEE likely contributes to these pharmacological effects.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

ACKNOWLEDGMENTS:

The authors express their sincere appreciation 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 necessary infrastructure and facilities that enabled the successful execution of this research study. Additionally, the authors wish to extend their heartfelt thanks to Dr. Hemantkumar A. Thakur, Head of the PG Department of Botany at GES's HPT Arts and RYK Science Institution, Nashik, for his invaluable expertise and assistance in authenticating the plant materials utilized in this investigation.

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Received on 02.12.2024 Revised on 19.03.2025

Accepted on 15.05.2025 Published on 13.01.2026

Available online from January 17, 2026

Research J. Pharmacy and Technology. 2026;19(1):466-471.

DOI: 10.52711/0974-360X.2026.00068

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