INTRODUCTION:

Asthma:

An inflammatory respiratory condition, asthma generally manifests as a chronic form. The symptoms include abrupt flare-ups of dyspnea, hacking, difficulty breathing and chest stiffness, commonly in the late evening or early morning. Additionally, it causes the bronchi to become a person's expiratory volume and airflow are usually diminished. hyperresponsive and irritates the respiratory tract. They are not limited to asthma sufferers. Among the several respiratory conditions, bronchial asthma is the most severe. Globally, the rate of both illness and mortality is steadily increasing. The number of individuals with asthma is increasing, necessitating more scientific research on different drugs, their mechanisms of action and the underlying mechanisms of asthma.¹

The ailment affects over 10–15% of people living in developed countries. Experts generally agree that its prevalence and severity are growing.²

Airway blockage is reversible which contributes to the remission of symptoms associated with asthma attacks. In rare cases, status asthmaticus can lead to death due to chronic airway blockage. Asthma causes airway blockage through smooth muscle spasm and micro-vascular leakage resulting in edema and buildup of inflammatory cells and by-products in the airways. These inflammatory alterations can exacerbate smooth muscle contraction and micro-vascular leakage. Asthma is characterized by airway inflammation including the invasion of leukocytes. Asthma sufferers experience airway inflammation which differs from other respiratory disorders with thicker basement mucosal membranes.³Asthmatic individuals' airways contains inflammatory cells which may contribute to airway blockage. Mast cells in the airways have a role in the early asthmatic response, releasing mediators after bridging with IgE receptors.⁴

Bronchial asthma is characterized by heightened sensitivity of the trachea-bronchial tree to various stimuli. Asthma's main symptoms are dyspnea, wheeze and coughing episodes that can range from mild and practically undetectable to severe and unrelenting. Hyperresponsiveness manifests as fluctuating airway blockage.

In fatal cases of asthma, the pre-terminal and terminal bronchioles exhibit five distinct structural changes.

Symptoms include like basophilic, polysaccharide-rich mucus plugs in terminal bronchioles, increased goblet cells, thickened and irregular basement membrane, hypertrophied smooth muscles in pre-terminal bronchioles, an inflammatory infiltrate of mononuclear cells, Eosinophilic granulocytes in the submucosa and between hypertrophied bronchioles.⁵ Between 100 to150 million individuals worldwide suffer from asthma. This portion of the data shows that over 190,000 people die each year from asthma-related causes and the number of patients with the ailment is rising globally. There are reportedly 15–20 million asthmatics in India. One of the reasons for the higher occurrence in children is genetic susceptibility.⁶Types of asthma included Extrinsic asthma Intrinsic asthma, Mixed⁷. Sida veronicafolia (Lam) is a well-known herb used in Ayurvedic and Siddha treatment. The plant is also referred to as Nagabala, Bhumibala, Shaktibala, etc. in Ayurveda.⁸ A little, thin, straggling plant that roots at nodes and frequently covers with the superficial soil is Sida Veronicafolia (Lam.), a member of the Malvaceae family. Measurements: 2–5cm long, up to 4cm broad, broadly oval, serrated. With unicellular and multicellular trichomes that irritate the skin, the stem and leaves are both highly hairy.⁹

MATERIALS AND METHODS:

Plant Material:

Leaves of Sida veronicafolia (Lam) was acquired in massive quantities from college campous area and authenticated by Department of Botany, Y.C. Institute of Science, Satara. The leaves of Sida veronicafolia (Lam) were washed with the distilled water to remove dirt and soil. It was further shade dried and then coarsely powered.

Preparation of Sida veronicafolia as a test drug:

The powder of leaves of Sida veronicafolia was subjected to successive soxhlet extraction with solvents of increased polarity. The ethanolic extract was selected for the present study. The extract was concentrated using rotary flash evaporator and stored at room temperature.

Route and drug administartion:

As earlies report stated that EELSV was safe and that there was no animal mortality. Therefore, using the fixed dosage approach of CPCSEA, 2500miligram per kilogram was determined to be the LD50 cut off value.¹⁰ Therefore for antiasthmatic activity, the dosage of 100, 200, 400milligram per kilogram per orally were selected.

Animals:

For the investigation, healthy albino mice weighing 20–30g and goat trachea were employed. The mice were kept under conventional circumstances, with 12 hours of light and dark cycles, 25±30⁰C temperature and 35–60% humidity, in polypropylene cages. They were feed and water ad libitum. The study was approved by the Institutional Animal Ethical Committee of YTC college of Pharmacy, Satara, Registered under CPCSEA, India. Animal ethical certificate no.-1915/PO/ReBi/S/16/CPCSEA 04/11/2016.

Screening Methods for asthma:

1. Isolated goat tracheal chain preparation:

Fresh Goat trachea was take from slaughter when the immediately cut the goat. Four tracheas in each drug sample were used for the study. The trachea was kept in aeration-by-aeration tube containing O_2.Aeration was continued till the trachea was cut in transverse series to form the chain then trachea was tied by thread. It was kept in bath suspended Contained Krebs’s solution maintained at 37±1⁰c stream of CO, in O₂ was bubble that passes through the organ tube. The speed of rotating drum was maintained at 0.12mm per sec. The load was given 400mg of china clay and tissue was given 45 minutes to equilibrate. The comparatively graph of histamine concentration 100µg/ml and the test drug i.e Sida veronicafolia at concentration 250µg/ml and 500µg/ml. The height of response was measured in cm.¹¹

2. Clonidine induced catalepsy in mice:

The bar test was used. To find out how test drug extracts, especially Sida veronicafolia, affect catalepsy induced on by clonidine, researchers employ the bar test. Five groups of mice, with six mice in each group. Group I animals received the vehicle (10 milligraml/kg, p.o.) and acted as the control group. Animals in group II were given standard drugs 10mg/kg of chlorpheniramine maleate, intraperitoneally. The ethanolic extract of Sida veronicafolia (Lam) leaves was given to the animals in groups III, Group IV, Group V at three different doses: (100, 200, and 400milligramg/kg per orally). A horizontal bar with a diameter of 1cm and a height of 3 cm above the table was used to hold the mice's forepaws, and each animal's time to remove its paws from the bar was recorded. Clonidine (1 milligramg/kilogramg, sub cutaneous.) was administered to all groups 1 hour following the test drug administration, and the time of catalepsy was recorded at 15, 30, 60, 90, 120, 150, and 180 minutes.¹²

Statistical Analysis:

Values were in mean±SEM for six animals in the each group and statistical significant differences between mean values were determined by two way of the analysis of variance (ANOVA) followed by a Tukeys test for the post-hoc multiple comparision tests. Graph pad prism instant 102.3(403) was used and p<0.05, p<0.01, p<0.001 was be the considered to be significant.

RESULTS AND DISCUSSION:

In tracheal chain preparation of goat, response as follows:

Table 1: Response of Histamine at con.100 µg/ml in isoalated goat tracheal chain preparation.

Sr. No.	Dose (ml)	Height (Cm)	Drug con(µg/ml)	% max. Response
1	0.1	0.4	100 µg/ml	21.05
2	0.2	0.5	100 µg/ml	26.31
3	0.4	1	100 µg/ml	52.63
4	0.8	1.7	100 µg/ml	89.47
5	1.6	1.9	100 µg/ml	100

Table 2: Response of Sida veronicafolia at concentration 250µg/ml in isoalated goat tracheal chain preparation.

Sr. No.	Dose (ml)	Height (Cm)	Drug con(µg/ml)	%max. Response
1	0.1	0.31	250 µg/ml	25.83
2	0.2	0.5	250 µg/ml	41.66
3	0.4	0.7	250 µg/ml	58.33
4	0.8	0.91	250 µg/ml	75.83
5	1.6	1.2	250 µg/ml	100

Table 3: Response of Sida veronicafolia at concentration 500 µg/ml in isoalated goat tracheal chain preparation.

Sr. No.	Dose (ml)	Height (Cm)	Drug con(µg/ml)	%max. Response
1	0.1	0.2	500 µg/ml	23.33
2	0.2	0.4	500 µg/ml	45.55
3	0.4	0.7	500 µg/ml	77.77
4	0.8	0.83	500 µg/ml	92.22
5	1.6	0.9	500 µg/ml	100

Figure 1: Graphical representation of Effect of ethanolic extract of leaves of Sida veronicafolia (Lam) and Histamine on tracheal chain preparation

Table 4: Clonidine induced catalepsy in mice:

Sr. No.

Group(s)

Time of catalepsy in mice (sec)

15min

30min

60min

90min

120min

150min

180min

Control

41.555

±0.35

48.19

±0.32

67.07

±0.39

72.31

±0.26

83.15

±0.28

106.78

±1.04

68.88

±0.38

Standard

17.42

±0.15***

18.50

±0.15***

21.01

±0.13***

23.59

±0.18***

27.62

±0.16***

12.04

±0.24***

8.29

±0.27***

EESV

100 µg/ml

24.19

±0.09***

38.61

±0.17***

35.57

±0.21***

36.01

±0.03***

45.28

±0.12***

48.33

±0.17***

37.95

±0.17***

EESV

200 µg/ml

26.75

±0.15***

39.24

±0.26***

31.92

±0.34***

30.44

±0.19***

37.47

±0.18***

30.62

±0.19***

21.70

±0.18***

EESV

400 µg/ml

26.36

±0.15***

33.02

±0.03***

35.25

±0.14***

35.41

±0.16***

36.55

±0.15***

25.47

±0.19***

23.49

±0.14***

N=5; values are expressed in mean±SEM, Control: Distilled Water (10ml/kg, p.o); Standard: Chlorpheniramine maleate (10mg/kg, i.p); EESV100: ethanolic extract of Sida veronicafolia (100mg/kg, p.o); EESV200: ethanolic extract of Sida veronicafolia (200mg/kg, p.o) EESV400: ethanolic extract of Sida veronicafolia (400mg/kg, p.o). Statistical analysis done by ANOVA followed by tukeys test. (**P<0.01, ***p<0.001) compared with control group

DISCUSSION:

Asthma is a widespread and chronic inflammatory disorder of the airways that is not fully understood. As a result of inflammation, the airways become hyper-responsive and narrow easily in reaction to a variety of stimuli. Symptoms of this condition include coughing, wheezing, chest tightness and shortness of breath which might worsen at night. Although airway narrowing is normally reversible, in certain people with severe asthma, inflammation can cause irreversible airflow restriction. Pathological aspects of the airway include inflammatory cells, plasma exudation, edema, smooth muscle hypertrophy, mucus clogging, and epithelial shedding.¹³

The present study deals with anti- asthmatic activity of ethanolic extract of leaves of Sida veronicafolia (lam), by using various screening methods like isolated goat tracheal chain preparation (In vitro) and clonidine induced catalepsy in mice (in vivo). The biological mediator histamine has a role in the development of allergies, inflammation, and primary bronchoconstriction. It narrows the lumen of the airways produces mast cells, leukotrienes, prostaglandins, and cytokines. Histamine muscarinic and beta-adrenergic receptors are present in the goat tracheal smooth muscle. The contractile agents employed in drug bioassays are acetylcholine, histamine and serotonin. Broncho-constriction can be directly caused by some mediators. For fast relief in situations like acute asthma episodes, spasmolytic drugs such as beta-adrenergic agonists, xanthine derivatives and cholinolytics are utilized. Inflammatory mediators such as histamine, acetylcholine, leukotrienes, and prostaglandins are released during the early stages of asthma and are brought on by allergens, irritants, cold air, or physical activity.¹⁴

In goat tracheal chain, ethanolic extract of Sida veronicafolia at dose 250μg/ml and 500μg/ml dose gives decrease in contractile response as compared to Histamine 100μg/ml. Four tracheas were used for each of drug sample and responses measured. The height of response compared and maximum contractile response measured. The 500µg/ml of EESV gave the decrease in response as compared to other two concentrations. The approach is used to study how antispasmodic drugs affect the tracheal musculature. The reason for this is that the tracheal and bronchial muscles have a strong anatomical and physiological relationship. Goat tracheal chain is far more sensitive and it is also simpler to handle and prepare.¹⁵

In catalepsy by clonidine, Animals that hold an imposed posture for extended periods of time before reverting to their natural posture are said to be suffering from catalepsy. In addition, catalepsy that block dopaminergic transmission or increase the inhibitory neurotransmitter histamine in the brain. α2-adrenoreceptor agonist clonidine induces dose-dependent catalepsy in mice, which is inhibited by histamine H1 receptor antagonists but not H2 receptor antagonists¹⁶. Intracerebroventricular (i.c.v.) injections of histamine deplete transmitter storage in nerve terminals, which alters pre-synaptic catecholamine processes in the central nervous system (CNS) of conscious mice. Rather than the H2 receptor blocking drug metiamide, the H1 receptor antagonist chlorcyclizine suppresses catalepsy. The brain has mast cells that produce histamine. There is no question that brain histamine helps to create the extra pyramidal motor symptoms associated with catalepsy. Clonidine's cataleptic action in mice may be mediated by histamine (via H1 receptors) released from brain mast cells when α2 adrenoreceptors are stimulated by the drug.¹⁷ The bar test is the most efficient way to evaluate how a test drug affects clonidine-induced catalepsy. The horizontal bar that is 3 cm above the table and has a diameter of about 1 cm must be where the mice's forepaws are placed. The amount of time it takes each animal to take its paws off the bar has to be noted both before and one hour after the test drug is given. Moreover, measurements of the catalepsy's duration must be taken every 15, 30, 60, 90, 120, 150, and 180 minutes. An assessment is conducted by contrasting the duration of catalepsy with the control. The test and standard groups are contrasted with the control group.¹⁸ The catalepsy in mice by clonidine induced was found significantly inhibited at 48.33±0.17s at 150 min and 37.47±0.18s, 36.55±0.15s respectively at 120 min after the administration clonidine respectively of Sida veronicafolia at concentration 100, 200,400 mg/kg orally) used respectively. 106.78±0.24s, 12.04±1.04s duration of catalepsy of control group and standard group, so by comparing the control and standard group with test which shows less time of catalepsy.

CONCLUSION:

Ethanolic extract of Sida veronicafolia gives decresed contractile response at dose con. of 250, 500µg/ml than the Histamine on the tracheal chain of goat. Also duration of catalepsy decresed as compared to control group. So by observed data obtained from the such models it can be concluded that the ethanolic extract of leaves of Sida veronicafolia possesses the significant anti-asthmatic activity.

ACKNOWLEDGMENTS:

The management and Principal of YSPM’s Satara, India, are appreciated by writers for providing the facilities needed to complete the paper.

CONFLICT OF INTEREST:

We confirm that there is no conflict of interest.

ETHICAL STATEMENT:

The study was approved by the Institutional Animal Ethical Committee of YTC college of Pharmacy, Satara, Registered under CPCSEA, India (Registration No. YSPM/YTC/PHARMA/04/2023-24)

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Received on 16.07.2024 Revised on 30.08.2024

Accepted on 02.10.2024 Published on 07.12.2024

Available online on December 30, 2024

Res.J. Pharmacology and Pharmacodynamics.2024;16(4):269-273.

DOI: 10.52711/2321-5836.2024.00046

In Vitro and In Vivo Evalution of Anti-Asthmatic Activity of Leaves of Sida veronicafolia (Lam)

1. Isolated goat tracheal chain preparation:

2. Clonidine induced catalepsy in mice: