Exploring Therapeutic Potentials of Wedelia chinensis: An in vitro Anti-Asthmatic Investigations

Asthma is a chronic respiratory disease that is characterized by episodic airway obstruction, which is reversible either spontaneously or with treatment, by bronchospasm, and mucus hypersecretion. The effect it causes in millions of people across the globe, in terms of symptoms ranging from wheezing, coughing, breathlessness, and chest tightness, has been noted. The symptoms of asthma are wheezing, a high-pitched whistling sound in the breathing, coughing- sometimes dry, other times productive, shortness of breath (dyspnea), tightness or constriction of the chest, trouble speaking or breathing. Allergic asthma is the most common type of asthma induced by exposure to certain allergens, including dust, mites, pollens, or pet dander. Non-allergic asthma, by contrast, is not provoked by allergens; instead, it is brought on by stimuli such as cold air, exercise, or tension. Mixed Asthma combines some features of both allergic and non-allergic types. Occupational Asthma is a form precipitated through workplace exposure to chemicals, dust, or other irritants. Exercise-induced bronchospasm is an asthma triggered by physical activity. Other classification of asthma is according to severity, i.e., from mild to severe.  The well-known therapeutic interventions are available, like beta 2 adrenergic, methyl xanthine’s, anticholinergics, mast cell stabilizers, and leukotriene antagonists (LTs) for symptomatic treatment and management of asthma. Currently available drugs have some limitations & adverse effects, including corticosteroids, etc.  Use of herbal drug remedies or phytoconstituents from plant origin is comparatively safer and has similar efficacy with slower response. The use of the plant Wedelia chinensis, better known as the Chinese Wedelia, is one of the flowers belonging to the family Asteraceae as a perennial plant. It generally occurs in most parts of Asian countries, notably in China and India, from the tropics to the subtropics. Wedelia chinensis, used traditionally in the folk medicine industry, has anti-inflammatory, antipyretic, and antispasmodic property¹⁴. The variety of bioactive molecules like flavonoids, alkaloids, and terpenoids of this plant is reported for their medicinal properties. Wedelia chinensis has received recent attention due to its potential as a medicinal plant for possible modern therapeutic applications, including anti-asthmatic, anti-allergic, and anti-inflammatory activities; antimicrobial, antioxidant, and hepatoprotective activities were also recently reported. In general, Wedelia chinensis is a medicinal plant of great value with much traditional medicinal usage and promising modern applications. The Wedelia chinensis leaves are beneficial in managing asthma due to their anti-inflammatory, antispasmodic, and bronchodilator properties. Its bioactive compounds, including flavonoids and terpenoids, may be responsible for its anti-asthmatic properties, making it a potential complementary therapy for asthma management, which could ultimately reduce the need for conventional medications and improve the quality of life for asthma patients.

2. METHODS

2.1 Reported Methods for Evaluation of Anti-asthmatic Activity:

2.1.1 Preclinical Evaluation (In-vitro Methods)

1. Receptor binding assay in vitro β2-adrenergic receptor
2. Enzyme inhibition assay of phosphodiesterase
3. Cellular assays of histamine release from mast cells
4. Animal model of asthma, mouse model, ovalbumin-induced asthma

2.2 In vivo Methods

1. Animal models of asthma, such as ovalbumin-induced asthma in mice.
2. Animal model for goat tracheal preparation.
3. Bronchial provocation tests, such as methacholine challenge.
4. Pulmonary function tests, such as spirometry, plethysmograph
5. Inflammatory cell counting, such as eosinophil count.

3. MATERIALS & METHODS

3.1 Animal model:

The chicken ileum preparation is one of the most used in vitro models for studying smooth muscle physiology and pharmacology. It is the final section of the small intestine in chickens.

3.2 Procedure for Tissue Preparation: Fresh chicken ileum was brought from a slaughterhouse, then it was cleaned using a syringe with the help of Tyrode physiological salt solution (PSS). Then made the small fragments (pieces) of 2-3 cm and tied them to the oxygen tube using thread was suspended in an organ bath containing Tyrode's solution with continuous aeration by one bubble using an aerator.

3.3 Preparation Of Plant Extract:

A dried coarse powder of Wedelia chinensis plant material was ground using a mixer grinder. A 25g of coarse powder was accurately weighed and dissolved in 100 ml of distilled water for maceration. The mixture was heated gently at 50-80°C consecutively for 30 mins. Later, the filtrate was separated and collected from the plant extract using Whatman filter paper. The concentrated liquid extract was dried using a rotary evaporator. The extracts were collected for further in vitro experimentation and stored in the refrigerator.

4. Experimental Procedure ^[6]:

4.1 Dose-response curve of acetylcholine:

The chicken ileum was brought from the slaughterhouse (as shown in Fig. 2) and isolated tissue i.e. chicken ileum, was cut into small pieces, with the removal of excess connective tissue and fat. (as shown in Fig. 3). The isolated tissue preparation was mounted in the organ bath containing a physiological solution (e.g., Tyrode solution) at 37°C and pH 7.4 (as shown in Fig. 4). The 30 sec baseline taken by applying tension of 500mg, the standard drug solution was prepared by with accurately weigh 0.01gm of acetylcholine, and added in 20ml of distilled water.  Standard acetylcholine solution in to the tissue organ bath in increasing concentrations- 500 (e.g. 0.1 0.2, 0.4, 0.8, 1.6ml doses were added) (as shown in fig.5). The DRC were performed with a contact time for 60sec, and the speed of the drum is 0.25mm/sec was maintained. The responses were measured and recorded in dose dependent manner with double dose (as shown in Fig. 6). After the DRC, the graph was plotted against the log concentrations (log scale) to % response (tension or contraction force). The results were calculated and data were analyzed by measuring height of responses, % response, and other parameters as required (as shown in Fig. 7). Same responses were repeated and verified: The experiment was repeated multiple times to ensure consistent results and verified the findings⁵.

4.2 PA₂ value of Wedelia chinensis by using acetylcholine:

The PA₂ value was determined by performing DRC (Dose-response curve) for acetylcholine was added cumulatively in the organ bath in an increasing concentration (e.g., 10^-8 to 10^-4 M). The concentrations were recorded for both acetylcholine as standard drug and Wedelia chinensis extract as a test drug at a fixed concentration (e.g., 0.1, 0.2, 0.4ml). The DRC of acetylcholine was performed and observation was recorded in the presence of the plant extract and the steps were repeated till the ceiling effect was obtained. The calculation of pA₂ value and determined by considering the log concentration of Ach vs. drug response (contraction) for both curves (without and with extract) and graph was plotted. The pA₂ value is the negative logarithm of the concentration of extract that reduces the Ach response by 50%.

5. Observations:

The contractile effect of acetylcholine was prevented by Wedelia chinensis on chicken ileum tissue preparation, and we obtained the opposite effects to those of acetylcholine on chicken ileum preparation. Normally, acetylcholine (Ach) causes contraction of the chicken ileum smooth muscle by increasing the tone and contractility of the ileum muscle, by stimulating the muscarinic receptors (M₃) in the ileum, leading to increased intracellular calcium and muscle contraction. Compared to that of Ach, the plant extract of Wedelia chinensis produced relaxation of the ileum smooth muscle by blocking the acetylcholine-induced contractility. Thus, plant extract dose samples decrease the tone of the ileum muscle and block the muscarinic receptors (M3) in the ileum, leading to decreased intracellular calcium and muscle relaxation. In the presence of Wedelia chinensis, the effects of acetylcholine are antagonized, and the ileum muscle relaxes. These results may be due to the phytoconstituents present in the plant extract binding to the muscarinic receptors (M₃), blocking the action of acetylcholine. The standard preparation of Acetylcholine produces a contraction of chicken ileum tissue preparation and Wedelia chinensis shows the relaxation, which indicate the antagonistic property of Wedelia chinensis on the same isolated tissue preparation. These results suggested the antiasthmatic property.

6. Preparatory Thin Layer Chromatography (TLC) ^[20]:

The preparatory TLC plates were prepared and cleaned the plate and a thin layer of Silica gel was applied onto the plate. At the bottom 1cm above the edge of the plate made a marking line was made by pencil¹².  A small quantity of the sample drops (Wedelia chinensis, Ach.) was applied using a capillary tube just over the baseline, onto the TLC plate. The TLC plates were kept in the developing chamber with solvent ethyl acetate, the solvent system: formic acid: butanol (5:3:1). The samples of extract and standard acetylcholine, atropine allowed to move into the previously saturated TLC chamber by capillary action. The clear spots of separation were visualized after removal of the TLC plate from the chamber and allowed to air dry. The separated spots of both Wedelia chinensis and acetylcholine were visualized and detected in an iodine chamber. Finally, the Rf value of individual spots were calculated.

Figure 6.1.1: Noticeable interaction between Acetylcholine and Wedelia chinensis. Extract is observed on the TLC plate

6.2 Observation table:

7. RESULTS:

According to the results of log molar concentration compared to that of standard acetylcholine solution (Table 4), we found a lower pA₂ value of Wedelia chinensis against acetylcholine-induced contractions than 6.8 using chicken ileum isolated tissue preparation. Therefore, these results indicate that Wedelia chinensis has a considerable antagonistic effect against acetylcholine-induced contractions using isolated chicken ileum tissue preparation, meaning that this is a low-potency antagonist for acetylcholine. Results of TLC analysis for both acetylcholine and Wedelia chinensis extract revealed the presence of active phytoconstituents, with the calculated Rf value considered for the presence of active phytoconstituents, i.e., the flavonoid or phenolic compound apigenin could be responsible for antiasthamatic therapeutic benefits.

Figure 7.1: Dose-response curve of Acetylcholine and effect of Wedelia chinensis.

Table 2: Effect produced by acetylcholine on isolated chicken ileum preparation.

Table 3: Effect produced by Wedelia chinensis (extract) in the presence of acetylcholine on isolated chicken ileum preparation.

Graph:

Figure 7.2: Estimation of PA₂ value of Wedelia chinensis using Acetylcholine as agonist.