The Therapeutic Role of Aloe Barbadensis Miller Leaf in Managing Asthma and Inflammatory Conditions

Asthma is a serious allergic disorder of the respiratory system, marked by inflammation and narrowing of airways. This is a great responsibility for medical treatment. Current therapy for asthma has many serious side effects. Therefore, searching for a new therapeutic agent with no or fewer side effects is needed while most inexpensive for the patients. Due to the availability of strong bioactive chemicals, anti-asthmatic herbal remedies have been employed in traditional medicine and have demonstrated potential therapeutic anti-asthmatic efficacy. Breathlessness, chest tightness, and wheezing during the paroxysmal attack of bronchial asthma are symptoms brought on by a paroxysmal narrowing of the bronchial airways. Asthma is characterized by inflammation (The airway lining becomes red, swollen, and narrow), obstruction (airway muscles become tightened and it may cause difficulty to get air in and out of the lungs), and hyper-responsiveness (muscles encircled and rapid response on small allergens and irritants) of the airway. Generally, patients cough more during sleep and when awake in the morning¹. Asthma affects 10% of the population. Epidemiology will help us study asthma's prevalence, morbidity, and mortality rate. Around 235 million individuals around the world have been affected. Many people die from this deadly disease in many countries due to its high prevalence rate. The number of asthma patients has been increasing day by day². Millions of people have died from asthma. The highest number of patients is in New Zealand, Australia, and the United Kingdom, and the lowest patient rate is in China and Malaysia. About 7% of adults and 15% of children have asthma. In several individuals, inflammation causes frequent episodes of wheezing, shortness of breath, coughing, and chest tightness, particularly at night or in the early morning or after exposure to allergens, cold air, and exercise³. Nowadays asthma is considered similar but differs only based on disease severity. Therefore, treating patients with asthma requires differences in dose, route of intake, or frequency of taking the ?2-adrenoceptor agonist, and corticosteroids essential to manage asthma disease. However, asthma sub-phenotype identification has challenged this view in modern medicinal systems. Over the past two decades, research has identified the fundamental source of asthma is the allergic pathways. As per the National Institute of Health, Asthma is an inflammatory disease that targets the airway’s narrowing, resulting in the change of eosinophils, mast cells, lymphocytes, and cytokine levels. The patient with asthma is well known to have greater levels of IgE that bind to the receptor of mast cells and inflammatory products⁴. The antigen-antibody interaction results in the activation of inflammatory cellular reactions⁵ thereby releasing the mediator such as histamine, a prostaglandin that ultimately leads to the contraction of airway smooth muscles. Mast cell plays a key role in the pathophysiology of asthma and is found in large amounts in the airways of asthmatic patients. Now the inhaled allergens activate sensitized mast cells by binding with IgE molecules present on the surface of mast cells storing lease various bronchoconstrictor mediators. Dendritic cells also process the allergens after being conditioned by the thymic stromal lymphopoietin (TSLP) released by the mast and epithelial cells to release several of the chemokines that draw T helper 2 cells, these cells in turn, induce B cells to produce and secrete IgE antibodies that sensitize mast cells, induce eosinophil mediated inflammation and stimulate mast cell proliferation ⁶. Aloe barbadensis Miller belongs to the Liliaceae family with thick, green to yellowish green lance-shaped, sticky-juicy, sharp, and edged leaves⁷. Aloe vera grows in dry regions of Africa and the eastern European continents. Aloe vera is probably the most used medicinal plant commercially and the most popular plant around the world⁸. Ethanolic leaf extract of the plant contains vitamins, minerals, enzymes, sugars, anthraquinones, phenolic compounds, lignin, saponins, sterols, amino acids, and salicylic⁹. Aloe vera has many therapeutic effects in wound healing, as well as being anti-inflammatory, antioxidant, etc.¹⁰. This dissertation investigated the anti-asthmatic and Anti-inflammatory activity of the ethanolic extract of Aloe barbadensis Miller leaf (EEAB) in experimental animal models namely carrageenan-induced paw edema in rats and also asthma effect in clonidine-induced catalepsy in mice and histamine induce convulsions in guinea pigs.

MATERIAL AND METHOD:

Chemicals and reagents:

Histamine, Clonidine, chlorpheniramine maleate, ketotifen, ethanol, saline water, carrageenan, diclofenac sodium.

Experimental Animals:

The Albino mice (25-30 g), Wistar rats (150–170 g), and guinea pig (350–400 g) were, housed in the polypropylene cages at a temperature of 22 ± 2 °C, with 12 h day-light cycle. During whole experiments, animals were fed with a balanced diet and water ad libitum. The experimental protocol of the study was approved by the Institutional Animal Ethical Committee (CPCSEA/IAEC/CP-PL/10/2023) of Sudhakarrao Naik Institute of Pharmacy, Pusad.

Collection and authentication of Plant material:

The plant was collected from the kitchen gardens of the Umarkhed city of Yavatmal district in December and January. The plant was authenticated by Dr. Ramkrishna Sakhre, Department of Material Science, Ayurvedic College, pusad, yavatmal445204.

Preparation of the extract:

The fresh Aloe barbadensis miller leaves were washed thoroughly 2-3 times with distilled water and air-dried. A few of these leaves were chopped into small pieces and ground into a fine powder with the help of a grinder machine, and a mortar pestle and polyethylene bags were used to store them. These bags were kept in the oven for further analysis. The dried powder was extracted with 70% ethanol by using the Soxhlet apparatus.

Phytochemical Screening:

The obtained ethanolic extract was tested for various chemical constituents with the help of qualitative chemical tests ¹¹.

Acute Toxicity Study:

The acute oral toxicity study on the whole leaf and green rind extracts of A. vera was conducted according to the ‘Up and Down’ method described in OECD guideline 425. Nulliparous, non-pregnant female Wistar albino rats were used. For each extract, the animals were randomly divided into 4 groups, each having 3 rats. A single animal was administered a starting dose of 175mg/kg and observed for a period of 48h. The animal was observed continuously for the first 30min post-administration, every 1h for 4h, then once daily for 48h for signs of toxicity including bodyweight variation, salivation, tremors, convulsions, diarrhea, changes in the skin, fur, eyes, and mucous membranes, or death before the next doses of 550, 1750 and 5000mg/kg were administered. In addition, the animals were monitored daily for an additional 12 days for delayed signs of toxicity and mortality. The body weights were taken on day 0, day 7, and day 14. Finally, the LD50 was determined¹².

Evaluation of Anti-Asthmatic Activity:

1. Clonidine-induced catalepsy in mice-

Albino mice will be divided into four groups (n=3). The inducer control group received distilled water (10ml/kg p.o.), The standard group received pheniramine malate (10mg/kg. I.p.). The third and fourth groups will receive EEAB at doses of 200 and 400 mg/kg body weight. p.o. respectively.All the groups will receive Clonidine (1mg/kg s c.) 1 hr after the drug administration and the duration of catalepsy will be measured at (15,30, 60, 90, 120, 150, and 180 min). Evaluation: A decrease in the duration of catalepsy is calculated versus control. Standard and test groups are compared with the control group¹³.

2. Histamine-induced bronchospasm in guinea pigs-

In this method, guinea pigs were kept in a closed chamber and exposed to an aerosol of 0.5% histamine using the nebulizer. The time required to develop pre-convulsive dyspnoea (PCD) was recorded from the time of aerosol exposure to the onset of dyspnoea leading to the appearance of convulsions¹⁴. As soon as pre-convulsive dyspnoea was induced, animals were removed from the chamber and placed in fresh air to recover. Guinea pigs were divided into 4 different groups, with six animals in each group. The control group was orally treated with, 1% tween-80, at the dose of 5 ml/kg, body weight. Test groups received oral treatment of EEAB at doses 200 and 400 mg/kg, body weight, and ketotifen at a dose of 1 mg/kg, body weight orally was administered to the standard group. All the groups were given a single dose treatment for seven days. The time for the onset of PCD was recorded on day 0 before treatment and on day 7, 2 h after the last dose. The increases in PCD onset time by the animals were calculated by the following formula¹⁵.

Percent increase in PCD

time = [1 ? T1/T2 × 100]

Where,

T1 = Time for onset of symptoms before treatment.

T2 = Time for onset of symptoms after treatment.

3. Evaluation of Anti-Inflammatory Activity Study–

1. Carrageenan-induced rat paw edema:

The anti-inflammatory reaction is readily produced in rats in the form of paw edema using carrageenan. The rats were divided into four groups, each consisting of 3 animals. Group I: Control animals received distilled water 10ml/kg p.o. Group II: Animals received standard Diclofenac sodium at the dose of 5mg/kg p.o. Group III: Animals received EEAB at the dose of 200 mg/kg p.o. Group IV: Animals received an EEAB at the dose of 400 mg/kg p.o. After 30 min of extract administration, 0.1ml of 1% w/v carrageenan was injected into the right hind paw sub-plantar region of each rat. The left paw served as a control for comparison. The paw volumes of both paws of control and extract-treated rats were measured at 30, 60, 90, and 120 min after carrageenan administration". The percentage of edema inhibition for each rat and each group was obtained by using the formula:

Anti-inflammatory activity (%) = [(VC-Vt)/VC] × 100

Where,

VC = inflammatory increase in paw volume in the control group.

Vt = increase in paw volume in drug-treated animals.

Statistical Analysis:

The result were analysed with Prism Software by GraphPad (version 8.4.2). The results for each category will be expressed as the mean± SEM for each group. Statistical variations were evaluated using One-way variance analysis (ANOVA) accompanied by Dunnett's t-test. Where**** represents significant at p?0.0001 as compared with Normal control.

RESULT AND DISCUSSION:

Table 1- Phytochemical analysis of an ethanolic extract of Aloe barbadensis Millar

All the extracts caused no mortality or behavioral changes in the rats throughout the study. Generally, there was an increase in the animals’ body weight throughout the study period. Therefore, the LD50 was estimated to be above 5000mg/kg for all the extracts.

The percent yield of ethanolic extract of Aloe barbadensis Millar:

Table 2 - Percent yield of ethanolic extract of Aloe barbadensis Millar

Table 3 -Clonidine-induced catalepsy in mice
       
           
       

^**Statistically significant at (p<0> ***Statistically significant at (p<0>

 ^##Statistically Significant at P<0>

ns indicate non significate.
       
           
       

Fig no. 2- Histogram of the Effect of EEAB on histamine-induced bronchospasm in the rat.

^*Statistically significant at (p<0>**Statistically significant at (p<0> ***Statistically significant at (p<0>

^##Statistically Significant at P<0>

ns indicate non significate.

Table 5 - Carrageenan-induced rat paw edema

^*Statistically significant at (p<0> **Statistically significant at (p<0> ***Statistically significant at (p<0>

^#Statistically Significant at P<0>##Statistically Significant at P<0>

After exposure to histamine aerosol animals started showing PCT. Animals in the control group showed Pre-convulsion at around 2.83 seconds. A high dose of 400 mg/kg shows a significant p<0>