Evaluation of Acute Oral Toxicity and Mast Cell Degranulation of an aqueous ethanolic extract of Tritium aestivum Linn.

 

Vibhor K. Jain^1*, Dheeraj Ahirwar¹, Bindu Jain¹, Bharati Ahirwar²

¹School of Pharmacy, Chouksey Engineering College, Bilaspur (C.G), India

 

ABSTRACT:

Aim: The present study was aimed to determine LD₅₀ and to evaluate the safety of aqueous ethanolic (50:50) extract of the grass of Tritium aestivum by acute oral toxicity study in female mice as per OECD guideline 420. The percentage inhibition of peritoneal mast cell degranulation in the rat by the plant was also studied to establish its potential in the management of asthma.

Materials and Methods: The plant was extracted with 50% aqueous ethanol using cold percolation method. Phytochemical analysis of the extract was carried out to identify the phytoconstituents present in it. The protocol of Limit test at a dose of 2000 mg/kg was used to study the acute oral toxicity. Five nulliparous, non-pregnant albino mice were used for the study. Animals were administered the extract at a dose of 2000 mg/kg body weight. They were observed individually for mortality, wellness parameters, and body weight for 14 days. The in vitro percentage inhibition of mast cell degranulation was studied using the peritoneal fluid, mast cell activating compound 48/80 and the extracts at the concentration levels of 25 mg/ml and 50 mg/ml.

Results: The aqueous ethanolic extract of grass of Tritium aestivum at the limit dose of 2000 mg/kg did not produce any mortality. No change in wellness parameters and body weight were observed for the extract. The LD₅₀ of the extract was greater than 2000 mg/kg. Qualitative phytochemical screening of the aqueous ethanolic extract showed the presence of important phytochemical constituents like alkaloids, flavonoids, tannins, phenols, saponins, terpenoids and anthraquinones. The extract of this plant was found to be potent and had significant (P < 0.05) inhibitory effects on compound 48/80 induced mast cell activation degranulation.

Conclusion: We conclude from this study that the grass of Tritium aestivum is safe and can be used for the treatment of various allergic diseases like asthma. The efficacy of the plant to inhibit mast cell degranulation could be due to the contribution of different phytochemical constituents present in it.

 

 

 

INTRODUCTION:

Asthma is a chronic inflammatory lung disease which causes narrowing of the airways.  It is associated with bronchospasm, cellular infiltration, and bronchial hyperactivity.  Asthma is characterized by recurring episodes of wheezing, chest tightness, coughing and shortness of breath. [1].

 

It causes impairment of the airways. [2] Various factors like dust, cold air, emotions allergens, drugs, respiratory infection, chemicals etc are known to cause asthma. [3]

Different types of cells like mast cells, eosinophils, neutrophils, basophils macrophages, T cell, B cell and other are involved in asthma. Mast cells act as a link between IgE and airway hyperresponsiveness (AHR) and play a major role in the pathogenesis of asthma. [4, 5] In asthmatic patients the mast cell degranulation is greatly increased. [6, 7] In these patients the mast cells are present in the bronchial smooth muscles and bronchial epithelium. The mast cell infiltrates in airway mucous glands in subjects with asthma. Mast cells play a key role in the regulation of mucus secretion. Therefore researchers are looking for potent agents which could be helpful in the stabilization of mast cells and specific in inhibition of the degranulation of mast cells.[8] Bioactive compounds like flavonoids, saponins, tannins, steroids and polyphenols found in plants have shown interesting results in various target specific biological activities such as bronchodilation, mast cell stabilization, inhibition of leukotrienes etc. in the treatment of asthma. The synthetic drugs provide prompt relief from symptoms of asthma but they are associated with serious side effects like hematemesis, ulcers, drying of mouth and respiratory secretions, liver damage etc. Therefore, herbal drugs are becoming choice of treatment because they are believed to be relatively safe for the treatment of diseases.

 

Natural products are believed to cause less damage to the body. [9] However, the herbal medicines may also cause adverse outcomes due to the presence of toxic impurities in them. [10, 11]

 

Therefore, it is important to assess the safety of herbal compounds under development. The safety of a compound is routinely evaluated using acute oral toxicity. [12] Shoot of Triticum aestivum Linn. is called as a wheatgrass. It belongs to the family: Gramineae. Triticum is a genus of annual and biennial grasses, yielding various types of wheat, native to south-west Asia and the Mediterranean region. T aestivum Linn or common bread wheat is widely cultivated almost all over the world. The plant has many health benefits due to its high nutritional value and is used in the treatment of many diseases. The whole wheat, which includes bran and wheat germ, therefore, provides protection against diseases such as constipation, ischaemic, heart disease, disease of the colon called diverticulum, appendicitis, obesity, diabetes etc. [13] Due to the presence of various phytochemical constituents Triticum aestivum  has high therapeutic value  and serves as an effective herbal medicine for the treatment of serious diseases.[14]

 

On the basis of literature and scientific evidence of this plant, the present study was undertaken to evaluate the acute oral toxicity and the percentage inhibition of mast cell degranulation for its use in the treatment of asthma.

To the best of our knowledge, this is the first study of its kind.

 

CHEMICALS:

The chemicals used in this study were purchased from different suppliers. Ferric Chloride, Sodium hydroxide, Sodium Chloride, Glucose, Sodium bicarbonate, Potassium Chloride, Sodium dihydrogen phosphate, gelatin, Magnesium Chloride, Bovine serum albumin, HEPES, Compound 48/80, were purchased from Sigma-Aldrich.  Ketotifen fumarate, Ethanol, Mayer’s reagent, Dragendroff’s reagent, Sulphuric acid, ammonia, chloroform, lead ethanoate, acetic acid, hydrochloric acid, Million’s reagent, Molisch’s reagent, Magnesium chips were purchased from Merck.

 

METHODOLOGY:

PLANT MATERIAL:

Cultivation and Collection of the Grass:

Grass of Tritium aestivum was grown indoors in earthen pots. For this, the seeds of the plant were soaked for 12 hours and evenly spread in the pots containing soil and compost. After ten days the grass was collected.

 

Extraction of Plant Material:

The grass was washed with water to remove any soil or dirt adhering to it. It was then dried in shade and the completely dried material was reduced to a coarse powder with the help of a mechanical grinder. The powder was labeled and stored in an airtight container for further use. The powdered plant material was extracted using aqueous ethanol (50:50) Briefly, 100 g of powdered material was macerated with 500 ml of solvent for 48 hours with subsequent filtration. The solvent from extract was evaporated to dryness using a rotary evaporator (BUCHI, Switzerland) at 40°C temperature and reduced pressure. The dried extract was weighed and the percentage yield was calculated. The crude extract was dried in a freeze drier and preserved at +4°C for further studies.

 

PHYTOCHEMICAL SCREENING:

Qualitative phytochemical screening of the extract was done using standard methods to detect the presence of various phytoconstituents. [15, 16 and 17] The test solution was prepared by dissolving 3 g of extract in the solvent and filtering the solution through Whatman No. 1 filter paper.

 

Alkaloids:

Mayer’s Test:

To the extract solution 2 drops of Mayer’s reagent was added from the sides of the test tube. Formation of a white precipitate indicated the presence of alkaloids.

 

Dragendroff’s Test:

1-2 ml Dragendroff’s reagent was added to 1 ml of extract solution. Formation of a yellow precipitate showed the presence of alkaloids.

 

Anthraquinones:

Borntrager’s Test:

Dilute H₂S0₄ was added to the extract solution in a test tube and the mixture was boiled. The resulting solution was filtered and chloroform was added and shaken well in a separating funnel. To the organic layer, ammonia solution was added slowly. The appearance of pink/ red/ violet color in the lower layer (ammoniacal) indicated the presence of anthraquinones.

 

Carbohydrates:

Molisch’s Test:

To the extract solution taken in a test tube few drops of Molisch’s reagent was added to it. Further, 1 ml of concentrated H₂S0₄ was added from the side of the test tube. After 2-3 min 3 ml of distilled water was added to it. The appearance of a red or pale violet color at the interface indicated the presence of carbohydrates.

 

Flavonoids:

Fecl₃ Test:

1 ml extract solution was taken in a test tube and few drops of 10% Fecl₃ solution was added. Formation of a greenish-blue or violet color indicated the presence of flavonoids.

 

Lead Ethanoate Test:

3 ml extract solution was treated with equal volume of lead ethanoate solution. Formation of a buff-colored precipitate indicated the presence of flavonoids.

 

Sodium Hydroxide Test:

1 ml extract solution was taken in a test tube and 2 ml of 10% NaOH solution was added to it. The yellow color of the solution disappeared upon the addition of dilute HCl which indicated the presence of flavonoids.

 

Shinoda’s Test:

To the extract, 1 ml of ethanol was added. The solution was warmed and filtered. To the filtrate, 2-3 pieces of magnesium chips were added. Then few drops of concentrated HCl were added to it. Formation of pink/ orange/ red to violet color indicated the presence of flavonoids,

 

Saponins:

Foam Test:

Extract solution was diluted with 2 ml distilled water, shaken and kept aside for 2-3 minutes. Persistent frothing upon warming the solution indicated the presence of saponins.

 

Steroids :

Libermann-Buchard’s Test:

3 ml acetic acid was added to the extract solution. The reaction was cooled by keeping the test tube in ice, then 1 ml of concentrated H₂S0_4­ was added to the solution. The appearance of a violet to blue or bluish green color indicated the presence of steroids.

 

Terpenoids :

Salkowski test:

5 ml of extract solution was mixed in 2 ml of chloroform, and 3 ml of concentrated H₂S0₄ was carefully added to form a layer. Formation of a reddish brown color at the interface showed the presence of terpenoids.

 

Tannins:

Ferric Chloride Test:

To 2 ml extract solution, 1 ml 10% FeCl₃ was added. Formation of a bluish black or bluish-green precipitate indicated the presence of tannins.

 

Glycosides:

To the extract solution 2 ml distilled water was added. To this 1 ml, dil. NaOH solution was added. Formation of a yellow color confirmed the presence of glycosides.

 

Phenols:

To 1 ml of extract solution, 2 ml distilled water was added followed by addition of a few drops of 10% FeCl₃ solution. Formation of a blue or green color indicated the presence of phenols.

 

Proteins:

To the extract solution 5-6 drops of Million’s reagent was added. Formation of a precipitate which turned red on heating indicated the presence of proteins.

 

ACUTE TOXICITY STUDY:

Animals:

A total of ten healthy young adult Swiss Albino mice non-pregnant and nulliparous, weighing between 25-30 gm (8-12 weeks old) were used for the experiment. The experimental protocol was approved by Institutional Animal Ethical Committee as per the guidance of CPCSEA, Ministry of Social Justice and Empowerment, Government of India. (IAEC No. 1275/PO/Re/09/CPCSEA).

 

Housing and feeding conditions:

The animals were housed in standard conditions of temperature (22 ± 2°C), relative humidity (55 ± 5%) and light (12 hrs light/dark cycles). They were fed with commercial mice feed and water ad libitum.

 

Preparation of animals:

The animals were randomly selected, marked appropriately for identification and kept in clean propylene cages for seven days prior to the experiment to allow them for acclimatization to the laboratory conditions. Prior to dosing animals were fasted (food but not water was withheld overnight).  Following fasting the animals were weighed again and the dose was calculated according to their body weights. After the dose was administered food was withheld for 4 hours.

 

Preparation of doses:

The extract was dissolved in distilled water and filtered through Whatman No. 1 filter paper. The dose of the extract was prepared shortly prior to the administration.

 

Administration of dose:

Animals were divided into two groups

Group I. The control group (n=5) was given distilled water.

 

Group II. Test group (n=5) was given a single dose of 2000 mg/kg of 50:50 aqueous ethanolic extract of Tritium aestivum, through oral gavage with the help of an intubation cannula. [18-21]

 

Table 1: Dose of the extract and frequency of administration

Extract Dose	Vehicle	Route of administration	Frequency of administration
Aqueous ethanol (50:50), 2000 mg/kg	Distilled water	Oral route using an intubation cannula	Single Dose

 

Observations:

Animals were observed individually at least once during the first 30 min after dosing and periodically during the first 24 hours with special attention given during the first 4 hours. Animals were observed daily for two weeks from the commencement of the experiment. Observations included changes in skin and fur, eyes and mucous membranes, behavioral pattern and attention was given to observations of tremors, convulsions, salivation, diarrhea, lethargy, sleep, coma, and mortality. All the observations of individual animals were systematically recorded and compared with that of control animals.

 

Body weight:

Individual weights of the animals were determined prior to the administration of the test extract. All the animals were re-weighed on the 7^th and 14^th day of the experiment and changes in their weights were recorded.

 

MAST CELL DEGRANULATION:

Isolation of Mast Cells:

The animals were anesthetized using ether and 20 ml of Tyrod Buffer B (137 mM NaCl, 5.6 mM glucose, 12 mM NaH[CO₃], 2.7 mM KCI, 0.3 mM Na[H₂][PO₄], and 0.1% gelatin),  was injected in the peritoneal cavity. The abdomen was gently massaged and the cavity was carefully opened. With the help of a Pasteur pipette, the peritoneal fluid containing the peritoneal cells was aspirated. The fluid was centrifuged at 150 rpm for 10 min at room temperature and resuspended in Tyrod Buffer B. It was again centrifuged at room temperature for 15 mins at 400 rpm. The cells in the pellet were washed and resuspended in the Tyrod Buffer A (10 mM HEPES, 130 mM NaCl, 5 mM KCI, 1.4 mM Ca[Cl₂], 1 mM Mg[Cl₂], 5.6 mM glucose and 0.1% BSA) containing calcium. [22]

 

METHODOLOGY:

The mast cell of isolated from peritoneal fluid of 6 animals were divided into 5 groups with 6 replicate of each.

Group I:               0.1 ml Mast cell suspension.

Group III:             0.1 ml Mast cell suspension + 0.1 ml of 2µg/ml Compound 48/80+ 10µl/ml

Ketotifen fumarate

Group IV:     0.1 ml Mast cell suspension + 0.1 ml of 2µg/ml Compound 48/80 + 0.1 ml of

25 mg/ml Extract in normal saline

Group V:      0.1 ml Mast cell suspension + 0.1 ml of 2µg/ml Compound 48/80 + 0.1 ml of 50 mg/ml Extract in normal saline

 

After incubation, two to three drops of the suspensions were taken on a glass slide and fixed using a solution of formaldehyde and methanol (1:3). The mast cells were stained with toluidine blue (0.1%) and slides were observed under a microscope at 40X. The percent degranulation of mast cells was calculated using the following formula. [3, 23]

 

% inhibition of mast cell degranulation =

(1- no. of degranulated mast cells/ total no. of mast cells) X 100

 

STATISTICAL ANALYSIS:

The data were analyzed using GraphPad Prism (7.03), the software. The results were expressed as Mean ± Standard Deviation. One way ANOVA followed by Tukey’s multiple comparision test was used to analyze the data between the groups and p<0.05 was considered to be significant.

 

RESULTS:

The grass of Tritium aestivum was extracted with aqueous ethanol (50:50) using cold percolation method and the result of percentage yield is given in Table 2.

 

Table 2: Percentage yield of the extract

 

Phytochemical screening of aqueous ethanolic (50:50) extract of Tritium aestivum revealed the presence of various phytochemical constituents like alkaloids, flavonoids, glycosides, steroids, anthraquinones, saponins, tannins, terpenoids, and phenols. The result of phytochemical analysis of the extract is presented in Table 3.

Table 3: Phytochemical Screening of the extract

S.No	Constituents	Test	Inference
1.	Alkaloids	Dragendroff’s	+
		Mayer’s
2.	Anthraquinones	Borntrager’s	+
3.	Carbohydrates	Molisch’s	+
4.	Flavonoids	Ferric Chloride	+
		Lead Ethanoate	+
		Sodium Hydroxide	+
		Shinoda’s	+
5.	Saponins	Foam	+
6.	Steroids	Libermann-Buchard’s	+
7.	Tannins	Ferric Chloride	+
8.	Terpenoids	Salkowski	+
9.	Glycosides	Dil NaOH	+
10.	Proteins	Million’s Reagent	+
11.	Phenols	Ferric Chloride	+

 

Mortality:

No mortality was observed in test group at the limit dose of 2000 mg/kg body weight.  Therefore the plant is considered to be safe and the LD₅₀ value of Tritium aestivum extract is greater than 2000 mg/kg body weight.

 

Wellness Parameters and Bodyweight:

No significant change was observed in the body weight and wellness parameters of control and treated groups. The result of the observations in the control and test group is shown in Table 4 and Table 5. It signifies that consumption of food and water was normal and there was no metabolic impairement.

 

 

Table 4: Observations after dosing in control and test group

C : Control group, T : Test group, + : Normal, - : NIL

 

Table 5: Effect of treatment with extract on the body weight of animals (dose 2000 mg/kg) on Day 1 (before treatment), and Day 14 (after treatment)

Group	Treatment	Body weight (gm)
		Day 1 (Mean±SD)	Day 14(Mean±SD)
Control	Distilled Water	26.44 ± 1.29	27.44 ± 1.36a
Treated	Aqueous ethanolic (50:50) extract	26.66 ± 0.92	27.62 ± 0.92b

Values were expressed as Mean± Standard Deviation, (n=5), a, b = Not Significant at p<0.05

 

The aqueous ethanolic (50:50) extract of Tritium aestivum significantly (p<0.05) inhibited the degranulation of peritoneal mast cells induced by mast cell activator compound 48/80. The result of percentage inhibition is shown in Table 6. The percentage inhibition of mast cell degranulation of all test groups is compared as shown in figure 1.

 

Table 6: Percentage inhibition of mast cell degranulation

Values represented as Mean±SD, n=6, *(p<0.05) was considered significant when compared to negative control

 

Figure 1: Comparison of % inhibition of mast cell degranulation between test groups

 

DISCUSSION:

The aqueous ethanolic (50:50) extract of Tritium aestivum was safe at a dose regimen of 2000 mg/kg body weight. Mast cell degranulation caused by the compound 48/80 is inhibited by the extract significantly. The stabilization of mast cell is responsible for reducing the airway inflammation by inhibiting the various inflammatory mediators and thus may find its use for the management of asthma. [24] Phytochemical screening of the extract showed the presence of phenols, flavonoids, steroids etc. These phytoconstituents are known to have potent anti-inflammatory and antioxidant activity. The phytoconstituents may be responsible for the inhibition of mast cell degranulation induced by the compound 48/80. [25]

 

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Accepted on 24.12.2017        © RJPT All right reserved

Research J. Pharm. and Tech 2018; 11(2):643-648.