Biological Efficacy of Desmostachya bipinnata Grass against Allergy and Hypersensitivity using different Experimental Models

 

Harish Gupta1, Girendra Kumar Gautam2*

1Department of Pharmacy, University Campus, Bhagwant University, Ajmer (R.J.).

2Director, Shri Ram College of Pharmacy, Muzaffarnagar, Uttar Pradesh, India.

*Corresponding Author E-mail: dr.girendra@gmail.com

 

ABSTRACT:

The objective of the study is to evaluate antiallergic and antihypertensive activities of ethanolic extract of Desmostachya bipinnata. Oral administration of 250 and 500mg/kg of D. bipinnata extract was used to study the effects of the test drug on animal models of allergic reactions, including milk-induced eosinophilia and leukocytosis, compound 48/80-induced mast cell degranulation, and active and passive anaphylaxis. Additionally, the effects of D. bipinnata extract on sensitised guinea pig ilea (ex-vivo) and tracheal chain preparations were tested and evaluated (in-vitro). Results showed that compound 48/80 in the mesenteric area reduced mast cell degranulation and allergic reactions significantly after treatment with D. bipinnata extract at 500mg/kg dose. Further, studies shown that D. bipinnata can prevent the contractions generated by acetylcholine, histamine, and antigen in the ileum of sensitive Guinea pigs. Results showed that test drug can also neutralise free radicals (in vitro). Anti-allergic and anti-anaphylactic effects of D. bipinnata extract may be due to the presence of phytoconstituents on mast cell membranes.

 

KEYWORDS: Histamine, Allergy, Anaphylaxis, Immunity, Medicinal plant.

 

 


INTRODUCTION: 

Anaphylactic illnesses caused by an immediate hypersensitive reaction are a major public health concern, worldwide. Anaphylaxis and other life-threatening allergic reactions imposes heavy loss to an individual for health and wealth. Additionally, whealing-and-flare reactions can cause a variety of skin problems including hives. IgE-stimulated mast cell degranulation is the hallmark of anaphylaxis reaction, it is an acute hypersensitivity event.1 However, inflammatory mediators are produced when antigens stimulate mast cells, contributing to the inflammatory response near the site of mast cell activation. Reduced activation of IgE-sensitive mast cells could help treat or prevent an allergic reaction. Mast cells,2-3 are one of the most important effector cells that have been connected to the underlying causes. In the case of allergies, mast cells amplify symptoms. Immunoglobulin E (IgE) is produced by β-cells as a reaction to sensitization.

 

The phospholipid enzyme phospholipase C (PLC) is activated, intracellular inositol (1,4,5)-triphosphate (IP3) is raised, protein kinase C (PKC) is stimulated and Ca2+ levels rise. Mast cells and basophils degranulate and release cytokines and chemokines upon reexposure to an allergen.

 

Mast cell degranulation is triggered by an increase in intracellular Ca2+ concentrations. Immunotherapy and antihistamines can be used to treat anaphylactic symptoms. All first-generation antihistamine side effects, such as mental impairment, sedative action and anticholinergic effects are associated with the use of drugs.4 Numerous adverse effects on several organs have been reported with this class of drug.5 Due to the detrimental consequences of conventional medicine, several studies have been carried out to find out more natural approaches to alleviate the symptoms of allergy. Interestingly, certain natural remedies may be able to help to relieve the symptoms of allergy and hypersensitivity. 

 

Desmostachya  bipinnata (L.) Stapf, Poaceae, as a sacred plant has been used in India since ancient times. 'Kusha' is the Sanskrit word for the plant in India. 6-7 This plant is native to northeast and west tropical, and northern Africa, as well as nations in the Middle East, and tropical Asia. Salt reed-grass is another common English term used for the plant. It has been used in folk medicine for a variety of ailments. Researchers found that this herb contains diuretic, antidiarrheal, analgesic, antipyretic, and antiinflammatory properties. Other potential benefits include its antioxidant and anti-ulcerogenic characteristics, hepatoprotective effects, treatment of diarrhoea, menstrual cramps, and jaundice.8 Thus, the protocol of the study was designed to evaluate efficacy of D. bipinnata grass against allergic and anaphylactic symptoms reactions in experimental model.

 

MATERIALS AND METHODS:

Drugs and chemicals:

Ethanol (R.L. fine chemicals), Acacia (Rankem chemicals), Compound 48/80 (Sigma Aldrich) and Disodium cromoglycate (Solar Pharmaceuticals) were procured.

 

Collection and extraction of D. bipinnata grass:

D. bipinnata grass was collected by researchers from Raigarh Pharmacy College in Raigarh, India, in April 2020. The taxonomic validity of the plant was confirmed by Mr. R.S. Jayasomu, a Senior Principal Scientist in New Delhi. A voucher specimen of D. bipinnata can be found in the herbarium of the same department (NISCAIR/RHMD/Consult/2020/3710-11-2). Twenty days of room temperature air drying in the shadow of D. bipinnata grass were required. Grinding and sieving dry grass in a mechanical grinder produced a range of 50 to 150 mm particles. Dried D. bipinnata grass powder was used in the Soxhlet device and extracted with 90 percent ethanol for 48 hours. The extract was concentrated in a vacuum evaporator at 40°C and reduced pressure, and its solvent was removed. Glass jars with the concentrated extract were then stored at 4°C until needed.

 

Phytochemical analysis of D. bipinnata extract:

Drug samples were examined for active phytoconstituents such as phenols, flavonoids, saponins, proteins, carbohydrates, tannins, reducing sugar, lipids, and alkaloids.

 

Drug preparation:

Ibuprofen, dexamethasone, ketotifen fumarate, and carboxy methyl cellulose (CMC) suspensions of 1 percent w/v were used to administer the drugs.

 

Experimental animals:

The study included albino Wistar rats, Swiss albino mice, and Dunkin-Hartley guinea pigs of both sexes. The animals were housed at a temperature of 25±2°C and a relative humidity of 55% in an environment where they had access to regular meal pellets and water.

 

Acute toxicity studies:

The acute toxicity of D. bipinnata ethanolic extract was evaluated in accordance with OECD 425 criteria. The animals were observed on every 30 minutes for the first four hours. No deaths had been reported after 24 hours. No abnormalities in the animals' behaviour, coloration or texture of their fur, or the functioning of their respiratory or circulatory systems or central nervous systems were seen by our staff. Additionally, the presence of tremors and convulsions was observed and noted. The drug was found non-toxic upto 2000 mg/kg.

 

Assessment of anti-allergic activity:

Effect of D. bipinnata ethanolic extract against leukocytosis and eosinophilia in mice:

The animals were randomly divided into five groups of six Swiss albino mice (20-25 g). Control animals received saline, however, boiled and refrigerated milk were administered subcutaneously (s.c.) to positive control group, while dexamethasone (0.27 mg/kg) and D. bipinnata extract 250 and 500 mg/kg were administered to groups III, IV, and V, respectively. One hour later, all mice groups were given hot milk (4 ml/kg, s.c.) as a follow-up experiment. Blood samples were obtained under a light ether anesthetic before and after the milk was delivered. Both groups' leukocytes and eosinophils were tallied and analysed.9

 

Anaphylaxis reactions in rats:

We employed 100 μg egg albumin (E.A.) adsorbed on 12 mg aluminium hydroxide (adjuvant) to sensitize rats on the first, third, and fifth days of exposure. Serum was isolated from blood samples taken on the 11th day. Five groups of six rats each were formed and treated with the drugs. Antiserum from rats was employed, injected subcutaneously to rats with freshly shaved dorsal skin in the amount of 100 μl. Dexamethasone (0.27 mg/kg) was given to group III animals, along with CMC suspension and D. bipinnata extract was given to group IV (250 mg/kg) and group V (500 mg/kg). 0.5 ml of a solution of Evan's blue was given to all rats after 30 minutes of drug/extract treatment (100 μg). The Vernier Caliper was used to measure the leakage area of blue dye in mm2.10

 

Effect of D. bipinnata extract on serum IgE in rats:

Rats were given extracts of D. bipinnata to test their immune system's response. Rats that had been previously sensitised were randomly assigned into four groups, each with six rats. Group I was control group; however, groups II, III, and IV, the oral doses of D. bipinnata extract were 250 and 500 mg/kg, and the i.p. dose of dexamethasone (0.27 mg/kg), respectively was administered starting on the fourth day and lasting until the tenth day. On the sixth, eighth, and eleventh days after sensitisation, serum IgE levels were measured in the blood of sensitised rats. Anti-rat IgE antibody was used as a capture reagent and a biotin-conjugated anti-rat IgE antibody as a detection reagent in a sandwich ELISA. Then, peroxidase-extravidin and OPD-H2O2 were introduced. 0.15-20 ng/ml IgE standard was used to dilute samples. Analysing diluted serum from egg albumin-immunised rats revealed anti-egg albumin IgE.

 

Active anaphylaxis in rats:

Horse serum comprising 0.5% was administered intravenously to a group of 30 rats, those were already sensitised. Six extremely sensitive rats served as the test animals for each of the four groups were selected. Group I received no treatment. CMC (10 millilitres per kilogram orally) was administered to Group II animals. For ten days, dexamethasone at a 0.27 percent oral dosage was administered to groups III. Group IV and Group V were administered  D. bipinnata extract in oral doses of 250 and 500 mg/kg, respectively. Rats were given 0.25 ml of horse serum diluted 1:1 in normal water on the 10th day, two hours after treatment. A high respiratory rate, respiratory distress, cyanosis, and dyspnea have been linked to allergic reactions. Allergic reactions could be evaluated for their severity using a respiratory severity scale. An evaluation method based on the following criteria was chosen: an elevated respiratory rate for at least ten minutes; dyspnea; cyanosis; respiratory collapse and death.

 

Histamine-induced paw oedema:

Group 1 and group II were control and induced groups, respectively. D. bipinnata extract at doses of 250 mg/kg and 500 mg/kg, as well as 50 mg/kg ibuprofen, were given to group III, IV and V, respectively. They were used to divide the rats into five groups of six animals each. Thirty minutes before histamine was administered, each rat received a 1% weight-per-volume (w/v) dose of ibuprofen and D. bipinnata extract. The volume of the paws was measured using a plethysmometer (Orchid scientific, Nashik).

 

Effect of D. bipinnata extract on compound 48/80 induced mast cell degranulation in rats:

One milligram per kilogram of compound 48/80 was injected subcutaneously into the rats. Randomly generated groups of six sensitised rats were formed. Test groups received D. bipinnata extract and ketotifen fumarate was given daily for seven days. The rats were given 10 millilitres of cold phosphate-buffered saline on the seventh day (i.p.). The peritoneal fluid from the animals was collected and placed in a silicon test tube with 7-10 millilitres of phosphate buffer after they were euthanized (pH 7.4). Mast cells were excluded from the sample during the exclusion test using a diluted version of trypan blue dye (0.4 percent). The mast cells were separated using percoll density centrifugation. A concentration of five micrograms per millilitre of compound 48/80 was added to the rehydrated mast cell pellets, and they were incubated for ten minutes at 37℃. The cells were stained with toluidine blue after centrifugation. Degranulated and undamaged mast cells were counted using high magnification microscopy and the fraction of degranulation was calculated.

 

Statistical analysis:

All data was represented using the Mean ± SEM (n=6). Statistics were performed via one-way ANOVA using GraphPad Prism software (version 5.01). The results of an ANOVA and a Dunnett multiple comparison test showed that it was statistically significant. Compared to a control group, the results are statistically significant (*P< 0.05).

 

RESULT:

Phytochemical analysis:

D. bipinnata grass extract showed presence of Flavonoids, phytosterols, lipids, carbohydrates, proteins, alkaloids, and tannins as phytoconstituents.

 

Acute toxicity study:

Oral administration of D. bipinnata extract to mice at doses up to 2000 mg/kg showed no effect on side effect or mortality. As a result, an LD50 of more than 2000 mg/kg was established. Acute toxicity data from doses of 250 and 500 mg/kg of D. bipinnata extract in anti-allergy and anti-anaphylaxis  investigations were selected for this extract.

 

Anti-allergic activity screening:

D. bipinnata extract reduced milk-induced eosinophilia, leukocytosis, and differential leukocyte count. Rats receiving subcutaneous injections of whole milk had significantly increased levels of eosinophils, white blood cells (WBCs), and different leukocytes (neutrophils and monocytes). Eosinophilia, leukocyte, monocyte, and neutrophil counts were considerably lowered in milk-fed animals pre-treated with 250 mg/kg and 500 mg/kg D. bipinnata extract. However, dexamethasone treatment had a significant role in reducing blood cell counts (Table 1).


 

Table 1: Effect of D. bipinnata grass extract on milk-inducible eosinophilia, leukocytosis as well as leukocyte differentiation in mice

Treatment (mg/kg)

Eosinophilis per cu. mm (10c/µl)

Differential Leukocytes (×10c/µl)

Total Leukocytes

per cu. Mmc

Neutrophils

Monocyte

Normal control

72.54 ± 1.95

2.02 ± 0.27

30.03 ± 1.11

6027.20 ± 29.84

Positive control

158.11 ± 4.04

8.94 ± 0.26

49.59 ± 1.21

9853.11 ± 42.04

Dexamethasone (0.27, i.p.)

81.74 ± 2.88

5.3 ± 0.29

35.05 ± 1.02

7513.22 ± 24.18

DBE 1 (250, p.o.)

96.94 ± 3.05

6.5 ± 0.22

39.84 ± 1.20

8184.11 ± 44.27

DBE 2 (500, p.o.)

85. 12 ± 3.05

5.4 ± 0.30

36.88 ± 1.32

8316.05 ± 22.19

DBE = D. bipinnata extract

 

Table 2: Effect of D. bipinnata grass on anaphylaxis (passive and active) and compound 48/80-induced mast cell degranulation

Treatment

(mg/kg, orally)

mm3 of blueing

reaction

The onset of behavioural symptoms

Changed behaviour in min

(% Mortality)

Respiratory score

(% Mortality)

Intact

Degranulated

(% Protection)

Normal control

0

Normal

0 (0)

0 (0)

100

0

Positive control

75.94 ± 2.74

Respiratory distress/collapse

3-5 min (65)

8.19 ± 1.11 (60)

23.99 ± 1.19

72.33 ± 2.23

Dexamethasone (0.27)

41.28 ± 2.03

Normal (0)

0 (0)

1.83 ± 0.13 (10)

-

-

DBE 1 (250)

59.22 ± 1.94

Dyspnea

10-12 min (25)

4.74 ± 0.60 (30)

30.33 ± 1.03

55.85 ± 1.02

DBE 2 (500)

49.32 ± 1.88

Normal

0 (0)

2.04 ± 0.38 (20)

39.84 ± 1.32

53.43 ± 2.04

Ketotifen (1)

-

-

-

-

58.43 ± 1.26

1.48 ± 2.01

DBE = D. bipinnata extract

 

Table 3: IC50 values of D. bipinnata extract on histamine and Ach-induced contractions

Antagonist

IC50 values of

The guinea pig ileum's histamine response in the presence of an antagonist

Response of the guinea pig tracheal chain to acetylcholine in the presence of antagonist

Nil

14 (12.5-17.9) µg/ml

[100 % contraction]

151 (144-178) µg/ml

[100 % contraction]

Chlorpheniramine maleate

0.27 (0.22-0.29) µg/ml

-

Atropine maleate

-

2.71 (2.2-3.1) µg/ml

DBE 2

410 (341-485) µg/ml

249 (228-275) µg/ml

DBE = D. bipinnata extract

 


Effect of D. bipinnata extract on anaphylaxis and compound 48/80-induced mast cell degranulation:

Pretreatment with D. bipinnata extract reduced passive cutaneous responsiveness in a dose-dependent manner. Anaphylactic reactions were minimised (lower the respiratory score) when rats were pre-treated with D. bipinnata extract and dexamethasone. Compound 48/80 and D. bipinnata extract sensitised rats to mast cell degranulation at a concentration of 1mg/kg, s.c.. To counteract mast cell degranulation, treatment with 1 mg/kg, p.o. of the standard drug ketotifen fumarate showed better results than D. bipinnata extract (Table 2).

 

Effect of D. bipinnata extract on histamine and acetylcholine (Ach) induced contractions:

Histamine caused a dose-dependent contraction, which was significantly alleviated by the combination of chlorpheniramine maleate and extract of D. bipinnata. For histamine reactions, the chlorpheniramine maleate IC50 was calculated to be 0.27µg/ml, while the D. bipinnata extract was 410µg/ml. An increase in acetylcholine-induced tracheal contraction in the presence of atropine and D. bipinnata extract maleate reduced the acetylcholine-induced contractions in guinea pigs. Calculated IC50 values for atropine maleate and D. bipinnata extract were 2.71µg/ml for atropine maleate and 249µg/ml for D. bipinnata extract (Table 3).

 

Effect of D. bipinnata extract on serum IgE in rats

Antibody (IgE) production was suppressed by both doses of D. bipinnata extract (250/500mg/kg). An increase in D. bipinnata extract's showed ceiling effect at 500mg/kg, the action was not increased after this dose (Figure 1).

 

Figure 1: D. bipinnata extract grass on serum IgE antibodies in rats

Suppression of histamine-induced swelling by the extract of D. bipinnata:

Histamine injections into the subplantar region of rats' hind paws resulted in oedema, which peaked one hour following the injection. Dose-dependent changes in guinea pig ileum contractions were observed in rats pre-treated with Ibuprofen and D. bipinnata extract (Figure 2) and disodium cromoglycate in perfusion when these two medications were combined.

 

Figure 2: Effect of D. bipinnata extract on histamine-induced hind paw oedema in rats

 

DISCUSSIONS:

A bilobed nucleus is present, as well as massive acidophilic granules, in these cells. Allergies and asthma are impacted by them. Eosinophil degranulation is the cause of cow's milk allergy. The presence of elevated eosinophils indicates a serious allergic condition. Eosinophilia and leukocytosis are biomarkers for asthma and allergy sufferers. A rise in bone marrow-derived lymphocytes and T cells could explain elevated eosinophilia and leukocytosis in the milk.11 Different allergy presentations (histamine, leukotrienes C4 and D4, cytokines, basic proteins) create inflammatory mediators (dilated blood vessels, edoema, eosinophilic infiltration) that lead to various allergic reactions.12-13 DBE therapy lowers milk-induced cellular immunological reactions (eosinophilia and leukocytosis) in mice, it may have therapeutic implications for type-I hypersensitivity reactions and asthma.

 

Mast cell histamine production, in turn, enhances vascular permeability and is mediated by IgE in allergic reactions induced by PCA and polybasic compound 48/80. Skin and 48/80 allergies were lowered by D. bipinnata extract therapy. Type I IgE-mediated allergic skin responses are inhibited by D. bipinnata extract. Mast cell degranulation may be suppressed by D. bipinnata extract if membrane receptor contacts are reduced. A plant extract rich in flavonoids has been shown to reduce anaphylactic shock provoked by IgE.14-15 Upon membrane breach, compound 40/80 stimulates mast cell production of cytokines and chemokines that promote membrane permeability.16 mast cells may be stimulated by membrane-based mechanisms linked to chloride channels or intracellular cAMP.17

 

By stabilising the lipid membrane and stimulating G-proteins through intracellular Ca2+ release, D. bipinnata extract may attenuate PCA and compound 48/80 IgE-mediated type I allergic reactions.18 Anti-muscarinic and H1 antagonistic properties of D. bipinnata were validated in experiments on guinea pigs tracheal chains and ileum. Anaphylaxis may be reduced and survival rates may be increased by D. bipinnata extract.19 OVA-induced allergic rat models show that coca (0.2 percent w/w) polyphenols reduce IgE production and myricitrin (a leaf extract rich in flavonols) myricitrin decrease total IgE,20 and apple polyphenols are employed in the treatment of seasonal allergy.21-22 These plants contain flavonoids, triterpenoids and steroidal saponins that stabilise mast cell membranes and combat asthma.23

 

CONCLUSION:

Present study shown that D. bipinnata extract reduces the generation of IgE and chemical mediators produced from mast cells in diverse ways. This in turn supports the traditional use of D. bipinnata extract in treating allergic responses. The extract of D. bipinnata was effective as anti-allergy herbal drug. The human clinical trials involving the extraction of active phytoconstituents, either singly or in combination, and testing these extracts on human subjects are necessary in future research.

 

CONFLICT OF INTEREST:

The authors declared no conflict of interest.

 

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Received on 03.11.2022            Modified on 09.02.2023

Accepted on 07.04.2023           © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(10):4543-4548.

DOI: 10.52711/0974-360X.2023.00740