Anti-Bronchitis Effects of Grewia hirsuta Vhl Extract in Cigarette Smoke Model

S.A. Rathod¹, T.R. Deasi², Pravin Tirgar³

School of Pharmacy, R K University, Rajkot, Gujrat, India.

*Corresponding Author E-mail: smjadhav2511@gmail.com

ABSTRACT:

Bronchitis is a chronic obstructive pulmonary disease. It can be either acute or chronic, depending on its severity. The main Etiology of this disease is cigarette smoking. So for this present bronchitis activity we induced bronchitis in wistar rats by Cigarette smoke model. Smoke has a number of toxic substances leading to impairment of ciliary function, increase the airway resistance, mucosal gland hypertrophy leadings to increase mucosal discharge, produces inflammatory changes by recruiting neutrophils and macrophages. And its activate the transcription of nuclear factor and IL-6. The Hydro alcoholic extract of Grewia hirsuta Vhl whole shrub were screened for anti-bronchitis activity by using cigarette smoke in-vivo animal model on rats. The outcomes of these investigations showed that the 200 mg/kg dose of Grewia hirsuta was more significantly (p<0.001) efficacious as an anti-bronchitis activity. The anti-bronchitis properties of the extract attributed to a decrease in IL-6 levels, total WBC count, C-reactive protein, neutrophil and lymphocytes infiltration. Additionally, the cytology of the lungs and BALF were examined to see whether the negative control group had higher lymphocyte infiltration than the treatment group. The histopathology investigation revealed that the treatment group of the animals had less leucocyte infiltration and bleeding than the negative control group. For the Grewia Hirsuta vhl comparative investigation, a Bronchodilator 2 mg/kg salbutamol was employed.

KEYWORDS: Bronchitis, Cigarette smoke, Grewia Hirsuta, Salbutamol, IL-6, Total WBC count.

1. INTRODUCTION:

Acute bronchitis usually caused by viruses, mycoplasma and bacteria. May last several days or weeks. Physical and chemical agents such as dust, allergens, strong fumes and those from chemical cleaning compounds or tobacco smoke may be cause it. The disease may be catarrhal, membranous or putrid. In COPD the major obstructive disorders are Chronic bronchitis, Emphysema, bronchial asthma, bronchiectasis and bronchiolitis.

Main symptoms seen in this disorders are Cough with sputum, Breathlessness and chest infection. Other than smoking infections like Adenovirus or HIV infection is associated with emphysema, Alpha 1antitrypsin deficiency, atmospheric pollution, occupation, immunological factors, some drugs and gastro-esophageal reflux. Chronic bronchitis characterized by hypersecretion of mucus accompanied by a chronic productive cough. There are three factors contributes to the increased thickness of the bronchial wall 1. Infiltration of the submucosa by chronic inflammatory cells 2. Marked hypertrophy of mucosal smooth muscle cells and 3. Marked hyperplasia of the mucus glands.¹

2. MATERIAL AND METHODS:

2.1 Formation of G. hirsuta Extract (GHE):

G.Hirsuta extract(GHE) was prepared by whole shrub of plant were crushed into powder, extraction takes place by cold maceration process with hydro alcoholic solvent 70:30 ratio for 7 days. Obtained (GHE) dissolved in methanol. This dose mixture was sonicated for 10 min in an ultrasound water bath (temperature at 24 ◦C) and filter. The resulting solution (GHE) was then administered orally in a volume of 100 mg/kg as a low and 200 mg/kg as high dose per rat as per their body weight.

2.2 Preliminary Phytochemical screening:

The crude hydro alcoholic extract was used to screen constituents such as alkaloids, tannins, glycosides, steroids, terpenoids, flavonoids, saponins, and phenol using different standard methods.

2.3 Animals:

Healthy wistar rats with a weight range of 180–200 g were procured from Kusum life science A—3 MIDC, Wasmat, Dist—Hingoli. The animals were held in cages and maintain the laboratory conditions, with a 12-hour dark-light cycle, a temperature of 23 ± 3 ◦C, and 46–54% humidity. They were provided with standard food pellets and water. The entire experimental process was approved by an Institutional Animal Ethics Committee registered under the "Committee for the Purpose of Control and Supervision of Experiment on Laboratory Animals" (CPCSEA), Ministry of Environment and Forests, Government of India. Approval number—CPCSEA/IAEC/P-01/2023.

2.4 Chemicals:

Cigarette(flake) from shop, Salbutamol was purchased from medical shop (Nagpur), Grewia Hirsuta (whole shrub) was purchased from shree shail (Nagpur, India). 4% formalin, 99% alcohol ,80% ethanol. Staining agent like H/E, PAP and Gimsa.

2.5 Formation of Dose of GHE:

Different Dosages of GHE Extract Was Freshly Prepared and Administered orally in a Volume of high and low dose as per Rat body weight. Salbutamol was Freshly Prepared by dissolving in Aqueous Solution of Saline and Administered Orally at a Dose of 2 mg/kg was Administered as per Body Weight of rats. Normal Control Animals Received 0.9% saline solution (1ml)

2.6 Induction of Bronchitis:

In induction group Exposure of wistar rats to cigarette smoke for 7 weeks. 5 days’ exposure per week. Means exposed total 4 cigarettes i.e. 2 at morning and 2 at afternoon per day. Total 20 cigarettes per week exposed for induction of bronchitis continue for 7 weeks. For exposure we made smoke exposure chamber on lab level with proper ventilation and partition between burn cigarette container and animals and container close during CS exposure. (Figure 1) In treatment group with high and low dose of GHE and STD drug salbutamol given from first day of CS Exposure for 30 days. After 6^th week of exposure weight the animals. (Figure 2) And the animals were anesthetized using ketamine (1.25 g/kg) for collection of blood and Broncho alveolar Lavage (BAL) fluid for biochemical evaluation. After that the animals were sacrificed, their lungs were isolated for further analyses. Scrape smear from BALF and Lungs.

Figure 1: CS Container

Figure 2: Timeline of the CS model

Groups:

A. Normal Control—0.9% saline solution

B. Negative control- CS Exposure

C. Standard— Salbutamol (2 mg/kg) oral

D. Treatment group - CS+GHE (100 mg/kg) oral

E. Treatment group - CS+ GHE (200 mg/kg) oral

2.7 Determination of Cell Count in Blood:

Blood up to 3ml was collected in a EDTA contain tube from each animal under anesthesia Xylazine (0.8 mg/kg)) and used for hematology, Biochemical, Cytokine(IL-6) and serology study. These samples were collected from retro- orbital plexus of rats. Every sample was centrifuged at 1000 rpm for 10 min at 2 ◦C by cold centrifugation; the cells in the pellet were washed in 0.5 ml 0.9% saline, and total cells were counted using Auto analyzer. After stained with Field’s stain identify different types of leucocyte cells.

2.8 Collection of Broncho alveolar Lavage Fluid (BALF):

0.9% Saline solution (0.5 mL) was instilled through trachea into the lungs and BALF was collected (total volume 1.5 mL) via tracheal cannulation. Every BALF sample was centrifuged at 500-1000 rpm and the supernatant layer collected was used for biochemical estimation and cytology study.

2.9 Biochemical Estimation (Total Protein and Albumin):

The activity of albumin in serum and BALF were measured using commercially available reagent kits (Delta Diagnostics, India). Blood was centrifuged at 6000 rpm for 10 min. Serum samples were collected and frozen at −80⁰C until the time of analysis.

2.10 Serology test (C-Reactive protein):

Blood without anticoagulant get clot and centrifuged at 2500 rpm for 15 min by cold centrifugation under the temperature 2⁰C and supernatant layer was collected.

2.11 Enzyme-Linked Immunosorbent Assay (ELISA):

To determine the levels of cytokines in vivo, BALF and lung homogenate, samples were collected. IL-6, were assayed with commercially available ELISA kits (Delta Diagnosis, India)

2.12 Cytology study:

To see the infiltration of lymphocytes and neutrophils cell by scrape smear from lugs and BALF over the slides and fixed in 99% alcohol contains coplin staining jar. Stain with Hematoxylin and Eosin (H & E), PAP and Gimsa. But here Gimsa staining was dry slides.

2.13. Histopathological Examination:

At the end of the study rats from each group were sacrificed and their lungs were removed, washed and cleaned with 0.9% saline and kept in 4% formalin and given for histopathological examination by staining it with Hematoxylin and Eosin (H & E)¹². The prepared slides were examined under compound microscope and photomicrographs were taken at 45X.

2.14 Statistical Analysis:

The statistical evaluation was accomplished with the help of Graph Pad prism 5 for 64 bit Windows Version. All the experimental groups were compared to evaluate the statistical significance using One-way analysis of variance (ANOVA). Each test was followed by the Dunnett’s multiple comparison. The data are characterized as mean ± SEM values and p values < 0.05 were measured as statistically significant.

3. RESULT:

3.1 Effect of Treatment in Body Weight and Relative Lungs Weight in the current study, negative control group were detected with reduced body weight and an increased relative lungs weight as compared to normal control group (*** p < 0.001). However, Standard group showed significant reduction in body weight as compared to normal control (*** p < 0.001). (Table 1)

3.2 Effect of Treatment group on Cigarette smoke induced Alteration in Hematological Parameters:

In the current study, significant changes occurred in hematological parameters of negative control rats were observed as compared to normal control rats. As compared to normal control group, a significant Increase in hemoglobin, red blood cells (RBC), WBC count, was observed, in negative control group (*** p < 0.001). Treatment groups (GH 100mg/kg and GH 200 mg/kg) significantly these alterations observed in hematological parameters as compared to negative control rats (** p < 0.01). Standard 2mg/kg salbutamol also decreased these mentioned hematological parameters as compared to negative control group (**p < 0.01). In case of WBCs, neutrophils and lymphocytes, a significant increase was observed in negative control group as compared to normal control group (** p < 0.01). When compared with normal control rats, Treatment group (GH 100mg/kg and GH 200 mg/kg) and standard group showed significant inhibition in differential WBCs, neutrophil and lymphocyte count (*** p < 0.001). (Table 2)

Table 1: Effect of treatment on CS induced alterations in Body Weight and Relative lungs weight.

Parameter	Normal Control	Negative Control	Standard	GH (100mg/kg)	GH (200 mg/kg)
Body weight (gm)	180.68 ± 17.09	190.01± 18.4	229.98±19.48	163.26 ±20.75	198.23 ±5.78
Relative Lungs weight (gm)	2.51 ± 0.06	3.28 ± 0.15^***	3.25 ± 0.08^###	2.38± 0.10	2.50 ± 0.09

Data were evaluated by one-way ANOVA followed by Dunnett’s Multiple Comparisons Test. Values are stated as Mean ± S.E.M. (n = 6). Statistical significance was evaluated as * p < 0.05, *** p < 0.001 vs normal control group and ### p < 0.001 vs. negative control group. Standard: 2 mg/kg, GH 100mg/kg and GH 200 mg/kg.

Table 2: Effect of treatment on CS induced alteration in hematological parameters and differential cell counts in BALF of rats.

Parameter	Normal Control	Negative Control	Standard	GH (100mg/kg)	GH (200 mg/kg)
Hb	13.18±0.16	18.11±0.26^***	13.96±0.37	15.38±0.47^***	14.50±0.26^*
RBC	4.96±0.16	9.44±0.18^***	5.40±0.41	6.76±0.36^**	6.51±0.30^**
WBC (%)	4640.0 ± 273.13	17817 ± 443.78^***	5416.7 ± 436.21	7307.0 ± 572.62^***	5916.7 ± 373.65
N (%)	25.83 ± 0.60	72.33 ± 0.88^***	29.833 ± 0.98^***	46.33 ± 3.37^**	35.50 ± 1.74
L (%)	25.66 ± 1.64	65.33 ± 3.56	39.33 ± 1.54	42.00 ± 2.11	40.33 ±1.25
E (%)	28 ± 0.47	7.8 ± 0.30^***	4.0 ± 0.36	6.3 ± 0.42^***	4.16 ± 0.30

Data were analyzed by one-way ANOVA followed by Dunnett’s Multiple Comparisons Test. Values are expressed as Mean ± S.E.M. (n = 6). Statistical significance was assessed as * p < 0.05, ** p < 0.01, **p < 0.05 vs. normal control group and vs. negative control group. N—Neutrophils, L—Lymphocytes, E— Eosinophils, Standard: 2 mg/kg salbutamol.

Table 3: Effect of treatment on CS-induced alteration in the levels of total protein and albumin in serum, BALF and lung tissues of rats.

Parameter	Normal Control	Negative Control	Standard	GH (100mg/kg)	GH (200 mg/kg)
CRP (Mg/L)	2.45 ± 0.35	16.83 ± 0.79^***	5.83 ± 0.79^*	12.0 0 ± 1.06^***	8.50 ± 0.56^***
BALF total protein (gm/dl)	7.03±0.20	11.250±0.77^***	7.65 ±0.20	8.2167 ±0.09	7.9167±0.10
BALF total albumin (gm/dl)	4.18±0.18	7.03±0.20***	4.90±0.11*	5.48±0.19***	4.91±0.06*

Data were analyzed by one-way ANOVA followed by Dunnett’s Multiple Comparisons Test. Values are expressed as Mean ± S.E.M. (n = 6). Statistical significance was assessed as * p < 0.05, ** p < 0.01 vs. normal control group and p < 0.05, p < 0.01 vs. negative control group.


Normal Control		Negative Control

STD (2mg/kg)	GH (100mg/kg)		GH (200mg/kg)

CYTOLOGY STUDY (Scrap smear from tertiary bronchi of Lung and BALF)

1. Control


No Lymphocyte Infiltration in BALF	No Lymphocyte Infiltration in Lung

2. Negative Control


Lymphocyte Infiltration in BALF	Lymphocyte Infiltration in lung

Photomicrograph of sections of lungs of:


Normal control	Negative Control

STD	GH(100mg/kg)

GH(200mg/kg)

4. DISCUSION:

This present study was carried out to evaluate Anti bronchitis activity of grewia hirsuta vhl whole shrub by cigarette smoke induced in vivo model in wistar rats. Following a pilot analysis of the induction model, we find that a 30-day CS exposure time is adequate to induce bronchitis in rats. On the basis of phytochemical reports of this hydro-alcoholic extract, Alkaloid, flavonoids and phenol main constituents were observed. After induction, some morphological changes were observed in lugs of negative control groups animals, with edema and redness. As compared to Negative control, there was less changes were observed in STD and treatment group. GH(200mg/kg) shown mild redness and edema as compared to GH(100mg/kg) dose. Bronchitis induction was increases the level of total WBC count, Neutrophils, eosinophils, lymphocytes, CRP and Cytokine (IL6) count. But in treatment group specially GH(200mg/kg) significantly (p<0.001) decrease the Cell counts, CRP level and inflammatory cell(IL-6). Also (p<0.01) Decrease BALF Albumin and total protein count. According to histopathology study; GH(200mg/kg) shown mild Eosinophilic and Lymphocytic infiltration as compared to GH(100mg/kg) dose. In treatment group cytology study of BALF and lung smear shown less Lymphocytic infiltration as compared to Negative control group. So on the basis of all above results and statistical data were conclude that GH(200mg/kg) dose more effective as compared to GH(100mg/kg) dose for anti-bronchitis activity by CS smoke induction model. Phenols and flavonoid contains of extract may show antioxidant activity which help to decrease the inflation in bronchi.

5. CONCLUSION:

Based upon the results obtained from the present study, the GH extract 200mg/kg and salbutamol exhibits its anti- bronchitis activity against CS induced bronchitis. The treatment decreased total and differential cell count in blood and BALF, thereby showing its anti-inflammatory property. The treatment also ameliorated total protein, albumin levels, cytokines (IL-6 and immune-inflammatory responses in lung tissues. thus exhibiting its immunomodulatory property. These results suggest that, GH200g/kg effects of extract and salbutamol can be considered as an “add-on therapy” for Bronchitis or could be used along with current available anti-bronchitis drugs.

6. REFRENCES:

1. A Textbook of Pathology, Mrinalini Sant, NCBA, Page no;403-406

2. Baek, E. B., Rho, J. hyung, Jung, E., Seo, C. S., Kim, J. H., and Kwun, H. J. Protective effect of Palmijihwanghwan in a mouse model of cigarette smoke and lipopolysaccharide-induced chronic obstructive pulmonary disease. BMC Complementary Medicine and Therapies. 2021; 21(1). https://doi.org/10.1186/s12906- 021- 03453-5.

3. Ferrera M.C., Labaki W. W., and MeiLan K. Han 2021 Division of Pulmonary and Critical Care Medicine, University of Michigan, Ann Arbor, Michigan 48109 Advances in Chronic Obstructive Pulmonary Disease https://doi.org/10.1146/annurev-med- 080919-112707

4. Fitria, V., Harun, N., Gartika, L., Kaharto, N. R., Anwar Hidayat, R. K., and Nandini, L. Phytochemical Screening and Test of Mucolytic Activity of Nira Stem Sente (Allocasia Macrorrhizos) by in Vitro. Journal of Physics: Conference Series. 2019; 1179(1): 012164. https://doi.org/10.1088/1742-6596/1179/1/012164

5. Kim, V., and Criner, G. J. Chronic bronchitis and chronic obstructive pulmonary disease. In American Journal of Respiratory and Critical Care Medicine. 2013; 187(3): 228–237. https://doi.org/10.1164/rccm.201210-1843CI

6. Khan AU, Khan M, Subhan F, Gilani AH. Antispasmodic, bronchodilator and blood pressure lowering properties of Hypericum oblongifolium--possible mechanism of action. Phytother Res. 2010 Jul; 24(7): 1027-32. doi: 10.1002/ptr.3067. PMID: 19960425.

7. Morgan DL, Jokinen MP, Price HC, Gwinn WM, Palmer SM, Flake GP. Bronchial and bronchiolar fibrosis in rats exposed to 2,3-pentanedione vapors: implications for bronchiolitis obliterans in humans. Toxicol Pathol. 2012 Apr; 40(3): 448-65. doi: 10.1177/0192623311431946.

8. Mariana A. Antunes and Patrica R. M. Rocco Elastase induced pulmonary emphysema: Insight from experimental models. 2011 May 19; 83(4): 1385-1395. doi10.1590/S0001-37652011005000039.

9. Nikula, K. J., and Green, F. H. Y. Animal models of chronic bronchitis and their relevance to studies of particle- induced disease. In Inhalation Toxicology. 2000; 12.

10. Owolabi OO, James DB, Sani I, Andongma BT, Fasanya OO, Kure B. Phytochemical analysis, antioxidant and anti-inflammatory potential of Feretia Apodanthera root bark extracts. BMC Complement Altern Med. 2018 Jan 12; 18(1): 12. doi: 10.1186/s12906-017-2070-z.

11. Postma DS, Bush A, van den Berge M. Risk factors and early origins of chronic obstructive pulmonary disease. Lancet. 2015 Mar 7; 385(9971): 899-909. doi: 10.1016/S0140-6736(14)60446-3.

12. Rahaman MM, Hossain R, Herrera-Bravo J, Islam MT, Atolani O, Adeyemi OS, Owolodun OA, Kambizi L, Daştan SD, Calina D, Sharifi-Rad J. Natural antioxidants from some fruits, seeds, foods, natural products, and associated health benefits: An update. Food Sci Nutr. 2023 Jan 13; 11(4): 1657-1670. doi: 10.1002/fsn3.3217.

13. Tuon, T., Valvassori, S. S., Lopes-Borges, J., Fries, G. R., Silva, L. A., Kapczinski, F., Quevedo, J., and Pinho, R. A. Effects of moderate exercise on cigarette smoke exposure-induced hippocampal oxidative stress values and neurological behaviors in mice. Neuroscience Letters. 2010; 475(1): 16–19. https://doi.org/10.1016/j.neulet.2010.03.030

14. Vogelmeier C.F., Gerard J. Criner, Fernando J. Martinez, Antonio Anzueto, Peter J. Barnes, Jean Bourbeau, Bartolome R. Celli Global Strategy for the Diagnosis, Management, and Prevention of Chronic Obstructive Lung Disease 2017 Report. Am J Respir Crit Care Med. 2017; 195(5): 557–582.

15. Yan W, Li T, Zhong Z. Anti-inflammatory effect of a novel food Cordyceps guangdongensis on experimental rats with chronic bronchitis induced by tobacco smoking. Food Funct. 2014 Oct; 5(10): 2552-7. doi: 10.1039/c4fo00294f.

16. Zheng H, Liu Y, Huang T, Fang Z, Li G, He S. Development and characterization of a rat model of chronic obstructive pulmonary disease (COPD) induced by sidestream cigarette smoke. Toxicol Lett. 2009 Sep 28; 189(3): 225-34. doi: 10.1016/j.toxlet.2009.06.850.

Received on 11.07.2024 Revised on 03.10.2024

Accepted on 22.01.2025 Published on 08.03.2025

Available online from March 12, 2025

Res.J. Pharmacology and Pharmacodynamics.2025;17(1):79-86.

DOI: 10.52711/2321-5836.2025.00013

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

Groups	Eosinophilic infiltration	Lymphocytic infiltration	Others Changes	Grade of Infiltration
Normal Control	Not seen	Not seen	Not seen	00
Negative Control	Mild	Moderate to Sever	Minimal degree Hemorrhage	5
Standard	-	Minimal	-	1
GH (100mg/kg)	Minimal	Mild to Moderate	Minimal Degree Hemorrhage	2
GH(200mg/kg)	Minimal	Mild	Minimal Degree Hemorrhage	1