Indian Journal of Ayurveda & Research

Immuno-modulatory Activity of Dushivishari Agada, an Ayurveda preparation in Allergic asthma induced experimentally in wistar rats.

Santosh  F. Patil1, S. K. Hiremath2 , Banappa Unger3 , Kalanghot P. Skandan

  1. Department of Agadatantra, KAHER’s Shri B. M. Kankanwadi Ayurveda Mahavidyalaya Belagavi , Karnataka India – 590003
  2. Department of Agadatantra, Taranath Government Ayurvedic Medical college. Bellary Karnataka, India – 533101
  3. I C M R – National Institute of Traditional Medicine Belagavi, Karnataka, India
  4. KAHER’s Shri B. M. Kankanwadi Ayurveda Mahavidyalaya, Belagavi, Karnataka, India – 590003

Address for correspondence:
Dr Santosh F Patil, Department of Agadatantra, KAHER’s Shri B. M. Kankanwadi Ayurveda Mahavidyalaya, Belagavi, Karnataka, India- 590003. Email : dr.santosh19@gmail.com

Financial support: None, Conflict of Interest: None

                                                                                      ABSTRACT.
Background: Asthma is chronic airway inflammatory disorder hallmarked with altered tracheo-bronchial response. WHO estimated three hundred million people suffer from Asthma all over the world and several medicines are available. In Ayurveda, the ancient system of Medicinal Science, this condition is well narrated and treatment offered is Dushivishari Agada (DVA) in powdered form.

Methodology: In this study we evaluated the effect of DVA in allergic asthma by experimentally inducing it in wistar rats. Animals were divided into four groups. Normal group (G I) was given orally normal saline for sensitization. In three experimental groups (G II,G III,G IV), asthma was induced by sensitizing with intra-peritoneal  Ovalbumin (OVA) and aluminium hydroxide and after 15 days they were challenged with OVA-PBS (Phosphate buffer solution) aerosols for 8 consecutive days. Animals in G II served as asthma control while G III and IV received DVA (250 mg) and Dexamethasone DXM (10mg/kg) respectively. In blood total and differential count, absolute eosinophil count (AEC), Serum IgE was done. Cellular count was done in Broncho-alveloar lavage fluid (BALF) and lungs underwent histological analysis.

Results DVA significantly reduced serum IgE (3.33 ± 1.08), AEC (145 ± 81.27) and inflammatory cells in BALF when compared to G II [Serum IgE (14.17± 1.16) and AEC (379.2 ± 206.4)] and was in comparison with G IV [Serum IgE (4.017± 0.68), AEC (50 ± 38.73)]

Conclusion DVA has shown immune-modulatory effect by bringing down inflammation in allergic bronchial asthma.

Keywords: Allergy, Asthma, Immuno-modulatory, Dushivishari agada

How to cite this article: Patil S F, Hiremeth S K, Unger B, Skandan K P. Immuno – modulatory activity of Dushiishari agada an Ayurveda preparation in Allergic asthma induced experimentally in wistar rats. Indian J Ayurveda Res 2018;1:19-24.

Introduction
Asthma incidence is approximated to increase by 100 million people by 2025, adding on to present prevalence of 300 million people worldwide (WHO 2004). It has clouded the patients by increasing the disability-adjusted life years (DALYs).[1]
Genetic predisposition is one of the factors of increased prevalence of asthma among children which may be responsible for it. External factors like environmental air (which contain subtle particulate matter, toxins and stable dust) and daily exposures to fumes or smoke emitted by the mosquito repellents etc causes airway hyper-responsiveness (AHR). In changed lifestyle, the available food and cold drinks are rich in omega-6 polyunsaturated fatty acids, preservatives (MSG, Sodium benzoate) and residual pesticides causing an imbalance between Th1/Th2 immune response.[2,3,4]
The treatment of asthma has evolved through times with administration of corticosteroids and bronchodilators ranging from oral, intravenous to inhalation route. However the treatment available is not fully satisfactory and come with their own local and systemic side effects.[5,6] Ayurveda, the oldest system of medical practice, believed the mechanism of asthma is developed by toxins (Dushivisha-DV). DV is type where toxin is in transformational stage which is more of like a pathophysiological presentation of disease produced in body. It is said that this toxin manifests its effects after sometime. Interestingly Ayurveda says that this DV presents or aggravates as a disease in episodic fashion when person gets exposed to environmental factors (like eastern winds, cloudy days, polluted climate and surrounding climate) or indigenous factors (indigestion, unwholesome diet fried foods). They also identified excessive sexual activity/arousal and sleep during day time aggravates the condition.[7,8]
Dushivishari Agada (DVA) is one such compound formulation indicated for DV condition which consists of 13 drugs [9] as given in Table 1. Fine powders of all the drugs were taken in equal proportion.

Table 1: Ingredients of Dushivishari Agada with Part used. 

Sl.no Dravya                      Scientific name Part used
1. Pippali Piper longum Linn. Phala (Fruit)
2. Pippalimula Piper longum Linn. Mula (root)
3. Dhyamaka Cymbopogon martinii (Roxb.) Wats. Patra (Leaves)
4. Jatamamsi Nardostachys jatamamsi DC(N. grandiflora) Mula (Root)
5. Lodra Symplocos racemosa Roxb. Twak (Stem Bark)
6. Ela Elettaria cardamomum Maton Phala (Fruit)
7. Suvarchika Tribulus terrestris Linn. Phala (Fruit), Mula (Root)
8. Katunnatum Oroxylum indicum (Linn) Benth.Ex Kurz. Mulatwak (Root bark)
9. Natam Valeriana wallichii D.C. Mula (Root)
10. Kusta Saussurea lappa C.B.  Clarke. Mula (Root)
11. Yastimadhu Glycyrrhiza glabra Linn. Mula (Root)
12. Rakhtachandana Pterocarpus santalinus Linn. f. Khandasara (Heartwood)
13. Gairika Red ochre

Individually some of these drugs have also shown anti-inflammatory, immune-modulatory, anti-histaminic or broncho-dilatory effects.[10,11,12,13,14]
Asthma is developed due to one or more factors of DV.  WHO advocated, reducing the disability brought about by morbidity and premature mortality caused by chronic disorders of respiratory system. It also warrants complimentary and alternate medicine having role in managing asthma.[15,16]
In the present experimentation an Ayurveda preparation, DVA was evaluated in  allergic asthma.

Materials and Methods
Preparation of DVA:  All the drugs of DVA were collected and authenticated at AYUSH approved Ayurveda Siddha & Unani drug testing Laboratory of Shri B M Kanakanwadi Ayurveda Mahavidyalaya, A constituent unit of KLE Academic of Higher Education and Research, Karnataka, India.  Tablets were prepared by following classical method of trituration to paste form with decoction of same drugs.[17]

Experimentation:
The experimental design was approved by the institutional animal ethical committee (BMK/IAEC/Res-07/2011). A total number of 24 male rats (Wistar, 250 ± 20 gm) were divided into four groups of six each.
Single dose intra-peritoneal injection of mixture 40 mg of Ovalbumin (OVA) (Sigma life sciences Chemical Co USA 62-88% purity analytical grade) and 2 mg of Aluminium hydroxide (SD Fine Chemicals, Mumbai) was given to immunize all animals in G II, III & IV. After fifteen days, for 8 consecutive days (day 15 – 22) animals were challenged with 1% OVA in phosphate-buffered saline (PBS) aerosol, in histamine chamber (aerosol flow rate is approximately 0.4ml/min and the particle size is app. 5um.) once daily for 30 min/day.  Animals of G III received DVA orally for 22 days at the dose of 1.08 g /kg/day in the split with Honey (0.54g/kg/day) as vehicle; G IV (Ova–DXM) animals received Dexamethasone (DXM) at the dose of 10 mg/kg on last three days of study (day 20 – 22), before the aerosol challenge. Control group animals (G I) were challenged with sterile saline aerosol. (Table 2). On 24th day of study the blood and tissue samples were collected.[18]

 Table no. 2: Showing the Schedule of inducing allergic asthma and treatment

Day 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
G I Sensitized on day 1 and rest till Day 15
(Normal Saline IP)
Challenge with normal saline aerosol  

 

 

 

R

E

S

T

 

S

A

C

R

I

F

I

C

E

GII Sensitized on day 1 and rest till Day 15
( Ovalbumin 40mg + Al2(OH3) IP)
Challenge with OVA + PBS aerosol
Treated orally with Normal saline till day 22
GIII Sensitized on day 1 and rest till Day 15
( Ovalbumin 40mg + Al2(OH3) IP)
Challenge with OVA+ PBS Aerosol
Treated with DVA (250 mg/day in divided dose) till day 22 (honey as vehicle)
GIV Sensitized on day 1 and rest till Day 15
( Ovalbumin 40mg + Al2(OH3) IP)
Challenge with OVA + PBS aerosol
DXM(10mg/kg) on days 20 to 22

Intermittently throughout the days of experimentation the subjective parameters like nasal irritation, crepts and wheezing (lung auscultation) and sleeping pattern were observed and noted down.

Parameters:
Blood was collected from retro-orbital plexus after giving light anaesthesia for total count, differential count, AEC and Serum IgE.
The rats were euthanized and then the trachea was cannulised. Right bronchi of all animals was secured by tying before lavaging and taken for histopathology study. Five point scoring system was used to score leukocyte permeation into Peri-bronchial space. Leukocyte score was given after examining three independent fields of lung section from each rat. Left lung was used for collection of Broncho-alveolar lavage fluid (BALF). 15 mL of ice-cold phosphate buffer saline (PBS) was used as lavaging fluid, for five times. Approximately 10 mL of BALF was recovered which was collected in a sterile test tube. Collected BALF was centrifuged at 4°C (1500 rpm) for 10 minutes for leukocytes collection. These cells were re-suspended in 100 μL of PBS for relative and total leukocyte counts using a haemocytometer which were counted in 10-square chambers.[19]
Data obtained and collected are expressed as Mean ± Standard Deviation of mean (SD). One way ANOVA followed by Newman-Keuls Multiple Comparison Test was applied for data analysis. P < 0.05 was considered to be significant.

Observations.
Gradual increase in subjective parameters like wheezing and crepts on auscultation was noted in Group II, III, and IV from 3rd day of aerosol exposure. Wheezing and crepts were comparatively less in Group III during challenge and reduced in G IV on day 24th after administration of DXM. Another notable observation of G IV group was that after 3 days of DXM administration weight loss was seen in rats which was not in G III. The normal sleeping and resting style of rats was changed during the aerosol challenge as depicted in image (Fig 1)

Results
Investigations are tabled of Blood investigation in Table 3, BALF in Table 4 and cell infiltration in lung tissue in Table 5.

Table no 3: Blood Total count, Differential count, Absolute eosinophil count and IgE expressed in Mean ± SD

Parameters Group I Group II Group III Group IV
Total count cells/mcl 13600 ± 3389 13267 ± 4070 12100± 3630 20700± 7366*
Neutrophil cells/mcl 17.33 ± 5.92 11.67 ± 2.42 20.33 ± 11.33 69.17±6.27
Lymphocyte cells/mcl 77.67 ± 8.04 84 ± 2.36 76.67 ± 10.88 27.17± 5.11‡
Eosinophil cells/mcl 1.833 ± 0.75 2.2 ± 0.83 1 ± 0.63* 0.3333 ± 0.51
Monocytes cells/mcl 3.167 ± 2.78 1.5 ± 0.83 2 ± 0.63 3.333 ± 1.86
AEC cells/mcl 216.7 ± 73.6 379.2 ± 206.4 145.8 ±81.27 50 ± 38.73
IgE UI/ml 12 ± 1.89 14.17 ± 1.16 3.333± 1.08‡ 4.017 ± 0.68
* p < 0.05 when Compared to Group II
†  p < 0.01 when Compared to Group II
‡ p < 0.001 when Compared to Group II

Table no 4: Broncho-Alveolar Lavage Fluid Total count, differential count.

Parameters Group I Group II Group III Group IV
Total count cells/mcl 120 ± 48.48 370 ± 224.4 479.2 ± 98 520.8 ± 227.2
Neutrophil cells/mcl 3.167 ± 2.13 5.5 ± 2.34 5 ± 2.82 8.667 ± 3.50
Lymphocyte cells/mcl 13.5 ± 4.88 16.67 ± 6.05 14.33 ± 5.88 20.17 ± 7.38
Eosinophil cells/mcl 0 ± 0 3 ± 1.67 1.667 ± 1.03 1.833 ± 0.98
Monocytes cells/mcl 83.33 ±  6.05 74.83 ± 9.36 79 ± 6.41 69.33 ± 11.08

Table no 5: Cell infiltration into Lung tissue.

Parameters Group I Group II Group III Group IV
Cells infiltrated 0.7 ± 0.10 2.367 ± 0.23 1.467 ± 0.10** 1.433± 0.19**

** p < 0.01 when Compared to Group II

 Discussion:
Dushivisha (DV) is denatured, attenuated, latent or vitiated poison. It is a state where poison originating from inanimate and animate sources or any concocted poison stays in the body after incomplete expulsion or which has undergone interim detoxification by anti-poisonous/antidote therapy or any poison that has been battered by forest fire, wind or sun externally and then enters the body or has inherent low potency and settles in body.[20]
According to Ayurveda, this low potency of poison is enveloped/ anchored dormant by the mechanism of immunity (Prakrut Kapha dosha) and stays in body for chronic period without causing any grave signs or sudden death.[21]
Ayurveda explains Prakruta kapha is immunity and it is considered as physical and physiological strength. If a change occurs in climate, region, place, diet or lifestyle, the immunity gets deranged that means body becomes susceptible to existing settled toxins. They in turn take over compromised immunity.[22] Ayurveda says low potent garavisha leads to DV which can lead to allergic respiratory presentations. In this study DVA was evaluated for its effect over DV by inducing allergic asthma in Wistar rats.
Asthma is a chronic airway inflammatory disorder characterized by inflammation, persistent hyper-responsiveness caused by antigen-antibody (IgE) complex on the surface of mast cells. This triggers eosinophil to release mediators to cause airway inflammation and spasm. Any drug to combat asthma needs a potent immune modulatory activity to establish its therapeutic utility in a long term rather than having a mere broncho-dilating action.[23]
Administration of DVA significantly reduced IgE, Absolute eosinophil count, BALF Eosinophil count in G III [(3.33 ± 1.08), (145 ± 81.27), (1.67 ± 1.03)]   which are allergic inflammation markers when compared to G II [(14.17± 1.16), (379.2 ± 206.4), (3 ± 1.67)] and was equivalent to standard G IV [(4.017± 0.68), (50 ± 38.73), (1.83 ± 0.98)].
Th2 cells orchestrates the pathophysiology of asthma by down signaling B lymphocytes to produce IgE mediated by IL-4 and through IL-5 to proliferate, activate, migrate and infiltrate airways. Thus DVA may be acting by inhibiting/down regulating Th2 lymphocyte activation and its downstream signaling.[24]
In G IV BALF neutrophil count (8.6±3.5) is on high side which accords with findings of Tanizaki et al [25] that there is increased number of BALF neutrophil in glucocorticoid treated patients. Corticosteroid therapy acts only on eosinophilic driven asthma while that of neutrophilic is non-responding and hence count remains high leading to further inflammation.[26] In G III both BALF Neutrophil (5 ± 2.8) and eosinophil count (1.67 ± 1.03) are reduced when compared to G II BAL fluid Neutrophil (5.5 ± 2.3 ) and eosinophil count (3 ± 1.67)  respectively suggesting that test drug may act on both the inflammatory cells.
Dexamethasone is known to cause immediate decrease in circulating lymphocytes (27.17 ± 5.11), Eosinophil (0.33 ± 0.15) and increase in neutrophils (69.17 ± 6.27) as also seen in Group IV. While in G III circulating lymphocytes (76.67 ± 10.88), eosinophil (1 ± 0.63) and neutrophil (20.33 ± 11.3) was not altered indicating that it may not suppress first line of defense and may not pave for opportunistic infections hence having advantage over standard drug dexamethasone. The rise seen in neutrophil may be immature cells put out by bone marrow to compensate lost cells in inflammatory reaction. This pathway could pave the chances for opportunistic infections.[27]
In BAL Fluid total count is raised in G III (479 ± 98) and IV (520.8 ± 227.2) when compared to G II (370 ± 224.4). Subsequent to inflammation, repair process is initiated to clear injured cells which involve multiple stages from cell proliferation to deposition. Common feature in inflammatory reactions is that of rise in leukocyte count which sometimes maybe extraordinarily high if bacterial infection is associated. Chronic inflammation may be insidious or a sequel of acute inflammation which is associated with presence of lymphocytes and macrophages.[28]Even if we see the results of lymhpocytes in BAL Fluid of G III (14.33 ± 5.88) in comparison with group II (16.67 ± 6.0) and G IV (20.17 ± 7.38) it indicates that test drug DVA not allowed inflammation to become chronic.
Administration of DVA also showed reduced infiltration of eosinophil at peribronchial and perivascular region in bronchial tissue (and BAL Fluid) in comparison with G II as evidenced by histopathology results in G III (Figure 2).
Conclusion
Precise mechanisms of chronic airway inflammation in asthma are yet to be understood and drugs formulated to combat the disease asthma needs to have a potent immunosuppressant activity without side-effect so as to establish its therapeutic utility in the long term rather than having a mere broncho-dilating action. Primary promising results of DVA warrants it’s utility in asthma as anti-inflammatory and anti-allergic.

Financial Support and Sponsorship.  
This experiment was not funded.

Conflicts of Interest.
Authors have no competing interests.
FIG 1 : Showing changed resting style  of rat during nebulization.

Figure 2 :Histopathology of Cell Infiltration in Lung: (Images Taken at 40X )

The right bronchi were obtained for histological studies day after the last OVA challenge Tissues were stained with hematoxylin and eosin. Peri-bronchial cells were counted using a five-point scoring system to estimate the severity of leukocyte infiltration. The leukocyte scoring was examined in three independent fields of lung section from each rat. Inflammatory cell infiltration in the lung tissues was scored as described in the method section.
Image A is Group 1 which was normal control, Image B- Group 2 Disease control, Image C- Group III Test drug DA, Image D- Group 4 Standard drug DXM.

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