Exploration of Solanum xanthocarpum Schrad. & Wendl. against Mycobacterium avium Subspecies paratuberculosis and Assessment of Its Immunomodulatory and Anti-Inflammatory Potential

Abstract

Mycobacterium avium subspecies paratuberculosis (MAP), being a dairy-borne pathogen, resistant of pasteurization and other sterilization techniques, is a major cause for development of inflammatory bowel disorders such as Johne's disease (JD) in dairy animals and Crohn's Disease (CD) in humans, for which no therapy is available to date. In the absence of effective therapy or a vaccine, management of CD has been accomplished by removal of the affected intestines. However, usually, even after removal of 2/3 of the intestine, CD reoccurs. Hence, there exists a need to develop an alternative therapy for such infection. The potential of herbals remains unexplored against MAP and related infections. Therefore, the conducted study is a novel initiative for the evaluation of anti-mycobacterial activity of bioactive extracts of Solanum xanthocarpum Schrad. & Wendl. against MAP infection. The said plant was authenticated according to the Ayurvedic Pharmacopoeia of India. Qualitative and quantitative evaluation of the extracts were done using chromatographic and spectroscopic techniques. Preliminary in vitro pharmacological assessments revealed the immunomodulatory and anti-inflammatory potential of the extracts. REMA assay was conducted to determine their anti-MAP activity along with determination of the best active extract. The hydro-alcoholic extract showed the best inhibition of MAP, providing a potential ray of hope against this emerging major pathogen of animals, and associated with Crohn's disease and other autoimmune disorders in human beings.

Keywords: Crohn’s disease (CD); Johne’s disease (JD); Mycobacterium avium subspecies paratuberculosis (MAP); Solanum xanthocarpum Schrad. & Wendl.; anti-inflammatory; immunomodulatory.

Figure 1

Microscopic evaluation of the whole plant of Solanum xanthocarpum Schrad. & Wendl. (A) Transverse section of stem which shows the presence cork (Ck) having thin-walled hexagonal cells followed by 6–7 layers of cork cambium (Cb). Phloem (Pb) shows the presence of fibres Xylem (Xy) shows the presence of tracheids, vessels, fibres and parenchymatous cells traversed by xylem rays. Pith (Pt) is wide, located in the centre. (B) Transverse section of fruit showing a single layer of epidermis (Ep) covered with a thin cuticle followed by a layer of collenchymatous cells (Co). The endosperm (En) shows the presence of oil globules. (C) Transverse section of leaf midrib having biconvex shape illustrating the presence of bicollateral vascular bundles (Vb), mesophyll cells (MP) along with palisade parenchyma (Pp) and spongy parenchyma (Sp). (D,E) Transverse sections of root depicting the presence of central pith (Pt), medullary rays (Mr), cork (Ck) comprised of thin-walled rectangular cells, pericycle (Pc), stone cells (Sc), calcium oxalate crystals (Ca) as sandy masses and simple starch grains (Sg) in the secondary cortex.

Figure 2

Assessment of free radical scavenging activity using the DPPH assay (% DPPH inhibition).

Figure 3

In vitro immunomodulatory assessment and action on pinocytes.

Figure 4

Confirmation of M. paratuberculosis at the molecular level. (A) Microscopy (Ziehl-Neelsen stain). (B) Conventional PCR (IS900 413 bp) where Lanes M–7 represents 1 kb DNA ladder, control, positive control, sample 1 (DNA from Solid culture of MAP strain ‘S5’ batch 1), sample 2 (DNA from solid culture of MAP strain ‘S5’ batch 2), sample 3 (DNA from liquid culture of MAP strain ‘S5’ batch 1), sample 4 (DNA from liquid culture of MAP strain ‘S5’ batch 2) and blank respectively. (C) Amplification of RT-PCR (IS900 413 bp); (D) Melt Peak of RT-PCR (IS900 413 bp).

Figure 5

REMA assay. (A) In vitro activity of test extracts, i.e., A.Q (aqueous), H.A (hydro-alcoholic) and Alco (alcoholic) extracts of Solanum xanthocarpum Schrad. & Wendl. (B) In vitro activity of standard anti-mycobacterial first line drugs, i.e., Rifampicin (225 mg), Isoniazid (150 mg), Pyrazinamide (750 mg), Ethambutol hydrochloride (400 mg) and second line drugs, i.e., Clarithromycin (250 mg), Ofloxacin (200 mg).

Figure 6

TLC photographic representation and quantitative estimation of biomarkers Solamargine, Solasonine Solasodine in Solanum xanthocarpum Schrad. & Wendl. aqueous (S1), ethanolic (S2) and hydro-ethanolic (S3) extracts respectively at 540 nm post derivatization using butanol: ethyl acetate: 10% acetic acid (5:3.5:1.5).