ABSTRACT
Ajowan (Trachyspermum ammi L.) spice has been used in food preparations and also as a traditional medicine in Ayurveda. Although a number of pharmacological activities have been attributed to ajowan, its role in immunomodulation is not known. The main objective of the present study is to examine the macromolecular immunomodulatory components. Macrophage activation was studied by nitric oxide (NO) release, phagocytosis and secretion of pro-inflammatory cytokines as the markers. Ethanol precipitate (fractional) of ajowan aqueous extract was subjected to conventional chromatography (Q Sepharose followed by Bio-Gel P-100). One of the proteins (30.7 kDa; ajowan glycoprotein or Agp) showed effective mitogenic activity towards splenocytes. Agp is a O-linked glycoprotein with the glycans contributing to one-third of the molecular mass. It has a high content of glutamic acid, serine, aspartic acid and proline whereas galactose (45.7%), arabinose (34.5%), glucose (7%), mannose (5%) and xylose (4%) are the constituent sugars. Secondary structure analysis indicated that Agp contains 79% α-helices and 21% random coil. Internal sequencing of the tryptic peptides did not show homology with the existing proteins in the database (BLAST). Agp at 1 µg/mL induced proliferation of B-cell enriched murine splenocytes and activated macrophages in releasing NO and promoted phagocytosis (p < 0.01). RAW 264.7 cells produced pro-inflammatory cytokines (IL-12, TNF-α and IFN-γ) at 1 µg/mL Agp (p < 0.01). Deproteinized Agp (dpAgp) failed to elicit activation of murine immune cells, whereas deglycosylated Agp (20 kDa; dgAgp) showed compromised efficiency. This is the first report of an immunomodulatory protein from ajowan.
Subject(s)
Carum/metabolism , Glycoproteins/chemistry , Glycoproteins/isolation & purification , Immunologic Factors/chemistry , Immunologic Factors/isolation & purification , Amino Acid Sequence , Animals , Carbohydrates/analysis , Cell Proliferation/drug effects , Circular Dichroism , Cytokines , Glycoproteins/metabolism , Glycoproteins/pharmacology , Glycosylation , Immunologic Factors/pharmacology , Macrophages, Peritoneal/drug effects , Macrophages, Peritoneal/metabolism , Male , Mice , Mice, Inbred BALB C , Nitric Oxide/metabolism , Phagocytosis/drug effects , Plant Extracts/chemistry , Protein Structure, Secondary , RAW 264.7 Cells , Rats, Wistar , Spleen/cytology , WaterABSTRACT
Biological activities of alkali extracted (Barium hydroxide: BE-480 kDa, Potassium hydroxide: KE1-1080 and KE2-40 kDa), purified arabinoxylans (AX) from the finger millet bran varying in their molecular weight, phenolic acid content, arabinose to xylose ratios were evaluated for their immune-stimulatory activities using murine lymphocytes and peritoneal exudate macrophages. All three purified AX displayed significant (p < 0.001) mitogenic activity and activation of macrophages including phagocytosis. Among these BE has shown higher enhancing lymphocyte proliferation (>2 fold) and macrophage phagocytosis than KE1 and KE2. The above results clearly documented that the immunostimulatory activity of arabinoxylans is directly proportional to the amount of ferulic acid content (0.11 mg/100 g), whereas molecular weight as well as arabinose/xylose ratio, did not have any bearing. Purified AX from the finger millet bran can be explored as a potent natural immunomodulator.
