Combination of Ononis hirta and Bifidobacterium longum decreases syngeneic mouse mammary tumor burden and enhances immune response

The resistance of solid tumors to conventional therapies has prompted the need for alternative therapies. To evaluate in vitro and in vivo effect of extracts from Ononis hirta against resistant mouse mammary gland cell line (66 cl-4-GFP) and to use a combination of Ononis hirta extract with Bifidobacterium longum to target resistant solid tumors in mice. Different solvent extracts of Ononis hirta were prepared and their in vitro antiproliferative activity was tested against 66 cl-4-GFP cell line using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Thin layer chromatography (TLC) and high-performance liquid chromatography (HPLC) were used to identify the active extracts. Balb/C mice were transplanted with 66 cl-4-GFP cell line and in vivo antitumor activity was assessed for the plant extract, Bifidobacterium longum, and a combination of plant extract and Bifidobacterium longum. Histological examination of tumors was performed using standard hematoxylin/eosin staining protocol while gram stain was used to detect the presence of anaerobic bacteria in these sections. A combination of Ononis hirta methanol extract and Bifidobacterium longum showed high ability in targeting solid mammary gland tumors in mice. It also induced extensive necrosis in these tumors. Thirty percent of mice treated with such combination were cured of their cancers. The mechanism underlying this anticancer activity involves immune system activation exemplified by the observed rejection of reinoculated tumors by cured mice. Chemical TLC analysis of the active methanol extract showed the presence of flavonoids, terpenoids, and alkaloids. HPLC analysis confirmed the presence of flavonoids and alkaloids in Ononis hirta methanol extract. The complete regression of the tumor is encouraging and shows that plant extracts in combination with Bifidobacterium longum is an inviting option to treat solid tumors.