Nutrients and phytochemicals characterisations, acute and sub-acute oral toxicity studies of BobyGuard C, a polyherbal nutraceutical with anti-breast cancer properties

Abstract

Background: In 2022, approximately 2.3 million new cases of female breast cancer and 670,000 related deaths worldwide despite significant advancements in conventional treatments. BobyGuard C (BGC) is a novel polyherbal nutraceutical formulated from five plants, selected for their antioxidant, anticancer, anti-inflammatory and nutritional properties to be used for breast cancer management. This study aimed to characterize its physicochemical, nutritional, and phytochemical properties as well as assess its safety through acute and sub-acute oral toxicity studies in Wistar rats.

Methods: Thecomposition of BGC was analyzed for macronutrients, minerals, and phytochemicals using standard methods. Antioxidant activity was assessed through DPPH, TAC and FRAP assays, while antiproliferative activity was evaluated using the MTT assay on MDA-MB 231 and MCF-7 breast cancer cell lines. Acute (single 5,000 mg/kg dose with 14 days observation) and sub-acute oral (daily administration of 784, 1,568, and 3,136 mg/kg for 28 days) toxicity studies in female Wistar rats followed OECD guidelines.

Results: BGC was found to be rich in proteins (38.36 g/100 g), carbohydrates (59.70 g/100 g), and essential minerals such as magnesium (60,066.67 µg/100 g), and it was free from toxic heavy metals. Several bioactive compounds, including diosgenin, diosbulbin H, β-carotene, Bafoudiosbulbin G and catechin were identified in BGC. Phytochemical analysis revealed high levels of phenols (9,783.48 mg GAE/100 g), flavonoids (47.72 mg QuE/100 g), and alkaloids (106.14 mg berberine eq/100 g), contributing to its strong antioxidant activity (DPPH inhibition: 90.39%). BGC exhibited significant antiproliferative effects on MDA-MB 231 cells, highlighting its potential anticancer activity. Acute toxicity tests showed no mortality at 5,000 mg/kg, with an LD₅₀ exceeding this dose. In the sub-acute 28-day repeated-dose oral study, doses up to 3,136 mg/kg/day resulted in some dose dependent hematological and biochemical changes but no histopathological abnormalities were observed indicating its safety at lower doses.

Conclusion: BGC is a nutritionally rich formulation with potent antioxidant and anticancer potential, demonstrating a favorable safety profile at lower dose (784 mg/kg).

Keywords: BobyGuard C; acute toxicity; nutrient; phytochemical; standardization; subacute toxicity.

FIGURE 1

(A) Base peak chromatogram for methanol extract in (−)-ESI mode, portraying annotated compounds from BGC. Growth of estrogen sensitive MCF-7 (B) and non-sensitive MDA-MB 231 (C) breast carcinoma cells treated with different concentrations of BGC after 24, 48 and 72 h; Cell proliferation of estrogen sensitive MCF-7 (D) and non-sensitive MDA-MB 231 (E) breast carcinoma cells. Cells were treated with BGC at 5, 10 and 20 μg/mL after 48 h. Controls remained untreated (n = 3). Treated cancer cell cultures were compared to non-treated control cultures of the same passage and cell numbers per well. ***p < 0.001 compared to control.

FIGURE 2

(A) Bodyweight of rats treated with a single dose of BobyGuard C (5000 mg/kg). (B) Weight change of BobyGuard (C) treated rats in sub-acute toxicity study. (C) Kidney, liver, heart, lung and spleen microarchitectures after treatment with BobyGuard C (784, 1,568 and 3,136 mg/kg). (D) Ovary, uterus and hippocampus microarchitectures after treatment with BobyGuard C (784, 1,568 and 3,136 mg/kg) BGC5000 (receiving BobyGuard C orally at a dose of 5,000 mg/kg daily). NOR (receiving 2% ethanol (vehicle)); BGC784 (receiving BobyGuard C orally at a dose of 784 mg/kg daily); BGC1568 (receiving BobyGuard C orally at a dose of 1,568 mg/kg daily) and BGC3136 (receiving BobyGuard C orally at a dose of 3,136 mg/kg daily). #p < 0.05, ##p < 0.01 and ###p < 0.001 compared to the normal control (NOR). Kidney: G = Glomerulus, Eu = Urinary Space, Liver: Vp = Portal Vein, He = Hepatocyte, Cb = Bile Duct, Ah = Hepatic Artery. Spleen: Pb = White Pulp, Pr = Red Pulp, Lung: Ep = Pulmonary Epithelium, Sa = Alveolar Sac. CA1: Nn = Normal Neuron; CA3: Nn = Normal Neuron, Dentate Gyrus: Cg = Granule Cell Layer, Cp = Polymorphic Cell Layer, Cm = Molecular Layer. Uterus: Lu = Uterine Lumen; En = Endometrium; St = Stroma.