Phytochemical Composition, Antioxidant, Antimicrobial and in Vivo Anti-inflammatory Activity of Traditionally Used Romanian Ajuga laxmannii (Murray) Benth. ("Nobleman's Beard" - Barba Împăratului)

Abstract

In the Romanian folk medicine, aerial parts of Ajuga laxmannii ("nobleman's beard," Romanian - "barba boierului" or "avrămească" or "creştinească") are traditionally used as galactagogue and anti-inflammatory agents. The present study aimed to evaluate the chemical composition (polyphenols, iridoids, and phytosterols), antioxidant, antimicrobial and in vivo anti-inflammatory activity of different extracts of A. laxmannii aerial parts. The major identified bioactive compounds were rutin, 8-O-acetylharpagide and β-sitosterol. The antioxidant activity of A. laxmannii extracts was evaluated using several methods, and the results showed good antiradical effects. Moreover, the antimicrobial evaluation showed a potent antifungal activity against C. albicans and P. funiculosum. Furthermore, the anti-inflammatory effect was determined by monitoring some parameters involved in the inflammatory process. The results obtained showed differences between the analyzed extracts; and therefore the importance of choosing the best solvent in order to extract the appropriate amount of bioactive compounds. A. laxmannii ethanol extract showed an anti-inflammatory effect by reducing total leukocytes, PMN, phagocytosis, and oxidative stress. Compared to diclofenac, only the 50 mg/mL A. laxmannii extract had better anti-inflammatory and anti-oxidative stress effects, and this could justify the importance of a correlation between the activity and the used concentration. These findings strongly suggest that A. laxmannii could be considered as a valuable source of bioactive compounds, which could be further valued as anti-inflammatory agents in the composition of several herbal drugs.

Keywords: Ajuga laxmannii; anti-inflammatory; antimicrobial; iridoids; polyphenols; sterols.

FIGURE 1

HPLC chromatogram of polyphenols from A. laxmannii aerial parts extract. The identified compounds: chlorogenic acid (1), isoquercitrin (2), rutin (3), quercitrin (4), luteolin (5), apigenin (6).

FIGURE 2

White blood cells (WBC) count. A.l. 100% – the animals received 5 mL/kg BW A. laxmannii ethanol extract 100% (100 mg dw/mL), A.l. 50% – the animals received 5 mL/kg BW A. laxmannii ethanol extract 50% (50 mg dw/mL), A.l. 25% – the animals received 5 mL/kg BW A. laxmannii ethanol extract 25% (25 mg dw/mL), Inflam – inflammation was induced by i.m. injection of turpentine oil (6 mL/kg BW), Diclo – the animals received 20 mg/kg BW diclofenac (p < 0.001).

FIGURE 3

Polymorphonuclear leukocytes (PMN). A.l. 100% – the animals received 5 mL/kg BW A. laxmannii ethanol extract 100% (100 mg dw/mL), A.l. 50% – the animals received 5 mL/kg BW A. laxmannii ethanol extract 50% (50 mg dw/mL), A.l. 25% – the animals received 5 mL/kg BW A. laxmannii ethanol extract 25% (25 mg dw/mL), Inflam – Inflammation was induced by i.m. injection of turpentine oil (6 mL/kg BW), Diclo – the animals received 20 mg/kg BW diclofenac (p < 0.001).

FIGURE 4

Total number of monocytes. A.l. 100% – the animals received 5 mL/kg BW A. laxmannii ethanol extract 100% (100 mg dw/mL), A.l. 50% – the animals received 5 mL/kg BW A. laxmannii ethanol extract 50% (50 mg dw/mL), A.l. 25% – the animals received 5 mL/kg BW A. laxmannii ethanol extract 25% (25 mg dw/mL), Inflam – Inflammation was induced by i.m. injection of turpentine oil (6 mL/kg BW), Diclo – the animals received 20 mg/kg BW diclofenac (p < 0.001).

FIGURE 5

Phagocytic activity (PA) of A. laxmannii extract. A.l. 100% – the animals received 5 mL/kg BW A. laxmannii ethanol extract 100% (100 mg dw/mL), A.l. 50% – the animals received 5 mL/kg BW A. laxmannii ethanol extract 50% (50 mg dw/mL), A.l. 25% – the animals received 5 mL/kg BW A. laxmannii ethanol extract 25% (25 mg dw/mL), Inflam – Inflammation was induced by i.m. injection of turpentine oil (6 mL/kg BW), Diclo – the animals received 20 mg/kg BW diclofenac (p < 0.001).

FIGURE 6

Phagocytic index (PI) of A. laxmannii extract. A.l. 100% – the animals received 5 mL/kg BW A. laxmannii ethanol extract 100% (100 mg dw/mL), A.l. 50% – the animals received 5 mL/kg BW A. laxmannii ethanol extract 50% (50 mg dw/mL), A.l. 25% – the animals received 5 mL/kg BW A. laxmannii ethanol extract 25% (25 mg dw/mL), Inflam – Inflammation was induced by i.m. injection of turpentine oil (6 mL/kg BW), Diclo – the animals received 20 mg/kg BW diclofenac (p < 0.001).

FIGURE 7

Total oxidative status (TOS). A.l. 100% – the animals received 5 mL/kg BW A. laxmannii ethanol extract 100% (100 mg dw/mL), A.l. 50% – the animals received 5 mL/kg BW A. laxmannii ethanol extract 50% (50 mg dw/mL) (p < 0.001), A.l. 25% – the animals received 5 mL/kg BW A. laxmannii ethanol extract 25% (25 mg dw/mL) (p < 0.01), Inflam – Inflammation was induced by i.m. injection of turpentine oil (6 mL/kg BW), Diclo – the animals received 20 mg/kg BW diclofenac (p < 0.001).

FIGURE 8

Total antioxidant response (TAR). A.l. 100% – the animals received 5 mL/kg BW A. laxmannii ethanol extract 100% (100 mg dw/mL), A.l. 50% – the animals received 5 mL/kg BW A. laxmannii ethanol extract 50% (50 mg dw/mL), A.l. 25% – the animals received 5 mL/kg BW A. laxmannii ethanol extract 25% (25 mg dw/mL), Inflam – Inflammation was induced by i.m. injection of turpentine oil (6 mL/kg BW), Diclo – the animals received 20 mg/kg BW diclofenac (p > 0.05).

FIGURE 9

Oxidative stress index (OSI). A.l. 100% – the animals received 5 mL/kg BW A. laxmannii ethanol extract 100% (100 mg dw/mL), A.l. 50% – the animals received 5 mL/kg BW A. laxmannii ethanol extract 50% (50 mg dw/mL), A.l. 25% – the animals received 5 mL/kg BW A. laxmannii ethanol extract 25% (25 mg dw/mL), Inflam – Inflammation was induced by i.m. injection of turpentine oil (6 mL/kg BW), Diclo – the animals received 20 mg/kg BW diclofenac (p < 0.001).

FIGURE 10

NO synthesis. A.l. 100% – the animals received 5 mL/kg BW A. laxmannii ethanol extract 100% (100 mg dw/mL), A.l. 50% – the animals received 5 mL/kg BW A. laxmannii ethanol extract 50% (50 mg dw/mL), A.l. 25% – the animals received 5 mL/kg BW A. laxmannii ethanol extract 25% (25 mg dw/mL), Inflam – Inflammation was induced by i.m. injection of turpentine oil (6 mL/kg BW), Diclo – the animals received 20 mg/kg BW diclofenac (p < 0.001).