Antiestrogenic and Anti-Inflammatory Potential of n-Hexane Fraction of Vitex negundo Linn Leaf Extract: A Probable Mechanism for Blastocyst Implantation Failure in Mus musculus

Abstract

The anti-implantation potential of different fractions of Vitex negundo Linn leaf extract was evaluated in female Swiss Albino mice. Animals from different groups were dosed orally either with 0.2% agar (vehicle) or with fractions of V. negundo leaf extract (n-hexane, chloroform, n-butanol, and remnant fractions) at 10:00 a.m., from day 1 to day 6 of pregnancy. The pregnant females from each group were sacrificed on different days of pregnancy (n = 6), and uterus was excised and used for estimation of lipid peroxidation and assay of superoxide dismutase activity as a marker for blastocyst implantation. Animals treated with n-hexane fraction showed altered level of superoxide anion radical and superoxide dismutase activity as compared to control animals. The probable mechanism by which this extract exhibits inhibition of blastocyst implantation is through the anti-inflammatory and antiestrogenic potential.

Figure 1

(a) TLC fingerprint profile of n-hexane fraction of Vitex negundo leaves. (1) Lupeol standard; (2) and (3) n-hexane fraction; (4) β-sitosterol standard. (b) TLC densitometric chromatogram of n-hexane fraction of Vitex negundo at 525 nm. a: Lupeol standard; b: n-hexane fraction; c: β-sitosterol. (c) Structure of lupeol and β-sitosterol.

Figure 2

Uterus of (a) control mice showing blastocyst implantation sites; (b) n-hexane fraction of Vitex negundo Linn leaf extract treated mice, not showing any implantation site; (c) chloroform fraction; (d) n-butanol fraction; and (e) remnant fraction of Vitex negundo Linn leaf extract treated mice shows partial inhibition of implantation.

Figure 3

Graph showing total number of implants in uterus of (A) control mice, (B) n-hexane fraction, (C) chloroform fraction, (D) n-butanol fraction, and (E) remnant fraction of Vitex negundo Linn leaf extract treated mice, respectively. ^* P < 0.05 was considered significant when compared to group of control animals.

Figure 4

(a) Superoxide dismutase (SOD) activity and lipid peroxidation (LPO; MDA levels) on different days of pregnancy (days 1–6) in uterus of control animals (n = 6) (r = −0.902). Selected area shows “window of implantation.” (b) Superoxide dismutase (SOD) activity and lipid peroxidation (LPO; MDA levels) on different days of pregnancy (days 1–6) in uterus of n-hexane fraction of Vitex negundo Linn leaf extract treated animals (n = 6) (r = 0.157).

Figure 5

Uterine weight in untreated, control (extract alone), ethinyl estradiol plus n-hexane fraction of Vitex negundo Linn leaf extract and ethinyl estradiol alone treated animals, respectively (n = 6). ^{#, ∗} P < 0.05 was considered significant when compared to ethinyl estradiol alone treated animals.