Pharmacological Potential of Hippophae rhamnoides L. Nano-Emulsion for Management of Polycystic Ovarian Syndrome in Animals' Model: In Vitro and In Vivo Studies

Abstract

The most common female endocrinopathy, polycystic ovarian syndrome (PCOS), generally affects women of childbearing age. Hippophae rhamnoides L. has been traditionally used to improve menstrual cyclicity. Gas chromatography by flame ionization detection analysis showed that it contained various phytoconstituents such as omega-3 fatty acid, phytosterols, palmitic acid, oleic acid, and linoleic acid. H. rhamnoides L. (HR) nano-emulsion was also formulated. HR and its encapsulated nano-emulsion (HRNE) were evaluated for the treatment of PCOS. Thirty-five healthy female adult albino rats were acquired and divided into seven groups (n = 5). Letrozole (1 mg/kg) was used for 5 weeks to induce the disease. To confirm disease (PCOS) induction, the animals were weighed weekly and their vaginal smears were analyzed daily under a microscope. After PCOS induction, animals were treated with metformin, HR, and HRNE with two different doses (0.5/kg and 1 g/kg, p.o.) for 5 weeks. At the end of the treatment, animals were euthanized, and blood was collected for hormonal assessment, lipid profiling, and liver functioning test assessment. Both the ovaries were preserved for histopathology and liver for the purpose of assessment of antioxidant potential. The results revealed that HR and HRNE at both doses improved the hormonal imbalance; follicle-stimulating hormone, estrogen, and progesterone levels are increased, while luteinizing hormone surge and testosterone level are controlled. Insulin sensitivity is improved. Ovarian histopathology showed that normal ovarian echotexture is restored with corpus luteum and mature and developing follicles. HR and HRNE also improved the lipid profile and decreased lipid peroxidation (MDA) with improved antioxidant markers (SOD, CAT, and GSH). Results were statistically analyzed by one-way analysis of variance and were considered significant only if p < 0.05. In conclusion, it can be postulated that H. rhamnoides L. proved effective in the management of PCOS and its nano-emulsion effects were statistically more significant, which might be due to better bioavailability.

Figure 1

GC-FID analysis of H. rhamnoides L. by GC-FID.

Figure 2

Structures of phytoconstituents present in H. rhamnoides L.

Figure 3

Characterization of H. rhamnoides L. Nano-emulsion (A) DSC, (B) TGA, and (C) FTIR of HR oil, PEG400, cinnamon oil, Tween 80, and HR nano-emulsion. DSC of nano-emulsion showed no endothermic peak, indicating the uniform distribution of HR oil, and TGA of the final formulation showed a two-step degradation. Characteristic peaks of HR oil also shown in the FTIR spectra of nano-emulsion indicated the chemical stability of formulation.

Figure 4

Effect of H. rhamnoides L. on body weight changes after disease induction. Values are expressed as mean ± SEM (n = 5). α designated a significant difference from N.C., while β designated a significant difference from the PCOS group (disease control). One-way analysis of variance (ANOVA) was used to evaluate the statistical significance between the groups, and multiple comparison was done by Tukey’s post-hoc test. N.C.: normal control group, HR: H. rhamnoides L., HRNE: H. rhamnoides L. nano-emulsion formulation.

Figure 5

Vaginal cytology of a normal control rat demonstrating the different estrous cycle phases. Vaginal smears of rats’ estrous cycle presenting different estrous phases. (A) In the proestrus phase, cornified epithelial cells were predominately nucleated (green arrows). (B) Cornified epithelial cells are classed as the estrus phase (_*). (C) The diestrus phase primarily consists of leukocytes (red arrows). (D) Cornified epithelial cells and nucleated cornified cells are all existing in the metestrus stage.

Figure 6

Vaginal cytology of diseased rats (PCOS group with the diestrus phase).

Figure 7

Microscopic examination of a cross section of the ovary of a rat stained by hematoxylin–eosin. (A) Ovarian section of control group rats exhibiting normal morphology of the rat ovary with different stages of ovarian follicles showed atretic follicles, developing follicles, and corpus luteum. (B) PCOS group showing typical cystic follicles and disorganized granulosa cells. (C) Ovarian section of the metformin-treated rat showed corpus luteum and less number of cystic follicles with developing follicles. (D) Ovarian section of rats treated with (H. rhamnoides L.) HR (0.5 g/kg) showed reduction in the number of cystic follicles and more developing follicles, primary follicles, and atretic follicles. (E) Ovarian section of HR (1 g/kg) treated rats showed the presence of developing follicles, primary follicles, and atretic follicles. (F) Ovarian section of HR 0.5 g/kg, nano-emulsion showed a good number of developing, primary, and atretic follicles. (G) Section of nano-emulsion (1 g/kg) showed corpus luteum, primary and developing follicles, and oocyte with a lesser number of cystic follicles. CF: cystic follicles, AF: atretic follicles, DF: developing follicles, CL: corpus luteum, PF: primary follicles, GF: growing follicles, Oo: oocyte.

Figure 8

Effect of H. rhamnoides L. on the serum hormone level: (A) FSH, (B) LH, (C) estrogen, (D) progesterone, (E) testosterone, and (F) insulin. Values are expressed as mean ± SEM (n = 5). α designated a significant difference from N.C., while β designated a significant difference from the PCOS group (disease control). δ designated a significant difference from the HR group. One-way analysis of variance (ANOVA) was used to evaluate the statistical significance between the groups, and multiple comparison was done by Tukey’s post-hoc test. N.C.: normal control group, HR: H. rhamnoides L., HRNE: H. rhamnoides L. nano-emulsion formulation.

Figure 9

Effect of H. rhamnoides L. on the lipid profile. Values are expressed as mean ± SEM (n = 5). α designated a significant difference from N.C., while β designated a significant difference from the PCOS group (disease control). δ designated a significant difference from the HR group. One-way analysis of variance (ANOVA) was used to evaluate the statistical significance between the groups, and multiple comparison was done by Tukey’s post-hoc test. N.C.: normal control group, HR: H. rhamnoides L., HRNE: H. rhamnoides L. nano-emulsion formulation.

Figure 10

(A–D) Effect of H. rhamnoides L. on oxidative stress and lipid peroxidation markers. Values are expressed as mean ± SEM (n = 5). α designated a significant difference from N.C., while β designated a significant difference from the PCOS group (disease control). δ designated a significant difference from the HR group. One-way analysis of variance (ANOVA) was used to evaluate the statistical significance between the groups, and multiple comparison was done by Tukey’s post-hoc test. N.C.: normal control group, HR: H. rhamnoides L., HRNE: H. rhamnoides L. nano-emulsion formulation. CAT: catalase, GSH: glutathione, MDA: malondialdehyde, SOD: superoxide dismutase.

Figure 11

Effect of H. rhamnoides L. on liver functioning tests (LFTs). Values are expressed as mean ± SEM (n = 5). α designated a significant difference from N.C., while β designated a significant difference from the PCOS group (disease control). δ designated a significant difference from the HR group. One-way analysis of variance (ANOVA) was used to evaluate the statistical significance between the groups, and multiple comparison was done by Tukey’s post-hoc test. N.C.: normal control group, HR: H. rhamnoides L., HRNE: H. rhamnoides L. nano-emulsion formulation, AST: aspartate aminotransferase, ALT: alanine transaminase.