Application of Raman spectroscopy in comparative study on the influence of ibuprofen and asparagus racemosus (herbal extract) on female reproductive hormones in Wistar rats

Abstract

Hormonal variation is a condition in which the body has less or more of a specific hormone which is a major cause of miscarriages, infertility, dysmenorrhea, breast cancer, among others in females. Measurements and detection of hormonal variation are currently achieved through the use of label-dependent and non-rapid methods such as Enzyme-Linked Immunosorbent Assay (ELISA), High-Performance Liquid Chromatography (HPLC), Gas Chromatography, and Mass spectroscopy. These methods are also expensive and involve complex sample preparation. In this work, the potentiality of the Raman spectroscopic technique in a comparative study on the influence of ibuprofen and Asparagus racemosus (herbal extract) on the levels of female reproductive hormones in the blood and vaginal fluid of Wistar rats is demonstrated. Portable Raman spectroscopic devices are also available and may be calibrated to perform hormonal level variation measurements. Here small volumes of blood samples (approximately 10 μL) obtained from the tail tip of the Wistar rats were applied onto conductive silver paint smeared microscope slides and Raman spectra measured upon 785 nm laser excitation. Raman spectra from the vaginal fluid of the same animals were also obtained. Using simulate samples consisting of blood from a male Wistar rat mixed separately with the four standard female hormones (Follicle Stimulating Hormone, FSH; Luteinizing hormone, LH: Progesterone and Estradiol) at different concentrations. The concentrations were within the ranges: (0.5 - 50 mlu/ml), (0.5 - 50 mlu/ml), (0.1 - 26.6 ng/ml), and (15 – 400 pg/ml) respectively. Using both ANOVA (Analysis Of Variance) and Principal Component Analysis (PCA) loadings plots, biomarker Raman bands of each of the hormones in blood were identified. The bands which exhibited significant intensity variation with concentration were centered at wavenumbers 1291 cm-1 for FSH, 682 cm-1 for estradiol, 1625 cm-1 for LH, and 1564 cm-1 for Progesterone. These bands were ascribed to CH2 twisting vibration, C=C stretching, CH2 stretching, and C=C stretching respectively. Using a chemometric data analysis tool (Artificial Neural Networks, ANN) on the Raman spectral data set, a model for each hormone was developed using the identified biomarker Raman bands, and the Limit of Detection (LOD) for each hormone estimated as 2.126 mlu/ml, 2.494 mlu/ml, 8.31x103 ng/ml and 8x10-3 pg/ml for FSH, LH, progesterone, and estradiol respectively. These LOD values were much lower than those reported for conventional detection techniques. The developed ANN models were also used to estimate the concentration levels of the respective hormones in blood samples obtained from untreated (normal), Asparagus racemosus (herbal extract) at low- (LD), and high-dose (HD), and ibuprofen (IBU) treated Wistar rats. In general, it was found that compared to the untreated rats (Normal), administration of a high dose of Asparagus racemosus resulted in increased levels of FSH and estradiol by 5.2%, that of LH increased by 5% while that of progesterone increased by 5.2% in blood. Ibuprofen on the other hand was found to decrease the levels of FSH and LH by 5% and 4.9% and increase those of Progesterone and Estradiol by 5% and 5.1% respectively. The reproductive hormone level modification away from the normal are known to influence fertility hence from these results, administration of Asparagus racemosus and ibuprofen influence fertility. These results showed the great potential of Raman spectroscopy together with chemometrics in reproductive hormone level determination in blood. The work has also shown that the same Raman profiles of the treated and normal rats can be obtained by examining the vaginal fluid. Since the vaginal fluid is obtained non-invasively, this may be another interesting way to determine hormonal level variations. With proper calibration and validation, Raman spectroscopy can be used as a rapid (1 minute) hormone level screening tool in blood and vaginal fluid.