Supercritical Fluid Extract of Putranjiva roxburghii Wall. Seeds Mitigates Fertility Impairment in a Zebrafish Model

Abstract

Putrajeevak (Putranjiva roxburghii Wall.; synonym Drypetes roxburghii (Wall.) Hurus) seeds have been used since ancient times in the treatment of infertility in the Ayurvedic system of medicine in India. In this study, the oil component of Putrajeevak seeds (PJSO) was extracted using the supercritical fluid extraction (SCFE) method using liquid CO₂ and the constituents were analyzed using gas chromatography-flame ionized detectorand high-performance thin-layer chromatography. PJSO contained trace amounts of β-sitosterol with oleic and linoleic acids as the major fatty acid constituents. Male and female zebrafish were mutagenized with N-ethyl-N-nitrosourea (ENU) and fish that produced less than 20 viable embryos were selected for the study. SCFE oil extracts from the P. roxburghii seeds were used in this study to reverse fertility impairment. The mutant fish were fed with PJSO for a period of 14 days and the rates of fertility, conception, and fecundity were determined with wild-type healthy fish as a breeding partner. Treatment with PJSO increased the ovarian follicle count as well as the number of mature eggs, while reducing the number of ovarian cysts. Sperm count as well as sperm motility were greatly enhanced in the ENU-mutagenized male zebrafish when treated with PJSO. The results obtained in this study demonstrate the effectiveness of P. roxburghii seed oil in reversing impaired fertility in both male and female zebrafish models.

Keywords: N-ethyl-N-nitrosourea mutagenesis; Putrajeevak seed oil; Putranjiva roxburghii; follicle development; impaired fertility; sperm motility; zebrafish; β-sitosterol.

Figure 1

High-performance thin layer chromatography of Putranjiva roxburghii seed oil (PJSO) supercritical fluid extract. (A) Chromatogram showing spectral peaks and area under the curve model for known concentrations of β-sitosterol standard (green lines) and PJSO (red lines) at 450 nm. Inset shows the linearity curve of standard at various concentrations. (B) Representative photograph of TLC plate developed after derivatization with anisaldehyde sulfuric acid reagent under white light. The concentrations of standard β-sitosterol were 1000 and 1200 µg/mL. The injection volume of PJSO was 1 µL. (C) Overlay spectral scans of the developed bands on a representative TLC plate at 350 to 700 nm range. β-sitosterol standard is represented by the green line and β-sitosterol from the PJSO sample is shown in red.

Figure 1

High-performance thin layer chromatography of Putranjiva roxburghii seed oil (PJSO) supercritical fluid extract. (A) Chromatogram showing spectral peaks and area under the curve model for known concentrations of β-sitosterol standard (green lines) and PJSO (red lines) at 450 nm. Inset shows the linearity curve of standard at various concentrations. (B) Representative photograph of TLC plate developed after derivatization with anisaldehyde sulfuric acid reagent under white light. The concentrations of standard β-sitosterol were 1000 and 1200 µg/mL. The injection volume of PJSO was 1 µL. (C) Overlay spectral scans of the developed bands on a representative TLC plate at 350 to 700 nm range. β-sitosterol standard is represented by the green line and β-sitosterol from the PJSO sample is shown in red.

Figure 2

Schematic representation of the design, timelines, and endpoints for the study. (A) Study design for the fertility-impaired female fish. (B) Fertility-impaired male fish. The dosage used for the standard drug letrozole was 0.036 µg/kg/day in the female zebrafish and clomiphene at 0.36 µg/kg/day in the males. The dosages used for the P. roxburghii seed oil (PJSO) were 0.2, 0.5, 1, 5, 10, and 100 µg/kg/day.

Figure 3

Treatment with PJSO reversed fertility impairment in female zebrafish. (A) Percentage of healthy eggs per spawning was calculated from different study groups. (B) Conception rate was calculated by the number of spawning events that formed a viable embryo to the total number of spawning events. (C) The number of spawning events that led to the birth of a viable larvae from embryos to the total number of successful spawning events was calculated as the fecundity rate. Total number of breeding pairs were 24 (n = 24). Data are represented as mean ± SD and one-way ANOVA followed by Tukey’s multiple comparison test was used to arrive at statistical significance; #### represents p < 0.0001 compared to the healthy control, **** represents p < 0.0001 compared to the fertility-impaired group.

Figure 4

PJSO induced recovery of ovarian morphology in fertility-impaired female zebrafish. Representative images from the various study groups were taken with a stereomicroscope under 10× objective magnification. (A) Healthy control, (B) Fertility-impaired group, (C) Fertility-impaired fish treated with 0.036 µg/kg/day letrozole, (D) Fertility-impaired females treated with 0.2 µg/kg/day PJSO, (E) Treatment group with 0.5 µg/kg/day PJSO, (F) Treatment group with 1 µg/kg/day PJSO, (G) Treatment group with 5 µg/kg/day PJSO, (H) Treatment group with 10 µg/kg/day PJSO, and (I) Treatment group with 100 µg/kg/day PJSO. There were no outliers in each of the study groups and minor or negligible amounts of variation were seen in all ovaries from each group.

Figure 5

Cytology smear of ovary from the various treatment groups stained with Hematoxylin–Eosin and representative images were captured at 40× objective magnification. (A) Healthy control group, (B) Fertility-impaired group, (C) Letrozole treatment group (0.036 µg/kg/day), (D) Fertility-impaired females treated with 0.2 µg/kg/day PJSO, (E) Treatment group with 0.5 µg/kg/day PJSO, (F) PJSO (1 µg/kg/day) treatment group, (G) PJSO (5 µg/kg/day) treatment group, (H) PJSO (10 µg/kg/day) treatment group, and (I) Treatment group with 100 µg/kg/day PJSO.

Figure 6

Identification and quantification of ovarian cysts in the ovary of the various study groups. Ovaries were imaged under the 40× objective of a stereomicroscope and the number of cysts was quantified (C—cyst; PG—Primary growth; PV—Pre-vitellogenic; EV—Early vitellogenic; MV—Mid-vitellogenic; FG—Full growth). Representative images from (A) Healthy control group, (B) Fertility-impaired group, (C) Treatment group with 0.036 µg/kg/day letrozole, (D) Fertility-impaired female zebrafish treated with 0.2 µg/kg/day PJSO, (E) 0.5 µg/kg/day PJSO treatment group, (F) 1 µg/kg/day PJSO, (G) 5 µg/kg/day PJSO, (H) 10 µg/kg/day PJSO, and (I) 100 µg/kg/day PJSO treatment group. (J) Data are represented as mean ± SD and one-way ANOVA followed by Tukey’s multiple comparisons test was used to determine statistical significance. ## represents p < 0.01 compared to the healthy control group, #### represents p < 0.0001 compared to the healthy control group, **** represents p < 0.0001 compared to the fertility-impaired group, n = 24.

Figure 7

Representative images of Hematoxylin–Eosin-stained cytology smears of ovaries from different study groups used for quantification of pelvic inflammation. Images were taken using a 40× objective. (A) Healthy control, (B) Fertility-impaired group, (C) Fertility-impaired fish treated with 0.036 µg/kg/day letrozole, (D) Fertility-impaired females treated with 0.2 µg/kg/day PJSO, (E) Treatment group with 0.5 µg/kg/day PJSO, (F) Treatment group with 1 µg/kg/day PJSO, (G) Treatment group with 5 µg/kg/day PJSO, (H) Treatment group with 10 µg/kg/day PJSO, and (I) Treatment group with 100 µg/kg/day PJSO. (J) Percentage of pelvic inflammation is represented as mean ± SD from 24 individuals and one-way ANOVA followed by Tukey’s multiple comparisons test was used to determine statistical significance. #### represents p < 0.0001 compared to the healthy control group, **** represents p < 0.0001 compared to the fertility-impaired group.

Figure 8

P. roxburghii seed oil reverses impaired sperm count and sperm motility in male zebrafish. (A) Sperm count per mL determined in different study groups (B) Percentage motility of sperm calculated from 1 × 10⁸ sperm. Data are represented as mean ± SD from 24 individuals and one-way ANOVA was used to determine statistical significance. #### represents p < 0.0001 compared to the healthy control group, and **** represents p < 0.0001 compared to the fertility-impaired group.

Figure 9

Testicular smears stained with Hematoxylin–Eosin to determine the recovery of impaired fertility in male zebrafish when treated with Putrajeevak seed oil. Images were captured using a 40× objective and representative images from the different study groups are shown. (A) Healthy control group, (B) Fertility-impaired group, (C) Clomiphene (0.36 µg/kg/day) treatment group, (D) 0.2 µg/kg/day PJSO treatment group, (E) PJSO 0.5 µg/kg/day treatment group, (F) PJSO 1 µg/kg/day treatment group, (G) 5 µg/kg/day PJSO treatment group, (H) 10 µg/kg/day PJSO treatment group, and (I) PJSO at 100 µg/kg/day treatment group.