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

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

Acharya Balkrishna 1,2, Pradeep Nain 1, Monali Joshi 1, Lakshmipathi Khandrika 1 and Anurag Varshney 1,2,*

1 Drug Discovery and Development Division, Patanjali Research Institute, NH-58, Haridwar 249 405, Uttarakhand, India; [email protected] (A.B.); [email protected] (P.N.); [email protected] (M.J.); [email protected] (L.K.) 2 Department of Allied and Applied Sciences, University of Patanjali, Patanjali Yog Peeth, Rorkee-Haridwar Road, Haridwar 249 405, Uttarakhand, India * Correspondence: [email protected]; Tel.: +91-1334-244107 (ext. 7458)

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

Citation: Balkrishna, A.; Nain, P.; count as well as the number of mature eggs, while reducing the number of ovarian cysts. Sperm Joshi, M.; Khandrika, L.; Varshney, A. count as well as sperm motility were greatly enhanced in the ENU-mutagenized male zebrafish when Supercritical Fluid Extract of treated with PJSO. The results obtained in this study demonstrate the effectiveness of P. roxburghii Putranjiva roxburghii Wall. Seeds seed oil in reversing impaired fertility in both male and female zebrafish models. Mitigates Fertility Impairment in a Zebrafish Model. Molecules 2021, 26, Keywords: Putranjiva roxburghii; Putrajeevak seed oil; N-ethyl-N-nitrosourea mutagenesis; impaired 1020. https://doi.org/10.3390/ fertility; zebrafish; β-sitosterol; follicle development; sperm motility molecules26041020

Academic Editor: Eva Tvrdá Received: 19 January 2021 1. Introduction Accepted: 9 February 2021 Published: 15 February 2021 Putrajeevak (Putranjiva roxburghii Wall.; synonym Drypetes roxburghii (Wall.) Hurus) seeds have been used in the traditional Indian system of Ayurveda since ancient times for

Publisher’s Note: MDPI stays neutral the treatment of infertility in both males and females. Many classical medicinal texts such with regard to jurisdictional claims in as Candra-Nighan. t.u (10th Century A.D.), Nighan. t.u´ses.a (12th Century A.D.), Saraswat¯ı published maps and institutional affil- Nighan. t.u(16th Century A.D.), and others have an extensive discussion on the use of iations. P. roxburghii in alleviating infertility. The plant belongs to the Euphorbiaceae family and is native to Southeast Asia, the Indian subcontinent, Japan, southern China, and New Guinea. The deseeded and powdered fruit is used against cough, cold, and celiac disease [1]. Its antipyretic and anti-inflammatory properties are thought to be useful in fertility and gynecological ailments [2]. Several different phytochemicals have been identified from Copyright: © 2021 by the authors. P. roxburghii Licensee MDPI, Basel, Switzerland. root, leaf, and stem including glycosides, saponins, triterpenes, and flavonoids. This article is an open access article Though the seeds are consumed, not much is known about the fatty acids and other distributed under the terms and constituents of the oil present in the seeds. This is the first time that the fatty acids conditions of the Creative Commons and phytosterol constituents of P. roxburghii seeds were extracted and identified. The Attribution (CC BY) license (https:// supercritical fluid (SCF) extraction method for oils is an environmentally safe technique creativecommons.org/licenses/by/ and has several advantages over traditional extraction processes [3]. Compared to the 4.0/). traditional solvent-based extraction system, SCF extraction is more robust due to less

Molecules 2021, 26, 1020. https://doi.org/10.3390/molecules26041020 https://www.mdpi.com/journal/molecules Molecules 2021, 26, 1020 2 of 23

variations from batch to batch. Supercritical fluids under pressure have a higher diffusivity compared to normal solvents and therefore can penetrate porous solid materials more effectively [4]. The use of food-grade liquid carbon dioxide in the extraction process at very mild temperatures protects the heat sensitive constituents from degradation and increases their yield in the final extract. However, one of the drawbacks of using CO2 as a solvent is that it is non-polar, and therefore, extraction of certain polar compounds may be suboptimal [5]. In our study, the oil from P. roxburghii (Putrajeevak) seeds extracted using the supercritical fluid extraction process was analyzed for its constituent fatty acids and sterols. The oil (Putrajeevak Seed Oil, PJSO) was then used for assessing its properties in reversing the fertility impairment seen in N-ethyl-N-nitrosourea (ENU) mutagenized zebrafish. Ze- brafish are versatile as a model organism due to their high reproductive rate and fecundity, and being vertebrates, they can be easily correlated with higher vertebrates. The causes for infertility are numerous and can vary from ovarian defects such as polycystic ovary syndrome, a diminished ovarian reserve of eggs that may be related to aging, and the presence of immature eggs. Though the underlying causes of such defects are not known, it can either be genetic or lifestyle-related. In males, the cause of infertility could generally be due to low sperm count or genetic disorders that can lead to lower sperm production or immotile sperm. ENU mutagenesis is a widely accepted method to induce random point mutations [6] that can be reversed and the method can be easily modified to reduce the mortality that is generally seen in such mutagenesis methods. The mutants that impact the fertility seem to target spermatogenesis in males, while altering the ovarian morphology and cytology in females [7]. In the current study, we have used ENU mutagenesis to generate fertility impairment mutations in both females and males. The impaired mutants that generated less than 20 embryos were selected for treatment with the Putrajeevak seed oil. Letrozole, an aromatase inhibitor, was used as a standard reference drug for the female infertility study group. Letrozole has been shown to have a better number of live births in women with polycystic ovary syndrome [8]. Clomiphene is an anti-estrogen thought to increase the quality of sperm in cases of male infertility [9] and was used as a reference drug for the male infertility study. The study was aimed at not only understanding the phytochemical composition of the supercritical fluid extract of the seeds but also to substantiate the traditional Ayurvedic knowledge of the use of the seeds to restore fertility. We have demonstrated the recovery of fertility, conception, and fecundity rates in fertility-impaired zebrafish upon treatment with Putrajeevak seed oil. We also show the recovery of ovarian and testicular cytology upon treatment.

2. Results 2.1. Fatty Acid Content in Supercritical Fluid Extract of Putrajeevak Seeds Several batches of extraction were performed and an average yield of 3% oil was obtained. The batch of oil used for these studies was obtained from powdered seeds (2 kg) of Putrajeevak (Putranjiva roxburghii Wall.) using a supercritical CO2 extraction procedure to obtain 60 gm of the oil extract (PJSO) with a yield of 3%. For identification and quantification of the fatty acid constituents present in the oil extract, the AOCS official method, Ce 2-66, was used to produce fatty acid methyl esters (FAME). The mixture from the PJSO was compared with a standard mixture (37 FAMEs) using GC-FID. The chromatography analysis identified four fatty acids present in high quantities and two others in relatively lower amounts (Table1). The other 31 fatty acids, according to the standard mix, were below the quantitation limits of the assay. Molecules 2021, 26, x FOR PEER REVIEW 3 of 25

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Molecules 2021, 26Molecules, 1020Table 2021 , 1.26 Fatty, x FOR acid PEER constituents REVIEW of the supercritical fluid extract of Putrajeevak seeds and their quantity as identified3 of3by 23of 25 Molecules 20212021,, 2626,, xx FORFOR PEERPEER REVIEWREVIEW 33 ofof 2525 Tablegas c hromatography1. Fatty acid constituents-flame ionized of the d etectsuperioncritical. The chemicalfluid extract structures of Putrajeevak were sourced seeds fromand theithe rRoy quantityal Society as identified of Chemistry by Tablegwebsiteas chromatography 1. Fatty(www.chemspider.com acid constituents-flame ionized of; J anuarythe detect superion 19,c.ritical 2021The chemical). fluid extract structures of Putrajeevak were sourced seeds from and the thei Royr quantityal Society as ofidentified Chemistry by gwebsiteas chromatography (www.chemspider.com-flame ionized; January detect 19,ion 2021. The). chemical structures were sourced from the Royal Society of Chemistry TableName 1. FattyMolecular acid constituents Formula of Molecular the super cWeightritical f luid extract of ChemicalPutrajeevak Structure seeds and their quantityContent as identified in PJSO by (%) Table 1. FattyTablewebsite acid constituents1. ( Fattywww.chemspider.com acid constituents of the supercritical ; ofJanuary the super 19, fluid c2021ritical extract). fluid of extract Putrajeevak of Putrajeevak seeds andseeds their and quantitytheir quantity as identified as identified by by NameTablegas chromatography 1. FattyMolecular acid constituents-f lameFormula ionized ofMolecular the detect superion Weightc.ritical The chemical fluid extract structures ofChemical Putrajeevak were sourcedStructure seeds from and thethei Royr quantityal SocietyContent as ofidentified Chemistry in PJSO by (%) gas chromatography-flamegasas cchromatography ionized--fflamelame detection. iionized d Theetectetect chemicalionion.. TheThe chemicalchemical structures structuresstructures were sourced werewere sourcedsourced from the fromfrom Royal thethe Roy Societyalal SocietySociety of Chemistry ofof ChemistryChemistry PalmiticNamewebsite Acid (www.chemspider.com Molecular C16H Formula32O2 ; MolecularJanuary256.4 19, Weight 2021 ). Chemical Structure Content in8.67 PJSO (%) website (www.chemspider.comwebsite (www.chemspider.com (accessed on;; JJanuaryanuary 19 January 19,19, 20212021 2021)).).). Palmitic Acid C16H32O2 256.4 8.67 Name Molecular Formula Molecular Weight Chemical Structure Content in PJSO (%) Name Molecular Formula Molecular Weight Chemical Structure Content in PJSO (%) NamePalmiticName Acid Molecular MolecularC16 FormulaH 32FormulaO2 MolecularMolecular256.4 WeightWeight Chemical Chemical Structure ContentContent in 8.67in PJSO PJSO (%) (%) Stearic Acid C18H36O2 284.48 8.67 Palmitic Acid C16H32O2 256.4 8.67 Stearic Acid C1618H3236O2 284.48 8.67 Palmitic Acid C16H32O2 256.4256.4 8.678.67 Palmitic Acid C16H32O2 256.4 8.67 Stearic Acid C18H36O2 284.48 8.67 Oleic Acid C18H34O2 282.5 49.29

Stearic Acid C18H36O2 284.48 8.67 Stearic AcidOleic Acid C18HC1836HO36342 O2 284.48282.5 8.6749.29 StearicStearic AcidAcid C18H36O2 28284.484.48 8.678.67

Oleic Acid C18H34O2 282.5 49.29 Oleic AcidOleic Acid C18HC1834HO342O2 282.5282.5 49.2949.29 18 34 2 Oleic Acid C18H34O2 282.5282.5 49.2949.29

Linoleic Acid C18H32O2 280.4 31.74

Linoleic AcidLinoleic Acid C18HC1832HO322O2 280.4280.4 31.7431.74

Linoleic Acid C18H32O2 280.4 31.74 Linoleic Acid C18H32O2 280.4 31.74 Linoleic Acid C18H32O2 280.4 31.74

Linolenic Acid C18H30O2 278.4 0.81

LinolenicLinolenic Acid Acid C18HC1830HO302 O2 278.4278.4 0.810.81

Linolenic Acid C18H30O2 278.4 0.81 Cis-11,14-Ei- Cis-11,14-EicosenoicLinolenic Acid Acid C20HCC183620HOH30236OO2 2 308.5278.4308.5 0.790.810.79 18 30 2 LinolenicCiscosenoic-11,14 Acid -AcidEi- C18H30O2 278.4278.4 0.810.81 C20H36O2 308.5 0.79 cosenoicCis-11,14 Acid-Ei- C20H36O2 308.5 0.79 cosenoicCis-11,14 Acid-Ei- Cis-11,14-Ei- C20H36O2 308.5 0.79 Cis-11,14-Ei- 2.2.20 PJSO36 2 Contains2.2. PJSO Trace Contains Amounts Trace Amounts of β-Sitosterol of β-sitosterol cosenoic Acid C20H36O2 308.5308.5 0.790.79 cosenoiccosenoic AcidAcid 2.2. PJSO Contains Trace Amounts of β-sitosterol High-performanceHigh-performance thin layer thin chromatography layer chromatography (HPTLC) (HPTLC) analysis analysis of PJSO of PJSO was was per- per-

formed with2.2.formed chloroform:methanolPJSOHigh with Contains-performance chloroform Trace Amountsthin:methanol (9:1)layer of as cβ hrom(9:1)- thesitosterol as mobileatography the mobile phase (HPTLC) phase (Figure (Figure analysis1 A). 1A). After of AfterPJSO derivati- derivatiza- was per- 2.2.formedtion PJSOHigh with with Contains- ptheerformance chloroform Anisaldehyde Trace Amountst:mhinethanol lSulayer fofuric (9:1)cβhrom-sitosterol acid asatography the reagent mobile (Figure (HPTLC) phase 1B), (Figure analysis a spectral 1A). of After scanPJSO derivatiza- (350 was to per- 700 zation with2.2. the PJSO Anisaldehyde Contains Trace SulfuricAmounts of acid β--sitosterolsitosterol reagent (Figure1B), a spectral scan (350 to formedtionnm) withHigh was with - theperformedperformance c Anisaldehydehloroform to t:detectmhinethanol Sullayer βfuric-sitoste c(9:1)hrom acid asrol atography reagentthe along mobile with(Figure (HPTLC) phase a standard 1B), (Figure analysisa spectral solution 1A). of Afterscan PJSO of (350knownderivatiza- was to per- 700con- 700 nm) was performedHigh-performance to detect thinβ -sitosterollayer chrom alongatography with (HPTLC) a standard analysis solution of PJSO of known was per- formedtionnm)centration Highwithwas with performed-thep erformance(Figure c Anisaldehydehloroform 1C). to detect t:Thehinmethanol Sullstandardayer β-fsitosteuric c(9:1)hrom acid curverol asatography reagentthealong showed mobile with (Figure (HPTLC) good phasea standard 1B), linearity (Figure analysisa spectral solution 1A).with of After scan 400 PJSOof knownto (350derivatiza- 1200was to per-con- ng700 of concentrationformed (Figure with 1cC).hloroform The standard:methanol curve (9:1) showedas the mobile good phase linearity (Figure with 1A). 400 After to 1200derivatiza- ng formedtionnm)centrationthe withwasstandard. with performedthe (Figure c Anisaldehydehloroform The 1C). band to Thedetect:m intensityethanol standardSul βf-uricsitoste (9:1)obtained acidcurverol as reagentthealong showedby mobile densitometric with (Figure good phasea standard linearity1B), (Figure analysis a spectral solution with 1A). was 400After scan of plotted to known derivatiza-(350 1200 toagainst ng con- 700 of of the standard.tion with The the band Anisaldehyde intensity obtainedSulfuric acid by densitometric reagent (Figure analysis 1B), a spectral was plotted scan (350 against to 700 tionnm)centrationthethe standard. withwas standard performedthe (Figure AnisaldehydeThe curve band1C). to to The detectintensity derive standardSul β f the-uricsitoste obtained con acidcurverolcentration reagentalong byshowed densitometric with (Figure of good βa- sitosterolstandard linearity1B), analysis a spectral insolution with the was PJSO400scan plottedof to known sample.(3501200 against to ng con- 700 Theof the standardnm) curvewas performed to derive to the detect concentration β-sitosterol along of β-sitosterol with a standard in the solution PJSO sample. of known The con- nm)centrationtheamount standard. standardwas performedwas (Figure curveThecalculated band1C). toto deriveThedetect intensity to bestandard β0.98% the-sitoste obtainedcon incurvecentrationrol the along sbyshoweduper densitometric withc ofritical βgood -asitosterol standardfluid linearity analysis extract in solution with the ofwas PJSOPutrajeevak 400 plottedof to sample.known 1200 against seeds.ng con- The of amount wascentration calculated (Figure to be 1C). 0.98% The standard in the supercritical curve showed fluid good extract linearity of Putrajeevak with 400 to 1200 seeds. ng of centrationtheamount standard. standard was (Figure calculated curveThe band1C). to The derive tointensity be standard 0.98% the obtained con in curve centrationthe s uperbyshowed densitometricc ritical of good β-sitosterol fluid linearity eanalysisxtract in with theof was Putrajeevak PJSO 400 plotted to sample. 1200 against seeds. ng The of the standard. The band intensity obtained by densitometric analysis was plotted against thetheamount standard. standard was calculated The curve band to derive tointensity be 0.98% the obtained con in centrationthe s byuper densitometricc ritical of β-sitosterol fluid eanalysisxtract in the of was Putrajeevak PJSO plotted sample. against seeds. The 2.3. P. roxburghiithe standard Seed Oil curve Increases to derive the Number the concentration of Positive of Spawning β-sitosterol Events in the in thePJSO sample. The theamount standard was calculated curve to deriveto be 0.98% the con in centrationthe superc ritical of β-sitosterol fluid extract in the of Putrajeevak PJSO sample. seeds. The Fertility-Impairedamount Femalewas calculated and Male to Zebrafish be 0.98% in the supercritical fluid extract of Putrajeevak seeds. amount was calculated to be 0.98% in the supercritical fluid extract of Putrajeevak seeds. Fertility impairment was induced in the zebrafish using ENU mutagenesis as de-

scribed in the methods section. Female fish that produced less than 20 embryos when used in breeding events with healthy males and vice versa were used in the study. A total of 550 females were exposed to the ENU mutagenesis protocol, out of which 542 survived. The fish were rested for 14 days, after which the fertility rate was assessed over two ovulation cycles. The number of female mutagenized fish with fertility impairment as judged by those producing less than 20 embryos was 413. Treatment with ENU followed by metronidazole in the male fish led to 519 survivors and 450 males that produced less than 20 embryos when mated with healthy females. Once the required number of mutagenized fish were randomized into eight groups with 24 fish each, they were treated with human relevant dosage of letrozole (reference drug for females) or clomiphene (reference drug for males) and six different dosages of the PJSO. A schematic of the study is shown in Figure2.

Molecules 2021, 26, 1020 4 of 23 Molecules 2021, 26, x FOR PEER REVIEW 4 of 25

A 450 nm

β - Sitosterol

(400 - 1200 μg/mL) PJSO Area (AU)Area

Track distance (mm)

B β - Sitosterol PJSO

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Figure 1. High-performance thin layer chromatography of Putranjiva roxburghii seed oil (PJSO) supercritical fluid extract. Figure 1. High-performance(A) Chromatogram thin showing layer spectral chromatography peaks and area of unPutranjivader the curve roxburghii model for known seed oilconcentrations (PJSO) supercritical of β-sitosterol ﬂuid extract. (A) Chromatogramstandard showing (green lines) spectral and PJSO peaks (red lines) and at area450 nm. under Inset shows the the curve linearity model curve forof standard known at various concentrations concentrations. of β-sitosterol (B) Representative photograph of TLC plate developed after derivatization with anisaldehyde sulfuric acid reagent under standard (greenwhite lines) light. and The PJSO concentrations (red lines) of standard at 450 nm.β-sitosterol Inset were shows 1000 the and linearity 1200 µg/mL. curve The injection of standard volume at ofvarious PJSO was concentrations.1 (B) RepresentativeµL. photograph(C) Overlay spectral of TLC scans plate of the developed bands after on a derivatization representative TLC with plate anisaldehyde at 350 to 700 nm sulfuricrange. β-sitosterol acid reagent under white light. Thestandard concentrations is represente ofd by standard the green lineβ-sitosterol and β-sitosterol were from 1000 the PJSO and sample 1200 isµ showng/mL. in red. The injection volume of PJSO was 1 µL. (C) Overlay spectral scans of the2.3. developed P. roxburghii bands Seed Oil on In acreases representative the Number of TLC Positive plate Spawning at 350 Events to 700 in nm the range.Fertility-β-sitosterol standard is represented by the greenImpaired line and Femaleβ-sitosterol and Male fromZebrafish the PJSO sample is shown in red. Fertility impairment was induced in the zebrafish using ENU mutagenesis as de- scribed in the methods section. Female fish that produced less than 20 embryos when used in breeding events with healthy males and vice versa were used in the study. A total of 550 females were exposed to the ENU mutagenesis protocol, out of which 542 survived. The fish were rested for 14 days, after which the fertility rate was assessed over two ovulation cycles. The number of female mutagenized fish with fertility impairment as judged by those producing less than 20 embryos was 413. Treatment with ENU followed by metronidazole in the male fish led to 519 survivors and 450 males that produced less than 20 embryos when mated with healthy females. Once the required number of mutagenized fish were randomized into eight groups with 24 fish each, they were treated with human relevant dosage of letrozole (reference drug for females) or clomiphene (reference drug for males) and six different dosages of the PJSO. A schematic of the study is shown in Figure 2.

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FigureFigure 2. 2.Schematic Schematic representation representation ofof thethe design,design, timelines, and endpoints for for the the study. study. ( (AA)) Study Study design design for for the the fertility fertility-- impairedimpaired female female fish. fish. (B ) ( Fertility-impairedB) Fertility-impaired male male fish. Thefish. dosage The dosage used for used the standard for the standard drug letrozole drug wasletrozole 0.036 µ wasg/kg/day 0.036 inµg/kg/day the female in zebrafishthe female and zebrafish clomiphene and clomiphene at 0.36 µg/kg/day at 0.36 µg/kg/day in the males. in the The males. dosages The dosages used for used the P.for roxburghii the P. roxburghiiseed oil seed oil (PJSO) were 0.2, 0.5, 1, 5, 10, and 100 µg/kg/day. (PJSO) were 0.2, 0.5, 1, 5, 10, and 100 µg/kg/day.

ThroughoutThroughout thethe 14-day14-day dosing dosing period, period the, the ﬁsh fish in in the the different different groups groups were were monitored moni- fortored mortality for mortality each day.each Alongday. Along with with the controlthe control group, group, the the fertility-impaired fertility-impaired group, group, the groupthe group treated treated with with letrozole, letrozole, and and the t differenthe differentP. roxburghii P. roxburghiiseed seed oil-treated oil-treated groups groups did did not shownot show any any mortality mortality and and were were 100% 100% viable. viable. TheThe numbernumber of positivepositive spawning events events that that resulted resulted in in the the formation formation of of embryos embryos waswas recordedrecorded andand expressedexpressed as a percentage. The The female female healthy healthy control control grou groupp had had 100% 100% spawningspawning events,events, while thi thiss was just just 83.3% 83.3% (Table (Table 22).). Treatment with either l letrozoleetrozole or or PJSOPJSO restoredrestored thethe numbernumber of positive spawning events to to 100%. 100%. TheThe controlcontrol males males showed showed a positivea positive spawning spawning rate rate of 100%,of 100% with, with all 24all breeding 24 breeding pairs producingpairs producing embryos. embryos. The fertility-impaired The fertility-impaired group group showed showed a lower a lower positive positive spawning spawning rate ofrate 75%, of 75%, with with only only 18 males 18 males producing producing viable viable spermatozoa spermatozoa to to produce produce embryos embryos (Table (Table2). Treatment2). Treatment with with clomiphene clomiphene showed showed a slightly a slightly better better positive posit spawningive spawning rate rate at 88%, at 88%, with 21with breeding 21 breeding pairs pairs showing showing positive positive spawning. spawning. This This increase increase was was statistically statistically signiﬁcant signifi- (cantp < 0.0001) (p < 0.0001)when when compared compared to the to fertility-impaired the fertility-impaired group. group. The differentThe different dosages dosages of PJSO of showedPJSO showed a dose-dependent a dose-dependent increase, increase, with the with 0.2 µtheg/kg/day 0.2 µg/kg/day dosage dosage showing show onlying a modestonly a

Molecules 2021, 26, 1020 6 of 23

increase with sperm from 19 males (79.1%) successfully forming embryos. Treatment with 0.5 µg/kg/day PJSO showed a positive spawning rate comparable to that of clomiphene at 91.6% (22 of 24 males). PJSO at 1 µg/kg/day had 23 with positive spawning (95.8%), while 5, 10, and 100 µg/kg/day showed complete recovery of the fertility impairment.

Table 2. Percentage of positive spawning events in the different study groups. The formation of an embryo irrespective of the health of the embryo was considered as a positive spawning event (n = 24, p < 0.0001).

Percentage of Positive Breeding Study Groups Female Male Control 100 100 Fertility-Impaired Model 83.3 ± 37.2 #### 75 ± 43.3 #### Reference Standard 100 87.5 ± 33.0 **** PJSO (0.2 µg/kg) 100 79.1 ± 40.6 **** PJSO (0.5 µg/kg) 100 91.6 ± 27.6 **** PJSO (1 µg/kg) 100 95.8 ± 19.9 **** PJSO (5 µg/kg) 100 100 PJSO (10 µg/kg) 100 100 PJSO (100 µg/kg) 100 100 #### represents signiﬁcance compared to the healthy control group; **** represents signiﬁcance compared to the fertility-impaired group.

2.4. Putrajeevak Seed Oil Increases the Fertility and Fecundity in Fertility-Impaired Zebrafish Model Female zebrafish showing impaired fertility were treated with letrozole (0.036 µg/kg/day) as a reference standard. Feed mixed with Putrajeevak seed oil (PJSO) at various concentrations (0.2, 0.5, 1, 5, 10, and 100 µg/kg/day) for 14 days was compared to the healthy control fish and the fertility-impaired model fish on the 15th day, to assess the recovery of fertility. Female mutant fish were allowed to spawn with healthy males and the percentage of healthy eggs was assessed per spawning. Healthy control fish showed a higher percentage of healthy eggs per spawning (92%) and this was significantly reduced (p < 0.0001) in the fertility-impaired group with only 27% of healthy eggs (Figure3A). Letrozole restored the healthy egg percentage to about 83%, which was significantly higher (p < 0.0001) than that of the fertility-impaired group. Treatment with PJSO showed a gradual and dose-dependent increase in the percentage of healthy eggs, and was significantly higher (p < 0.0001) than the fertility-impaired group, even with the lowest dosage of 0.2 µg/kg/day. This dosage was calculated to be 1/5th of the daily recommended dosage prescribed in humans. The human equivalent dosage per day restored the percentage of healthy eggs to about 65%, while the groups treated with higher dosages of PJSO had healthy egg percentages equivalent to that seen with letrozole at more than 82%. The rate of conception is calculated as the number of viable embryos to the total number of positive breeding events. In the healthy control fish, the rate of conception was observed to be 99.5% and this was drastically reduced to about 16% in the fertility- impaired group (Figure3B). When compared to the fertility-impaired group, treatment with letrozole showed a highly significant increase (p < 0.0001) in the rate of conception at 98.6%. The lowest dosage of PJSO showed only a slightly significant increase (p < 0.05) in the conception rate at about 21%. At 0.5 µg/kg/day and higher, there was a highly significant increase in the conception rate, which was restored to levels comparable to the healthy control group at 10 µg/kg/day (96.8%) and 100 µg/kg/day (97.4%). MoleculesMolecules 20212021, 26, ,26 x, FOR 1020 PEER REVIEW 7 of8 23of 25

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Figure 3. Treatment with PJSO reversed fertility impairment in female zebrafish. (A) Percentage of healthy eggs per Figurespawning 3. Treatment was calculated with PJSO from differentreversed study fertility groups. impairment (B) Conception in female rate zebrafish. was calculated (A) Percentage by the number of healthy of spawning eggs per spawningevents that was formed calculated a viable from embryo different to the study total number groups. of (B spawning) Conception events. rate (C )was The calculated number of spawningby the number events thatof spawning led to eventsthe birth that offormed a viable a viable larvae fromembryo embryos to the to tota thel totalnumber number of spawning of successful events. spawning (C) The events number was calculatedof spawning as the events fecundity that led to rate.the birth Total of number a viable of breeding larvae from pairs embryos were 24 ( nto= the 24). total Data number are represented of successful as mean spawning± SD and events one-way was ANOVA calculated followed as the fecundityby Tukey’s rate. multiple Total number comparison of breeding test was usedpairs towere arrive 24 at(n statistical = 24). Da significance;ta are represented #### represents as meanp ±< SD 0.0001 and comparedone-way ANOVA to the followedhealthy by control, Tukey’s **** representsmultiple comparisonp < 0.0001 compared test was toused the fertility-impairedto arrive at statistical group. significance; #### represents p < 0.0001 compared to the healthy control, **** represents p < 0.0001 compared to the fertility-impaired group. Of the healthy and viable embryos, the ones that survived to become larvae at 10 dpf wereOf used the tohealthy calculate and the viab fecundityle embryos, rate based the ones on thethat total survive numberd to of become fertile embryos.larvae at The10 dpf wehealthyre used control to calculate group showedthe fecundity a fecundity rate based rate of on 97.3%, the total while number the fertility-impaired of fertile embryos. group The healthyshowed control a highly group significant showed reduction a fecundity (p < rate 0.0001) of 97.3% at just, while 16.4% t (Figurehe fertility3C).- Interestingly,impaired group showedthe lowest a highly dosage significant of PJSO reduction showed only (p < a 0 modest.0001) at and just non-significant16.4% (Figure 3C). increase Interestingly, in the thefecundity lowest atdosage 18.8%. of Higher PJSO showed dosages showedonly a modest a dose-dependent and non-significant highly significant increase increase in the fe- × µ × µ cundity(p < 0.0001), at 18.8%. with Higher the 10 dosages(10 g/kg/day) showed a and dose 100-dep(100endentg/kg/day) highly significant dosages showing increase (p recovery of the fecundity rate to near normal levels, at 96.8% and 97.4%, respectively. In < 0.0001), with the 10X (10 µg/kg/day) and 100X (100 µg/kg/day) dosages showing recov- comparison, treatment with letrozole showed a slightly lower rate (93.9%), which was still ery of the fecundity rate to near normal levels, at 96.8% and 97.4%, respectively. In com- highly significant (p < 0.0001) when compared to the fertility-impaired group. parison, treatment with letrozole showed a slightly lower rate (93.9%), which was still highly significant (p < 0.0001) when compared to the fertility-impaired group.

Molecules 2021, 26, 1020 8 of 23

2.5. Treatment with Putrajeevak Seed Oil Showed Increased Egg Reserve and Recovery of Anatomical Structures in the Ovary The reproductive potential of any ovary is measured by the ovarian reserve of eggs, dependent upon both by the quality and quantity of eggs present in the growing oocytes during an ovulation cycle. A diminished ovarian reserve represents a loss of normal reproductive potential in the ovaries. Intact ovaries from all the fish in each group were gently dissected out and the follicles containing eggs in various stages of development were teased out onto a counting slide. The ovarian reserve at each stage of egg development was compared across the different groups in the study (Table3). The fertility-impaired group showed a significant reduction (p < 0.0001) in the number of follicles at the various stages of egg development such as Primary growth (PG), Pre-vitellogenic (PV), Early vitellogenic (EV), Mid-vitellogenic (MV), and Full growth (FG). In comparison, treatment with letrozole showed a significant increase (p < 0.0001) in the number of follicles at each developmental stage compared to the fertility-impaired model. When the total number of follicles was taken into account, the ovarian egg reserve was lower than that seen in the healthy control fish but was still significantly higher than that seen in the fertility-impaired group (Table3). Treatment with the lowest dosage (0.2 µg/kg/day) of PJSO had a modest but not significant increase in the PG, PV, and MV stages but showed a slightly significant increase (p < 0.05) in the EV stage and a highly significant increase (p < 0.0001) in the FG stage eggs. Overall, there was a highly significant increase (p < 0.0001) in the total number of ovarian egg reserves in this treatment group. The group of fish treated with PSJO at 0.5 µg/kg/day showed a significant increase (p < 0.0001) in the follicle count at all the stages of egg development except for the Mid-vitellogenic stage (p < 0.05), though the overall egg count was significantly higher than the fertility-impaired group. The total number of egg reserves showed a dose-dependent increase with an increasing dose of PJSO (Table3). The total egg count in the 100 µg/kg/day treatment group was higher than even the letrozole-treated group, suggesting a recovery from the dysfunctional ovary seen in the fertility-impaired group.

Table 3. Ovarian reserve of eggs determined by the number of ovarian follicles at different stages of development. Data are represented as mean ± SD of 24 individuals and one-way ANOVA followed by Tukey’s multiple comparison test was used to arrive at statistical signiﬁcance; ns represents no signiﬁcance, * represents p < 0.5 compared to the fertility- impaired group, #### represents p < 0.0001 compared to the healthy control, **** represents p < 0.0001 compared to the fertility-impaired group.

Follicle Count—Ovarian Reserve of Eggs Study Groups Primary Pre-Vitellogenic Early Mid-Vitellogenic Final Growth Total Growth (PG) (PV) Vitellogenic (EV) (MV) (FG) Control 114 ± 8.2 296 ± 8.1 311 ± 8.3 109 ± 7.8 483 ± 7.6 1313 ± 16.3 Fertility-Impaired 97 ± 6.8 #### 79 ± 8.9 #### 16 ± 6.8 #### 9 ± 7.6 #### 12 ± 6 #### 213 ± 15.3 #### Model Letrozole (0.036 µg/kg) 137 ± 7 **** 256 ± 7.7 **** 140 ± 7.4 **** 96 ± 8.4 **** 356 ± 7.6 **** 985 ± 18.0 **** PJSO (0.2 µg/kg) 101 ± 7.7 81 ± 7.4 23 ± 8 * 9 ± 6.2 98 ± 7.2 **** 311 ± 16.5 **** PJSO (0.5 µg/kg) 110 ± 6.8 **** 89 ± 8.4 **** 51 ± 6.8 **** 16 ± 8.6 * 168 ± 6 **** 434 ± 16.2 **** PJSO (1 µg/kg) 216 ± 7.4 **** 129 ± 8.2 **** 108 ± 7.5 **** 41 ± 7.7 **** 227 ± 8 **** 721 ± 14.4 **** PJSO (5 µg/kg) 233 ± 9.2 **** 229 ± 7.9 **** 141 ± 6.9 **** 52 ± 6.9 **** 301 ± 8.5 **** 956 ± 17.7 **** PJSO (10 µg/kg) 350 ± 6.7 **** 94 ± 7.8 **** 60 ± 7.8 **** 56 ± 7.5 **** 411 ± 8 **** 972 ± 19.4 **** PJSO (100 µg/kg) 353 ± 3 **** 96 ± 3.5 **** 64 ± 3 **** 59 ± 4.2 **** 434 ± 3.3 **** 1005 ± 8.4 ****

In a parallel experiment, the fish dosed similarly were used for dissecting the ovary for overall morphology and cytological examination. The ovary was dissected and observed under the 10× bright field objective of a stereomicroscope to study the general size and health of the ovary. The healthy individuals showed an intact ovary with a high volume of eggs in various stages of development (Figure4A) and there were no visible signs of hem- orrhage. The ovary dissected from the fertility-impaired group was less voluminous with a lower number of follicles and most of them were premature (Figure4B). A few follicular cysts were also identified by their opaqueness; other whitish fluid-filled follicles having Molecules 2021, 26, 1020 9 of 23

a very thin outer follicular epithelium that ruptures during handling were also observed. The presence of these abnormalities showed that the follicles have not matured properly in the fertility-impaired group. Ovaries from all the fish from each group were dissected out and there were very minor or negligible amounts of variations in overall appearance. There were no overt differences noticed possibly due to the selection criteria used for identifying fertility impairment in the fish. Treatment with letrozole showed well-defined follicles in various stages of development and the ovary was voluminous when compared to the fertility-impaired group (Figure4C). PJSO at lower dosages, 0.2 µg/kg/day (Figure4D) and 0.5 µg/kg/day (Figure4E), did not show much improvement in the gross anatomy of the ovary, with a less voluminous appearance as well as loosely packed follicles within the ovary sac. The follicles appeared to be covered in a very thin outer layer of parenchyma housing premature follicles. Treatment with medium dosages showed a gradual increase in the follicular count, with the 1 µg/kg/day (Figure4F) and 5 µg/kg/day (Figure4G) showing a slightly better follicular distribution. The high dosages of 10 µg/kg/day (Figure4H) Molecules 2021, 26, x FOR PEER REVIEWand 100 µg/kg/day (Figure4I) showed a better dose-dependent recovery of11 the of ovarian25

morphology as seen by an enriched follicle count at various stages of development.

FigureFigure 4. PJSO 4. inducedPJSO induced recovery recovery of ovarian of ovarian morphology morphology in fertility-impairedin fertility-impaired female female zebrafish. zebrafish. Representative Representative images from the variousfrom the study various groups study were groups taken were with taken a with stereomicroscope a stereomicroscope under under 10× 10Xobjective objective magnification. magnification. ( (AA)) Healthy Healthy control, control, (B) (B) Fertility-impaired group, (C) Fertility-impaired fish treated with 0.036 µg/kg/day letrozole, (D) Fertility-impaired fe- Fertility-impaired group, (C) Fertility-impaired fish treated with 0.036 µg/kg/day letrozole, (D) Fertility-impaired females males treated with 0.2 µg/kg/day PJSO, (E) Treatment group with 0.5 µg/kg/day PJSO, (F) Treatment group with 1 treatedµg/kg with/day 0.2 µPJSO,g/kg/day (G) Treatment PJSO, (E group) Treatment with 5 groupµg/kg/day with PJSO, 0.5 µ (g/kg/dayH) Treatment PJSO, group (F) with Treatment 10 µg/kg/day group withPJSO, 1 andµg/kg/day (I) PJSO,Treatment (G) Treatment group group with 100 with µg/kg/day 5 µg/kg/day PJSO. There PJSO, were (H )no Treatment outliers in group each of with the study 10 µg/kg/day groups and PJSO, minor andor negligible (I) Treatment groupamount with 100s ofµ variationg/kg/day were PJSO. seenThere in all ovaries were no from outliers each group. in each of the study groups and minor or negligible amounts of variation were seen in all ovaries from each group.

Molecules 2021, 26, 1020 10 of 23

Cytology smears of the ovary were stained with Hematoxylin and Eosin for de- termination of the cellular distribution and overall health of the ovary. Ovarian smear from healthy fish showed a well-defined nuclear staining and an eosinophilic cytoplasm with a high distribution of follicular epithelium and supportive parenchyma (Figure5A). The smear showed a good distribution of densely stained cells throughout. The fertility- impaired group showed a degenerative nucleus of the follicular epithelium (Figure5B). The cytoplasm was fluidic and, together with the degenerative nucleus, suggested the cytological features of a cyst rather than a healthy follicular epithelium, which was much reduced in its distribution. Overall, the cytological smear from the fertility-impaired group showed a higher presence of cysts rather than follicles. Treatment with letrozole restored most of the damage seen with fertility impairment, with visible yolk vesicles that are slightly eosinophilic (Figure5C). The cytological smear showed the presence of a number of post-vitellogenic follicles, suggesting a mature developmental stage of the ovary. PJSO at 0.2 µg/kg/day (Figure5D) showed a higher presence of degenerative nuclei of the follicular epithelium. The cytoplasmic staining was more basophilic, suggesting that the dosage was not enough to recover fertility impairment. Treatment with 0.5 µg/kg/day PJSO showed the presence of a few well-defined follicles with follicular epithelium and supporting parenchyma (Figure5E). A few eosinophilic yolk vesicles were also seen in the cytology smear, suggesting that a few matured follicles were also present. This suggests that the dosage was starting to show recovery though was not enough to restore the damage completely. The cytology smear from 1 µg/kg/day showed largely basophilic cells, suggesting that most of the follicles in this group are at a pre-vitellogenic stage of growth (Figure5F). Treatment with higher dosages at 5 µg/kg/day (Figure5G), 10 µg/kg/day (Figure5H), and 100 µg/kg/day (Figure5I) showed a well-defined nucleus present in the follicular epithelium of the cytology smears. A good distribution of eosinophilic yolk vesicles was noted in a dose-dependent manner, suggesting a rescue of the cytological features in parallel with that seen in the gross anatomy of the ovary.

2.6. Putrajeevak Seed Oil Reversed Ovarian Cyst Formation and Pelvic Inflammation A dissected ovary from each study group was observed under a bright field 40× objective to identify the presence of cysts in the ovary. They are identified as darker colored follicles without the presence of yolk in the cells. The ovary from the healthy control showed the presence of yolk-filled follicles at various developmental stages from the primary growth to fully mature eggs. The follicle stages were identified by their morphological features under a bright field microscope, including size and color. There was no evidence of cysts seen in this group (Figure6A). The fertility-impaired group showed the presence of fluid-filled cells with degenerative nuclei that lack yolk (Figure6B). This group showed a significant increase (p < 0.0001) in the number of cysts compared to the healthy control group (Figure6J). Treatment with letrozole showed a healthy ovary with a higher number of follicles in the full growth stage of development (Figure6C). In addition, there was a significant reduction (p < 0.0001) in the number of cysts present. Treatment with PJSO showed a dose-dependent reduction in the number of cysts. Treatment with low and medium dosages of PJSO at 0.2 µg/kg/day (Figure6D), 0.5 µg/kg/day (Figure6E), 1 µg/kg/day (Figure6F), and 5 µg/kg/day (Figure6G) still showed the presence of a good number of cysts. Though this number was significantly lower (p < 0.0001) than that seen with in the fertility-impaired group, it was still highly statistically significant (p < 0.0001) when compared to the healthy control group and was higher than that of the letrozole-treated group. Treatment with high dosages of PJSO at 10 µg/kg/day (Figure6H) and at 100 µg/kg/day (Figure6I) showed a significant reduction in the number of cysts compared to the fertility-impaired group. This was even lower than that seen with the letrozole-treated group but was still significantly higher (p < 0.01) than that of the healthy control group where no cysts were identified (Figure6J). Ovarian inflammation was quantified using the smear cytology of the ovary from the different groups. The smear was stained with Hematoxylin and Eosin and was observed for Molecules 2021, 26, 1020 11 of 23

the presence of degenerative cells and immune cells, which are used to mark the presence of inflammation. Cells in the control group showed well-defined nuclei and eosinophilic cytoplasm with a high distribution of follicular epithelium and supportive parenchyma (Figure7A). No immune cells were identified in the cytology smears, suggesting a complete lack of inflammation in the ovary. The fertility-impaired group showed an increased number of degenerative nuclei with loss of eosinophilic staining in the cytoplasm, suggesting inflammation of the ovary (Figure7B). A decrease in the distribution of the follicular epithelium was also observed, which is one of the reasons for the impaired fertility in this group. The percentage of inflammation calculated from triplicate fields was significantly higher (p < 0.0001) in the fertility-impaired group when compared to the healthy control group (Figure7J). The letrozole treatment group did not show any visible signs of inflammation in the ovary and the cytology showed the presence of yolk vesicles that are slightly eosinophilic, indicating rescue of the phenotype (Figure7C). Lower dosages of PJSO could not rescue the inflammation completely, but it was significantly less compared to the fertility-impaired group. Cytology smears of treatment groups with 0.2 µg/kg/day (Figure7D), 0.5 µg/kg/day (Figure7E), and 1 µg/kg/day (Figure7F) did not have any degenerative cells but still had the presence of immune cells within the ovary, suggesting that the dosage was not enough to completely rescue the fertility impairment. Higher dosages at 5 µg/kg/day (Figure7G), 10 µg/kg/day (Figure7H), and 100 µg/kg/day Molecules 2021, 26, x FOR PEER REVIEW 12 of 25 (Figure7I) did not show either the degenerative cells or the presence of immune cells, suggesting complete recovery from the inflammation.

A B C

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FigureFigure 5. 5.Cytology Cytology smear smear of of ovaryovary fromfrom the various treatment treatment groups groups stained stained with with Hematoxylin Hematoxylin–Eosin–Eosin and and representative representative images were captured at 40× objective magnification. (A) Healthy control group, (B) Fertility-impaired group, (C) Letro- images were captured at 40× objective magniﬁcation. (A) Healthy control group, (B) Fertility-impaired group, (C) Letrozole zole treatment group (0.036 µg/kg/day), (D) Fertility-impaired females treated with 0.2 µg/kg/day PJSO, (E) Treatment µ µ treatmentgroup with group 0.5 (0.036µg/kg/dayg/kg/day), PJSO, (F) ( DPJSO) Fertility-impaired (1 µg/kg/day) treatment females treatedgroup, ( withG) PJSO 0.2 (5g/kg/day µg/kg/day) PJSO, treatment (E) Treatment group, (H group) withPJSO 0.5 (10µg/kg/day µg/kg/day) PJSO, treatment (F) PJSO group, (1 µandg/kg/day) (I) Treatment treatment group group, with 100 (G µg/kg/day) PJSO (5 µ PJSO.g/kg/day) treatment group, (H) PJSO (10 µg/kg/day) treatment group, and (I) Treatment group with 100 µg/kg/day PJSO. 2.6. Putrajeevak Seed Oil Reversed Ovarian Cyst Formation and Pelvic Inflammation A dissected ovary from each study group was observed under a bright field 40× objective to identify the presence of cysts in the ovary. They are identified as darker colored follicles without the presence of yolk in the cells. The ovary from the healthy control showed the presence of yolk-filled follicles at various developmental stages from the primary growth to fully mature eggs. The follicle stages were identified by their morphological features under a bright field microscope, including size and color. There was no evidence of cysts seen in this group (Figure 6A). The fertility-impaired group showed the presence of fluid-filled cells with degenerative nuclei that lack yolk (Figure 6B). This group showed a significant increase (p < 0.0001) in the number of cysts compared to the healthy control group (Figure 6J). Treatment with letrozole showed a healthy ovary with a higher number of follicles in the full growth stage of development (Figure 6C). In addition, there was a significant reduction (p < 0.0001) in the number of cysts present. Treat- ment with PJSO showed a dose-dependent reduction in the number of cysts. Treatment with low and medium dosages of PJSO at 0.2 µg/kg/day (Figure 6D), 0.5 µg/kg/day (Fig- ure 6E), 1 µg/kg/day (Figure 6F), and 5 µg/kg/day (Figure 6G) still showed the presence of a good number of cysts. Though this number was significantly lower (p < 0.0001) than that seen with in the fertility-impaired group, it was still highly statistically significant (p < 0.0001) when compared to the healthy control group and was higher than that of the letrozole-treated group. Treatment with high dosages of PJSO at 10 µg/kg/day (Figure 6H)

Molecules 2021, 26, x FOR PEER REVIEW 13 of 25

Molecules 2021, 26, 1020 12 of 23 and at 100 µg/kg/day (Figure 6I) showed a significant reduction in the number of cysts compared to the fertility-impaired group. This was even lower than that seen with the letrozole-treated group but was still significantly higher (p < 0.01) than that of the healthy control group where no cysts were identified (Figure 6J).

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*MV *PG *MV *PV *EV *MV Figure 6. Identification and quantification of ovarian cysts in the ovary of the various study groups. Ovaries were imaged Figure 6. underIdentification the 40× objective and quantification of a stereomicroscope of ovarian and cyststhe number in the of ovary cysts w ofas the quantified various (C study—cyst; groups. PG—Primary Ovaries growth were; imaged under thePV 40—×Preobjective-vitellogenic of a; EV stereomicroscope—Early vitellogenic and; MV the—Mid number-vitellogenic of cysts; FG was—Full quantified growth). Representative (C—cyst; PG—Primary images from growth;(A) PV— Pre-vitellogenic;Healthy EV—Earlycontrol group, vitellogenic; (B) Fertility- MV—Mid-vitellogenic;impaired group, (C) Treatment FG—Full group growth).with 0.036 Representative µg/kg/day letrozole, images (D) Fertility from (A- ) Healthy 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, Molecules 2021, 26, x FOR PEER REVIEW 14 of 25 control group,(G) 5 µg/kg/day (B) Fertility-impaired PJSO, (H) 10 µg/kg/day group, PJSO (C) Treatment, and (I) 100 groupµg/kg/day with PJSO 0.036 treatmentµg/kg/day group. ( letrozole,J) Data are represented (D) Fertility-impaired as female zebrafishmean ± SD treated and one with-way 0.2ANOVAµg/kg/day followed PJSO,by Tukey’s (E) 0.5multipleµg/kg/day comparisons PJSO test treatment was used to group, determine (F) statistical 1 µg/kg/day signif- PJSO, (G) 5 µg/kg/dayicance PJSO,. ## represents (H) 10 µ pg/kg/day < 0.01 compared PJSO, to and the (healthyI) 100 µcontrolg/kg/day group, PJSO #### represents treatment pgroup. < 0.0001 ( Jcompared) Data are to representedthe healthy as mean control group, **** representssuggesting p < 0.0001 comparedthat the dosage to the f wasertility not-impaired enough group to completely, n = 24. rescue the fertility impairment. ± SD and one-way ANOVA followed by Tukey’s multiple comparisons test was used to determine statistical significance. Higher dosages at 5 µg/kg/day (Figure 7G), 10 µg/kg/day (Figure 7H), and 100 µg/kg/day ## represents p < 0.01 compared(Figure to theOvarian healthy 7I) di inflammationd controlnot show group, either was #### quantifiedthe degenerative represents using thep cells< s 0.0001mear or thecytology compared presence of the toof ov theimmuneary healthy from cells the control, group, **** represents p < 0.0001 compareddifferentsuggesting groups. tocomplete the The fertility-impaired recovery smear was from stained the group, inflammation. withn Hematoxylin= 24. and Eosin and was observed for the presence of degenerative cells and immune cells, which are used to mark the presence of inflammation. Cells in the control group showed well-defined nuclei and eosinophilic cytoplasm with a high distribution of follicular epithelium and supportive parenchyma (Figure 7A). No immune cells were identified in the cytology smears, suggesting a complete lack of inflammation in the ovary. The fertility-impaired group showed an increased number of degenerative nuclei with loss of eosinophilic staining in the cytoplasm, suggesting inflammation of the ovary (Figure 7B). A decrease in the distribution of the follicular epithelium was also observed, which is one of the reasons for the impaired fertility in this group. The percentage of inflammation calculated from triplicate fields was significantly higher (p < 0.0001) in the fertility-impaired group when compared to the healthy control group (Figure 7J). The letrozole treatment group did not show any visible signs of inflammation in the ovary and the cytology showed the presence of yolk vesicles that are slightly eosinophilic, indicating rescue of the phenotype (Figure 7C). Lower dosages of PJSO could not rescue the inflammation completely, but it was significantly less compared to the fertility-impaired group. Cytology smears of treatment groups with 0.2 µg/kg/day (Figure 7D), 0.5 µg/kg/day (Figure 7E), and 1 µg/kg/day (Figure 7F) did not have any degenerative cells but still had the presence of immune cells within the ovary,

Figure 7. FigureRepresentative 7. Representative images images of Hematoxylin–Eosin-stained of Hematoxylin–Eosin-stained cytology cytology smears smears of ovaries of ovaries from different from different study groups study groups used for quantification of pelvic inflammation. Images were taken using a 40× objective. (A) Healthy control, (B) Fertility- × used for quantificationimpaired group,of (C pelvic) Fertility inflammation.-impaired fish treated Images with were 0.036 taken µg/kg/day using letrozole, a 40 objective. (D) Fertility (A-impaired) Healthy females control, treated (B ) Fertility- impairedwith group, 0.2 µg/kg/day (C) Fertility-impaired PJSO, (E) Treatment fish treatedgroup with with 0.5 0.036µg/kg/dayµg/kg/day PJSO, (F) Treatment letrozole, group (D) Fertility-impaired with 1 µg/kg/day PJSO, females (G) treated with 0.2 µTreatmentg/kg/day group PJSO, with (E 5) µg/kg/day Treatment PJSO, group (H) Treatment with 0.5 groupµg/kg/day with 10 µg/kg/day PJSO, (F) PJSO, Treatment and (I) Treatment group with group 1 µ withg/kg/day 100 PJSO, µg/kg/day PJSO. (J) Percentage of pelvic inflammation is represented as mean ± SD from 24 individuals and one-way G µ H µ I ( ) TreatmentANOVA group followed with by 5 Tukey’sg/kg/day multiple PJSO, comparisons ( ) Treatment test was group used to with determine 10 g/kg/day statistical significance PJSO, and. (####) Treatment represents groupp with 100 µg/kg/day< 0.0001 PJSO. compared (J) Percentage to the healthy of control pelvic group, inflammation **** represents is represented p < 0.0001 compared as mean to ±theSD fertility from-impaired 24 individuals group. and one-way ANOVA followed by Tukey’s multiple comparisons test was used to determine statistical significance. #### represents p < 0.0001 compared to the healthy2.7. control Impaired group, Fertility **** Rate represents in Male Zebrafishp < 0.0001 Was compared Restored upon to the Treatment fertility-impaired with Putrajeevak group. Seed Oil The number of sperms produced per mL was measured to determine the functionality of the gonads. Males from the control group had little variation in sperm count between individual fish and had an average count of 5.2 × 107 sperm/mL (Figure 8A). There was a significant reduction (p < 0.0001) in the sperm count in the fertility-impaired group with only 1.3% of the healthy control group. Treatment with clomiphene restored the number of sperm to about 65% of the control number, which was highly significant when compared to the fertility-impaired group. Treatment with 0.2 and 0.5 µg/kg/day of PJSO showed only a modest and non-significant increase in the sperm count. Treatment with the other dosages showed a dose-dependent increase in the sperm count, which was statistically significant (p < 0.0001) when compared with the fertility-impaired group. The 1 µg/kg/day showed a sperm count 50% of the healthy control, the 5 µg/kg/day group had 63%, and the 10 and 100 µg/kg/day had a sperm count approximately 81% of the healthy control group.

Molecules 2021, 26, 1020 13 of 23

2.7. Impaired Fertility Rate in Male Zebrafish Was Restored upon Treatment with Putrajeevak Seed Oil The number of sperms produced per mL was measured to determine the functionality of the gonads. Males from the control group had little variation in sperm count between individual fish and had an average count of 5.2 × 107 sperm/mL (Figure8A). There was a significant reduction (p < 0.0001) in the sperm count in the fertility-impaired group with only 1.3% of the healthy control group. Treatment with clomiphene restored the number of sperm to about 65% of the control number, which was highly significant when compared to the fertility-impaired group. Treatment with 0.2 and 0.5 µg/kg/day of PJSO showed only a modest and non-significant increase in the sperm count. Treatment with the other dosages showed a dose-dependent increase in the sperm count, which was statistically significant Molecules 2021, 26, x FOR PEER REVIEW(p < 0.0001) when compared with the fertility-impaired group. The 1 µg/kg/day15 showed of 25 a

sperm count 50% of the healthy control, the 5 µg/kg/day group had 63%, and the 10 and 100 µg/kg/day had a sperm count approximately 81% of the healthy control group. X`

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 asF meanigure± 8. SDP. roxburghii from 24 individuals seed oil reverses and one-way impaired ANOVA sperm cou wasnt usedand sperm to determine motility statisticalin male zebrafish. significance. (A) Sperm #### represents count per p < 8 0.0001mL determined compared toin thedifferent healthy study control groups group, (B) Percentage and **** represents motility ofp sperm< 0.0001 calculated compared from to the1 × 10 fertility-impaired sperm. Data are group. represented as mean ± SD from 24 individuals and one-way ANOVA was used to determine statistical significance. #### represents p < 0.0001 compared to theIn healthy addition control to group, the sperm and **** count, represents the motilityp < 0.0001 ofcompared the sperm to the was fertility also-impaired analyzed. The group. motility assay helps in understanding sperm quality, which is essential for successful fertilization.In addition Lower to the sperm sperm motility count, the may motility indicate of the poorly sperm functional was also analyzed sperm and,. The inmo- turn, reducetility assay the ratehelps of in successful understanding formation sperm ofquality an embryo., which is The essential fertility-impaired for successful group fertili- had azation. drastically Lower low sperm motility motility percentage may indicate (5%) poorly when functio comparednal sperm to the and healthy, in turn control, reduce group (Figurethe rate8 B).of successful The clomiphene-treated formation of an embryo. group showed The fertility a significantly-impaired group higher had ( ap drasti-< 0.0001) motilitycally low percentage motility percentage compared (5%) to the when fertility-impaired compared to the group healthy but this control was group still lower (Figure at 22%. The8B). PJSOThe clomiphene treatment- groupstreated showedgroup showed a dose-dependent a significantly increase higher ( inp < the 0.0001) motility motility percentage, per- whichcentage was compared statistically to the significantfertility-impaired (p < 0.0001) group whenbut this compared was still lower to the at fertility-impaired22%. The PJSO group.treatment However, groups evenshowed with a thedose highest-dependent dosage increase (100 µ ing/kg/day), the motility the percentage percentage, which motility waswas onlystatistically 67%, unlike significant the 89% (p < motility 0.0001) seenwhen in compared the healthy to controlthe fertility group.-impaired group. However, even with the highest dosage (100 µg/kg/day), the percentage motility was only 2.8.67% Treatment, unlike the with 89% Putrajeevak motility seen Seed in Oil the Improved healthy control Testicular group. Cytological Features The smear cytology of the testis dissected from the various study groups was stained with2.8. Treatment Hematoxylin with andPutrajeevak Eosin to Seed assess Oil theImproved health Testicu of thelar tissue. Cytological The cytology Features from the healthy controlThe group smear showed cytology a of well-defined the testis dissected nuclear from morphology the various with study eosinophilic groups was cytoplasmstained andwith spermatogonia Hematoxylin and at various Eosin to stages assess of the maturity health of (Figure the tissue.9A). The The different cytology stagesfrom the such healthy control group showed a well-defined nuclear morphology with eosinophilic cytoplasm and spermatogonia at various stages of maturity (Figure 9A). The different stages such as spermatogonia, primary spermatocytes, secondary spermatocytes, spermatids, and spermatozoa were identified along with Sertoli cells, in the cytology from the control group. The fertility-impaired group of fish showed a remarkably lower number of spermatozoa, indicating a poor maturation cycle of the sperm (Figure 9B). In addition, a very low number of Sertoli cells and spermatocytes with a higher proportion of spermatogonia were detected. The clomiphene treatment group showed a normal tissue architecture of the testis with spermatogonia at various stages of maturation (Figure 9C). A moderate number of Sertoli cells were also detected. The 0.2 µg/kg/day PJSO treatment group showed a higher proportion of undifferentiated spermatogonia with poor eosinophilic staining, suggesting a functionally compromised gonad (Figure 9D). The groups treated

Molecules 2021, 26, 1020 14 of 23

as spermatogonia, primary spermatocytes, secondary spermatocytes, spermatids, and spermatozoa were identiﬁed along with Sertoli cells, in the cytology from the control group. The fertility-impaired group of ﬁsh showed a remarkably lower number of spermatozoa, indicating a poor maturation cycle of the sperm (Figure9B). In addition, a very low number of Sertoli cells and spermatocytes with a higher proportion of spermatogonia

Molecules 2021, 26, x FOR PEER REVIEWwere detected. The clomiphene treatment group showed a normal tissue architecture16 of of25 the testis with spermatogonia at various stages of maturation (Figure9C). A moderate number of Sertoli cells were also detected. The 0.2 µg/kg/day PJSO treatment group showed a higher proportion of undifferentiated spermatogonia with poor eosinophilic withstaining, 0.5 µ suggestingg/kg/day (Figure a functionally 9E) and 1 compromised µg/kg/day PJSO gonad (Figure (Figure 9F) 9showedD). The a groups very low treated num- withber of 0.5 spermatogoniaµg/kg/day (Figureand a higher9E) and number 1 µg/kg/day of spermatids PJSO,(Figure suggesting9F) showed an improvemen a very lowt in thenumber functionality of spermatogonia of the tissue. and aStudy higher groups number treated of spermatids, with PJSO suggesting at dosages an of improvement 5 µg/kg/day (Figurein the functionality 9G), 10 µg/kg/day of the tissue. (Figure Study 9H), groups and 100 treated µg/kg/day with PJSO (Figure at dosages 9I) showed of 5 µg/kg/day a higher (Figureproportion9G), of 10 spermatidsµg/kg/day, indicating (Figure9H), a andcomplete 100 µ recoveryg/kg/day of (Figure the functional9I) showed gonad. a higher The proportionsmear cytology of spermatids, showed the indicating ability of aP. complete roxburghii recovery seed oil of to the restore functional a fully gonad. functional The testissmear with cytology spermatogonia showed the at ability different of P. maturation roxburghii seed stages oil toand restore reverse a fully the functionalfertility impair- testis withment. spermatogonia at different maturation stages and reverse the fertility impairment.

A B C

D E F

G H I

Spermatocyte Spermatogonia Sertoli Cells Spermatids

Figure 9. 9. Testicular smears stained with Hematoxylin–EosinHematoxylin–Eosin to determine the recovery of impaired fertility in male male zebrafish when treated with Putrajeevak seed oil. Images were captured using a 40× objective and representative images 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 from the different study groups are shown. (A) Healthy control group, (B) Fertility-impaired group, (C) Clomiphene 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 µ µ µ (0.361 µg/kg/dayg/kg/day) treatment treatment group, group, (G) (5D µg/kg/day) 0.2 g/kg/day PJSO treatment PJSO treatment group, group, (H) 10 (E )µg/kg/day PJSO 0.5 PJSOg/kg/day treatment treatment group, group, and ((FI) PJSO 1atµ 100g/kg/day µg/kg/day treatment treatment group, 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. 3. Discussion This main aim of the study was to understand the role of P. roxburghii seed oil in rescuing the impaired fertility phenotype and to correlate the changes seen with the different phytoconstituents identified in the supercritical fluid extract. These studies demonstrated the effect of reversing the N-ethyl-N-nitrosourea (ENU)-induced impaired fertility in a zebrafish model. The use of the supercritical fluid extraction process for pharmacologically active phytocomponents would have ensured accurate and reproducible extraction of constituents that are present in trace amounts.

Molecules 2021, 26, 1020 15 of 23

3. Discussion This main aim of the study was to understand the role of P. roxburghii seed oil in rescuing the impaired fertility phenotype and to correlate the changes seen with the different phytoconstituents identified in the supercritical fluid extract. These studies demonstrated the effect of reversing the N-ethyl-N-nitrosourea (ENU)-induced impaired fertility in a zebrafish model. The use of the supercritical fluid extraction process for pharmacologically active phytocomponents would have ensured accurate and reproducible extraction of constituents that are present in trace amounts. Putrajeevak seed oil contains a mixture of fatty acids, with unsaturated fatty acids (82.6%) forming the bulk of the oil content. Oleic acid (C18:n9c) made up about half the fatty acid content at 49.2%, with linoleic acid (C18:2n6c) the next most abundant (31.7%). In addition to the fatty acids, β-sitosterol was found to be at 0.98% in PJSO. This phytosterol has a chemical structure similar to cholesterol and is widely distributed in the plant kingdom. Studies in American mink (Neovison vison) have shown a slight increase in litter size with dietary β-sitosterol supplementation for a period of 9 months [10]. In this study, the β-sitosterol content in PJSO was found to be 0.98% and even at a maximum dose of 100 µg/kg, the amount of β-sitosterol administered per day was 0.98 ng/kg/day. This was much lower than the maximal dose of nearly 50 mg/kg/day used in American minks. Studies have shown that only 4% of dietary β-sitosterol is absorbed [11]. Though studies in rats have shown that higher dosages (0.5 to 5 mg/kg in a day) given subcutaneously reduced sperm count and testicular mass [12], studies in fish have shown that β-sitosterol administration caused a decrease in the circulating levels of steroid sex hormones, and increased the production of vitellogenin expression [13,14]. These studies suggest that β-sitosterol may have a rescue effect on infertility at lower dosages. In our study, fertility in zebrafish was impaired using mutagenesis with N-ethyl- N-nitrosourea (ENU) for a period of 30 days. This causes mutations in both pre- and post-meiotic germ cells, and is an established mode of inducing infertility in both male and female zebrafish [15–17]. The accepted protocol for mutagenesis uses a longer duration of exposure (1h) in a smaller number of exposures (4 to 6) at weekly intervals. These protocols had a significant mortality in fish and therefore, the mutagenesis protocol was modified. Using ENU mutagenesis for 15 min per day for 30 days resulted in significantly higher viable fish (98.5%), with 76.1% of these showing fertility impairment as demonstrated by less than 20 embryos being produced. Though several different ratios of male and female fish are used for assessing the rate of fertility [18], we had the best possible rate of positive fertility when a 1:4 (male to female) ratio was used. In our study, the male zebrafish were also treated with metronidazole and only fish that were able to produce less than 20 fertile eggs when spawned with healthy females were used. Metronidazole leads to cell ablation and can cause complete testicular loss; however, in our study, a full-length treatment regimen was not employed and was paired with ENU mutagenesis to lead to infertility, where the result is a lack of the ability to produce functional semen. Mutagenized females that were able to produce less than 20 viable embryos when spawned with healthy males were chosen for the study. Mutations caused by ENU may lead to further downstream effects resulting in impaired fertility which may not be a direct result of the mutation [7]. The female zebrafish in the fertility-impaired group had an ovary that was smaller in size compared to the healthy control group and had very low follicular count with low egg reserve. Most of the follicles were at either the primary growth or the pre-vitellogenic stages of growth, suggesting that ENU mutagenesis resulted in an ovary with immature eggs. In addition, there was a significantly higher number of follicles that resembled cysts similar to polycystic ovarian syndrome. This suggests that ENU mutagenesis could result in dysfunctional ovaries, not only producing a lower number of mature oocytes but also having an increased number of unviable cysts. In the male zebrafish, this mutagenesis caused a drastic decrease in the sperm count as well as impaired motility of the viable sperm to a large extent. Anatomically, the testis had a significantly lowered number of spermatozoa, indicating a poor maturation cycle. Molecules 2021, 26, 1020 16 of 23

Letrozole is a known aromatase inhibitor which induces ovulation by reducing estrogens. This, in turn, may increase the production of follicle-stimulating hormone (FSH), thus increasing the production of ovarian follicles [19]. In our study, treatment with letrozole rescued most of the infertility seen in the ENU-treated zebrafish. There was an increase in the fertility rate as well as the fecundity rate, suggesting the increased production of viable larvae from this group. P. roxburghii seed oil at human relevant doses restored the fertility rate and the fecundity rate by more than 60% and this was increased to near the levels seen in the healthy controls by treatment at 5× the human dosage and higher. In addition, treatment with PJSO restored the gross anatomy and the cytology of the ovary to levels comparable with that of the healthy individuals. One of the major causes of female infertility is the presence of cysts in the ovary, similar to that seen in polycystic ovarian syndrome. A cyst is a fluid-filled follicle that cannot develop into a functional egg and letrozole has been used to improve fertility in such cases [20]. In this study, the fertility-impaired group had a significantly higher number of cysts compared to the healthy group and treatment with PJSO reversed cyst formation as well as inflammation seen in the ovary. The presence of cysts was not completely abrogated with letrozole but was significantly reduced, and even the highest dosage of PJSO could not completely reverse the formation of cysts. However, PJSO showed a comparatively better effect on the reduction in cyst formation. In addition to a reduction in the number of cysts, there was a drastic improvement in the number of follicles in the final growth stage of egg development with PJSO. This suggests that P. roxburghii seed oil was able to restore ovarian follicle development to near normal levels. The fatty acid analysis of PJSO showed that oleic acid and linoleic acid form the bulk of the fatty acid content. Experiments using radioisotope-labelled linoleic acid in rats showed a greater utilization of carbon from linoleic acid for cholesterol biosynthesis [21]. The higher levels of linoleic acid in PJSO may cause an increased biosynthesis of cholesterol which can, in turn, increase the levels of glucocorticoids and influence egg development and release [22]. Oligoasthenozoospermia, reduced sperm count and sperm motility, was seen in the zebrafish ENU mutants in this study. Anti-estrogens such as clomiphene have been shown to increase the secretion of gonadotropin-releasing hormone (GnRH) and thus, have been prescribed for empirical treatment of idiopathic male infertility [19,20]. Treatment with PJSO showed a significant increase in the number of spermatids and a reduction in the number of spermatogonia cells. This resulted in an increased number of mature sperm as well as increase in sperm motility, and subsequent increase in the number of positive spawning events. PJSO at human relevant dosage had a slightly lower but non-significant reduction in the number of sperm in comparison to the clomiphene treatment group. Significantly, the percentage of sperm was higher at human relevant dosage of PJSO compared to human relevant dosage of clomiphene, suggesting that PJSO was better at rescuing oligoasthenozoospermia.

4. Materials and Methods 4.1. Chemicals and Reagents All chemicals used in the study were of the highest grade and procured from Sigma- Aldrich (St. Louis, MO, USA) unless speciﬁed. Phosphate-buffered saline (8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4, and 0.24 g KH2PO4 to a ﬁnal volume of 1 liter with the pH adjusted to 7.4) and embryo medium (NaCl 294 g, KCl 12.7 g, CaCl2·2H2O 48.5 g, and MgSO4·7H2O 81.3 g made up to a liter) were made in house for everyday use.

4.2. Isolation of PJSO from P. roxburghii Seeds Putrajeevak seeds (Putranjiva roxburghii Wall.) were procured from Divya Pharmacy, Haridwar, India. The seeds were fully mature and were designated for clinical use. The Putrajeevak seeds were pulverized and made into a ﬁne powder and mixed with food- grade liquid CO2. Supercritical carbon dioxide (SC-CO2) was used in a supercritical Molecules 2021, 26, 1020 17 of 23

extractor—the SFE 5000 Bio-Botanical Extraction System (Waters Corporation, Milford, MA, USA) [23]. The extractor was equipped with a CO2 recycler and the backﬂow pressure ◦ was maintained at 450/80 and 55/4 Bar/ C and a CO2 ﬂow rate of 60 g/min over a period of 820 min. Extraction was performed in two stages with a static extraction phase followed by a dynamic extraction phase. The resultant extracted oily substance (batch number D4/CHM/SCFE15710919) was used for the biological experiments and chemical characterizations.

4.3. Gas Chromatography for Analysis of Fatty Acid Content The fatty acid content in PJSO was determined according to the method prescribed by the American Chemist’s Society [24]. Briefly, the supercritical fluid extract, PJSO, was mixed with 0.5 N methanolic NaOH and heated for 10 min to create fat globules. After this, BF3-methanol was added to the boiling mixture and was further heated for 2 min. To this, n-Heptane was added and boiled for a minute more. The mixture was removed from the heat, and saturated NaOH solution was added and mixed vigorously. After allowing the mixture to cool for 10 min at room temperature, the top layer of n-Heptane containing fatty acid mixture called the fatty acid methyl ester (FAME) was collected. Gas chromatography-flame ionized detection (GC-FID) was used to analyze the fatty acid mixture in the FAME using helium as a carrier (GC-2010 Plus gas chromatograph, Shimazdu, Kyoto, Japan). A standard mix for C4-C24 FAME containing a mixture of 37 FAMEs (Sigma-Aldrich, St. Louis, MO, USA) was used as a standard to ascertain the identities of the fatty acids present in the PJSO.

4.4. Identification of β-Sitosterol in PJSO Using HPTLC 4.4.1. Sample Solution Preparation The supercritical fluid extract of Putrajeevak seeds, PJSO (600 mg), was dissolved in 5 mL chloroform. The sample was sonicated for 20 min on ice (Fisherbrand Model 505, Hampton, NH, USA) to obtain a clear solution and then centrifuged at 8000 rpm for 5 min at room temperature to further clarify. The supernatant was transferred to a volumetric flask and the volume was made up to 10 mL with chloroform.

4.4.2. Preparation of Standard Solution Stock solution of β-sitosterol (Natural Remedies, Bangalore, Karnataka, India) was prepared by dissolving 10 mg in 1 mL of methanol. Working standards were further diluted in methanol to obtain solutions of 400 to 1200 µg/mL, which were then used for identiﬁcation and quantiﬁcation of β-sitosterol in PSJO.

4.4.3. HPTLC for Quantiﬁcation of β-Sitosterol in PJSO Standard β-sitosterol at different concentrations and known volumes of PSJO was spotted on aluminum-backed silica gel 60 F254 plates (Merck Millipore KGaA, Darmstadt, Germany). Chloroform:methanol (9:1) was used as a mobile phase and anisaldehyde sulfuric acid reagent was used for derivatization. Quantitation was by densitometry after scanning the derivatized samples at 450 nm. The concentration of β-sitosterol in the sample was estimated against a linearity curve from duplicate loadings.

4.5. Animal Ethics All animal handling protocols were followed under the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Govt of India, for Good Animal Practices. Approved protocols from the Institutional Animal Ethics committee (vide protocol number: 216/Go092019/IAEC) were followed throughout the animal study.

4.6. Zebrafish Husbandry Groups of zebrafish (Danio rerio) Wild-Type AB strain at 1.2 to 1.5 years of age, from the same spawning, were grouped together at 24 fish per group in polypropylene tanks Molecules 2021, 26, 1020 18 of 23

with 2 liters of water per ﬁsh with a light/dark cycle of 14/10 h. The water temperature was maintained at 27 ± 1 ◦C with constant aeration and water circulation to remove wastes and excess food particles. Water pH was checked every day and was adjusted accordingly if needed. Water exchange was performed every week by removing half of the existing water and replenishing it with aged de-chlorinated water.

4.7. Induction of Fertility-Impaired Zebrafish Fish in the study groups were treated with N-ethyl-N-nitrosourea (ENU) to induce infertility. The accepted mode of induction using ENU requires an hour long treatment with 3 mM ENU each week for a period of 4 to 6 weeks [15–17]. However, in our hands, this method resulted in an unacceptable level of mortality and, therefore, this method was modified to not only produce fertility-impaired mutants but also to reduce mortality. Briefly, adult fish, both male and female, were exposed to 5 mM ENU in water for 15 min each day for 30 days; they were then transferred to normal housing water. In the case of male zebrafish, the fish were further treated with 100 mM metronidazole by water dissolution for a further 20 days. Metronidazole causes cell ablation, leading to a lower number of functional cells and, in combination with ENU mutagenesis, reversible loss of semen production [25,26]. Groups of 6 male and female fish were randomly selected and were dissected to isolate the testes and ovaries for anatomical and pathological observation to confirm smaller sized glands and reduced spermatogonia in males and a lower number of ovarian follicles in female fish. Post mutagenesis, the fish were maintained in normal husbandry conditions for 14 days to allow the fish to overcome the stress. The female fish were allowed to spawn with healthy males (1:4 female to male ratio) to ensure a more positive fertility rate. Though lower ratios were also used, it was observed that a 1:4 (female to male ratio) improved the rate of fertility [18]. In the case of mutant males, they were allowed to spawn with healthy females (1:1 ratio). The spawning happened between 6.30 to 7.30 in the morning under controlled conditions at 28 ± 1 ◦C. Purposive sampling was employed to select mutant fish for the study; only females with an embryo count less than 20 and males that resulted in less than 20 embryos with healthy females were selected for the study.

4.8. Study Design Once the study population was selected, they were acclimatized to the study husbandry condition for 7 days. Fish were fed with normal Tetrabit flakes (a complete pet food for tropical fish from Tetra GmbH, Herrenteich, Germany) until the start of the experimental period. The fish were fed in a 24 h feeding cycle throughout the study period. A schematic of the study design is shown in Figure2.

4.8.1. Dosing and Preparation of Feed The seeds of P. roxburghii are directly consumed and the daily recommended quantity according to Ayurvedic texts is 2 to 3 g per day. For the calculation of human dosage for the supercritical fluid extracted oil, an average of 2.5 g was taken. The yield of oil was determined over multiple extractions and an average yield of 3% was taken. The equivalent dosage of the oil, calculated from the recommended weight of seeds to be consumed and the average yield of oil, was determined as ~75 mg/day. For the study, the human dosage of PJSO was taken as 70 mg/day. During the experimental period, the fish were fed with either the reference drugs or with different dosages of the P. roxburghii seed oil (0.2×, 0.5×, 1×, 5×, 10×, and 100× compared to relevant human doses). Different dosages of the compound were calculated for a known volume of feed. The study compound was mixed with the feed pellets and finely ground using a mortar and pestle. The feed mixture was extruded into pellets of standard size weighing 4 mg per pellet. The dosages were the same for both genders, and the translational doses for the different study groups were calculated according to the guidelines of USFDA [27] and are Molecules 2021, 26, 1020 19 of 23

given in Table4. Clomiphene was used as a comparator for male infertility, while letrozole was used in the female infertility study.

Table 4. Translational dose of standard drug and Putrajeevak seed oil derived from human equivalent dose per day.

Standard Drug Putrajeevak Seed Oil (PJSO) Study Groups Translational Translational Dose for Zebraﬁsh Drug Human Human Dose Dose for Dose Zebraﬁsh 0.2× 0.5× 1× 5× 10× 100× Male Clomiphene 25 mg/day 0.36 µg/kg 70 mg/day 0.2 µg/kg 0.5 µg/kg 1 µg/kg 5 µg/kg 10 µg/kg 100 µg/kg Female Letrozole 2.5 mg/day 0.036 µg/kg 70 mg/day 0.2 µg/kg 0.5 µg/kg 1 µg/kg 5 µg/kg 10 µg/kg 100 µg/kg

4.8.2. Endpoints with Mutant Female Zebrafish The dosing was for a period of 14 days, after which the fish were allowed to spawn with healthy males on the 15th day. The fertility rate, conception rate, and fecundity rate were assessed during the spawning, with a ratio of 1 mutant female to 4 healthy males. After the completion of a second cycle of ovulation, the ovary was dissected and the follicle count (ovarian reserve of eggs) was estimated. In a parallel experiment, after 14 days of dosing, the ovary was dissected on the 15th day and the overall morphology was assessed for the quantification of cysts. After this, the cytological smear was stained with Hematoxylin–Eosin and the percentage of inflammation was assessed.

4.8.3. Endpoints with Mutant Male Zebrafish After the acclimatization period of a week, the fish were fed with either clomiphene as a comparator or Putrajeevak seed oil at the same dosages as with the female fish for a period of 14 days. The fertility rate was estimated in spawning experiments with wild-type female fish (1:1 ratio) in standard breeding conditions on the 15th day and the sperm count per mL, motility of the sperm, and testicular cytology smear were assessed.

4.9. Dissection Fish were euthanized with 2 to 4 ◦C water [28]. The fish was dissected through an incision in the ventral side from the gills to the vent. The ovary and the testis were isolated by cutting open the viscera with a dissection knife. The ovary was identified by its large size, lobed morphology, cream color, and the presence of eggs within. The testis was identified as a semi-transparent, lobed structure and a large size.

Hematoxylin–Eosin Staining Cytology smears were prepared from either the ovary or the testis; these were used to replicate the clinical diagnostic criteria as close as possible, where biopsies are used to diagnose fertility impairment [29,30]. The smear was fixed on a glass slide and stained with Hematoxylin for 2 min followed by Eosin for a further 2 min. Excess stain was washed multiple times in PBS at pH 7.4 and viewed under 40× bright field objective using a Labomed l× 400 microscope (Labomed, Los Angeles, CA, USA). Images were captured using the image viewer software. Triplicate fields were randomly chosen and the cells per field were quantified using Qupath software [31].

4.10. Assessment of Study Endpoints in Female Zebraﬁsh 4.10.1. Fertility Rate The success of mating was assessed by the presence of embryos irrespective of their viability. The fertility rate was calculated based on the total number of positive spawning events that led to the formation of an embryo to the total number of breeding pairs.

4.10.2. Rate of Conception Conception afﬁrms that both fertilization has taken place as well as the presence of highly conﬂuent embryos that are viable. The conception rate was calculated as the number Molecules 2021, 26, 1020 20 of 23

of highly conﬂuent viable embryos per breeding event to the total number of embryos fertilized eggs.

4.10.3. Fecundity Rate Embryos collected from the breeding tank were transferred to 30 mL embryo medium for estimation of the number of viable larvae obtained from the embryos. The fecundity rate was calculated as the number of larval survivors from the viable embryos from each spawning event at 10 dpf.

4.10.4. Follicle Count—Ovarian Egg Reserve The ovary of an adult zebrafish is thick and lobular anatomically, and contains non- synchronously developing oocytes [32]. The four different stages of oocyte development before the formation of a mature egg (Full growth) are classified as Stage I—Primary growth (PG); Stage II—Pre-vitellogenic; Stage III—Early vitellogenic stage (EV); Stage IV—Late vitellogenic stage. The ovary was isolated from the dissected fish and was washed twice in PBS. The ovary was dispersed on a glass slide manually to release the follicles; the matured follicles were identified as yolk-filled, dense, morphological structures. The number of oocytes at different developmental stages was counted using a micro-columned slide to estimate the ovarian reserve of eggs. The yolk-filled follicles were counted using the 40× objective of a stereomicroscope (Labomed, Los Angeles, CA, USA).

4.10.5. Ovarian Cyst Quantification Cysts on the ovary were identified as a dark mass of cells that are irregularly shaped under 40× objective of a stereomicroscope. The presence of cysts is considered as a sign of immature follicles, leading to increased infertility in female zebrafish.

4.10.6. Quantification of Pelvic Inflammation To study inflammation, the ovary was isolated from euthanized fish and prepared for smear cytology. The smear was stained with Hematoxylin–Eosin and the presence of cellular debris along with increased presence of neutrophils and lymphocytes was recorded.

4.11. Assessment of Study Endpoints in Male Zebrafish 4.11.1. Sperm Count in Male Zebrafish A capillary tube capable of holding 2 µL volume was used for sperm collection. The fish were anesthetized in water at 17 ◦C until the operculum movement came to a stop. They were dried gently on a paper towel and placed in a holding chamber with the ventral side up. The cloacal region was gently wiped clean to ensure that all the moisture was removed as this could prematurely activate the sperm. The capillary tube with an attached aspirator was positioned at the cloaca and the sperm was collected by gently massaging the region with padded forceps to produce the ejaculate. This was collected into the capillary tube till the desired mark and then drained onto a glass slide for microscopy. Images were captured in three random fields and the total number of sperm was counted using Qupath software.

4.11.2. Motility of the Sperm The sperm was freshly collected as mentioned previously and the motility was assessed according to the protocol devised by Selvaraj et al. [33] with modifications. Briefly, 2.2 µL of the ejaculate was diluted to 20 µL using Hank’s Balanced Salt Solution (HBSS) at an osmolality of 300 mOsmol/kg, and was used immediately. The sample was centrifuged at 428× g for 5 min and resuspended in 1 mL of HBSS. After the number of sperm was counted, they were centrifuged again at 285× g and finally, resuspended at a concentration of approximately 1 × 108 sperm per µL of HBSS. Following this, 1 µL of sperm Molecules 2021, 26, 1020 21 of 23

was transferred to a clean glass slide and the motility of the sperm was recorded using a 10× objective. The raw video ﬁles were transferred to Image J and the scale was set as per the video resolution. The video was processed using Z project and was run through the wrMTrck plugin to track the number of moving objects [34]. The cumulative number of moving objects was considered as the progressive motility of spermatozoa.

4.12. Data Analysis and Statistics All data points are expressed as mean ± standard deviation (SD) of observations. Statistical analysis was performed using GraphPad Prism 7.04 (GraphPad Software Inc., San Diego, CA, USA). One-way ANOVA followed by Tukey’s multiple comparisons test was used to compute the signiﬁcance of the data. In the analysis, the fertility-impaired group was compared to the healthy control group, while the reference drug and PJSO treatment groups were compared to the fertility-impaired group. p-values less than 0.5 were considered signiﬁcant.

5. Conclusions Fatty acid analysis of P. roxburghii seed oil revealed oleic acid and linoleic acid as the major constituents. Trace amounts of β-sitosterol were also identified using HPTLC. ENU mutagenesis was successfully used to establish a fertility-impaired zebrafish model in this study. Putrajeevak seed oil has been shown to restore the ovarian reserve of eggs and the production of mature eggs, and restore fertility rate in a female infertility model. In addition, PJSO enhanced recovery of the follicular structures within the ovary as well as caused a reduction in the number of cysts. These results were comparable to the effect of letrozole in restoring the fertility in ENU-mutagenized female fish. PJSO also restored the sperm count and sperm motility in ENU-mutagenized male zebrafish similar to that seen with clomiphene. Overall, these results suggest that P. roxburghii seed oil administration is useful in reversing the impaired fertility in both the genders.

Author Contributions: A.B. provided broad direction for the study, identified and prepared the test formulations, generated resources, and gave final approval for the manuscript; P.N. performed the drug extraction and experiments; M.J. performed analytical chemistry experiments; L.K. performed data curing and wrote the manuscript; A.V. conceptualized and supervised overall studies, generated resources, critically reviewed and finally approved the manuscript. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. This presented work has been conducted using internal research funds from Patanjali Research Foundation Trust, Hardwar, India. Animal Ethics Committee Statement: The study was approved by the Animal Ethics Committee under the protocol number 216/Go092019/IAEC, approval date September, 2019. Informed Consent Statement: Not Applicable. Data Availability Statement: The data presented in this study can be made available upon request from the scientific community on the discretion of the corresponding author. Acknowledgments: We appreciate the zebrafish test facilities and experimentations at our CRO partner, Pentagrit Labs, Chennai, India. We thank Nemai Ghosh for GC-FID analysis, and Rajesh Mishra for Ayurvedic reference supports. We extend our gratitude to Priyanka Kandpal, Tarun Rajput, Gagan Kumar, and Lalit Mohan for their swift administrative support. Conflicts of Interest: The authors declare no conflict of interest. Sample Availability: Samples of the compounds are available from the author on request for suitable academic research. Molecules 2021, 26, 1020 22 of 23

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