Biological macromolecule cross linked TPP–chitosan complex: a novel nanohybrid for improved ovulatory activity against PCOS treatment in female rats

Abstract

Polycystic ovary syndrome (PCOS) is a relatively common endocrine disorder among young women and leads to metabolic problems associated with the onset of infertility. The present study was emphasized to investigate the protective role of biomolecule coated sodium tripolyphoshate–chitosan nanocomplexes (CNPs) by improving the ovulatory function and gonadotropic hormonal level changes in estradiol valerate (EV) induced PCOS female rats. Fabrication of biomolecule-loaded sodium tripolyphosphate cross-linked chitosan nanocomplex (CNP) by ionic gelation method using methanolic extracts of G. sylvestre leaves (GSLE) and C. zeylanicum (CZBE) barks was achieved and the synthesized GSCNPs and CZCNPs were characterized by FTIR, XRD, TEM and DLS. The protective effects of the synthesized nanocomplex against PCOS induced rats were proved with biochemical and histopathological analysis. Biomarkers essential for ovarian function, serum follicle stimulating hormone (FSH); luteinizing hormone (LH); prolactin (PRL); progesterone (PRG) and insulin (INS) levels were determined. The biomolecule-loaded chitosan nanoparticles treatment was significantly increased the follicle stimulating hormone and progesterone level while decreased significantly the luteinizing hormone, prolactin and insulin level in PCOS animal versus control. Histopathology results confirmed that the development poly cysts in the ovary containing atretic follicles with irregular estrous cycles were noticed in estradiol valerate induced rats. Further, it also enhanced the attenuated layer of granulosa cells in the cyst and destroyed oocytes when compared with the control. In conclusion, the present results suggested that the biomolecule-loaded chitosan nanoparticle (CNPs) treatment could revert estrous cycle back to normal in PCOS induced rats.

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Introduction

Polycystic ovary syndrome (PCOS), a complex endocrine disorder, affects 15% of women in their reproductive years and increases women's lifespan risk of severe disturbances in reproductive, endocrine and metabolic function.¹ About 70% of the patients with PCOS are being affected by common symptoms like irregular cycles, anovulation, infertility, hirsutism, the scalp hair thinning, acne and ovarian hyperandrogenism.² It is reported that insulin resistance (IR) which is categorised by a decrease in cellular ability to respond to insulin signalling appears to be an important pathophysiologic mechanism in the development of all metabolic complications involved with PCOS.³ Elevated insulin concentrations have been associated with lower levels of SHGB (Sex-Hormone Binding Globulin), leading to enhanced bioavailability of androgen.⁴ Altogether in connection with PCOS, insulin resistance and hyperandrogenemia may act together continuously, stimulates each other in a reciprocal fashion for the progressive development of both metabolic as well as reproductive complications.² However, advancement towards prevention of PCOS has been hindered due to incomplete knowledge of its aetiology.

Nanotechnology is a field of science that deals with production, manipulation and use of nanoscale/nanocomplex drugs in the medical field.^5,6 This has resulted in the advancement of numerous biomolecule–nanoparticle hybrids used in localized biomedical treatments.⁷ Recently, green synthesis of nanoparticles using medicinal plants has established for innovative drug delivery systems, including polymeric nanoparticles.^8,9 Synthesis of nanoparticles using biodegradable polymers such as chitosan has been promising due to their easy manipulation, rapid solubility effects, biocompatibility and less toxicity without causing any side effects.^10,11 Chitosan as nanoparticles draws attention for the development of innovative therapeutic drug release systems with enhanced biodistribution and decreased pharmacological toxicity. These applications are aided by the absorption-enhancing effect of chitosan for drug delivery.¹²

Plant derived drugs have increased recently due to the beneficial effects of herbal therapy that are safe with no side effects possessing preventive, curative, rehabilitative capacity with the presence of multiple active compounds leading to the potential effect.^13,14 Currently, different medicinal herbs are being considered as an important source of drugs for the treatment of PCOS (Table 1). According to Maharjan et al.,²² Aloe barbadensis gel treatment showed positive results on recovery of PCOS incidence in rats. Treatment with Matricaria chamomilla flower extract was enhanced the normal follicles development in PCOS rats.²³ Merrily and Winston²⁴ reported that G. sylvestre plant possess trophorestorative action of the beta cells of the pancreas and is well indicated for PCOS, due to its insulin modulating activity and the property of reducing the elevated triglycerides associated with the disorder. Gymnema may prove as one of the most significant herbs in the treatment of the underlying factor of insulin resistance connected with PCOS since the predictable medical models are focussing on pharmaceutical agents such as metformin to manage the syndrome.

Table 1 List of identified bioactive molecules and its chemical structure from various plant species

Name of the plant species	Bioactive compounds name	Chemical structure	References
Gymnema sylvestre	1. Saponin gymnemic acid		15
	2. Sterol stigmasterol		16
	3. Flavonol glycoside		17
Cinnamon zeylanicum	1. Cinnamaldehyde		18
	2. trans-Cinnamyl acetate		18
	3. Methyl hydroxy chalcone polymer		19
Aloe barbadensis	p-Coumaric acid		20
	Anthranol		20
	Aloesin		20
Matricaria chamomilla	Farnesene		21
	Tetracosane		21

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Cinnamon (Cinnamomum zeylanicum), has been used as preservatives, flavouring and pharmacological agents.²⁵ Earlier study has explained the effects of cinnamon on insulin, glucose and lipid metabolism associated with metabolic syndrome.²⁶ Jarvill-Taylor et al.²⁷ described that the effective component, MHCP (methyl-hydroxychalcone polymer) extracted from cinnamon was found to be useful to treat insulin resistance by increasing glucose utilization and improving the insulin receptor function in adipocytes. Wang et al.²⁸ reported that MHCP, a water soluble polyphenolic compound which is identified as insulin-sensitizing agent similar to troglitazone, may be considered as effective plant drug in the treatment of PCOS.

Synthesis of copper nanoparticles using aqueous and ethanol extract of leaves of G. sylvestre has also been demonstrated by Heera et al.²⁹ Sathishkumar et al.³⁰ studied the anti-bactericidal activities of the silver nanoparticles synthesized from C. zeylanicum powder and bark extracts. Although there are reports on synthesis and characterization of nanoparticles using these two plants, no report is published on the protective efficacy of biomolecule-loaded sodium tripolyphospate (TPP)–chitosan nanocomplex on PCOS induced rat model to date. Hence Gymnema and Cinnamon plants possessing potential pharmaceutical compounds (Saponins, MHCP) for insulin sensitizing ability that are associated with the treatment of PCOS, are selected to fabricate biomolecule-loaded nanocomplex for the present experiment.

The major goal of this study was to synthesize and characterize bioactive molecule incorporated TPP crosslinked-chitosan nanocomplex (CNPs) from G. sylvestre leaf (GSCNPs) and C. zeylanicum bark (CZCNPs) methanolic extracts and to evaluate their protective efficacy on estradiol valerate (EV) induced PCOS rat models by determining the gonadotropic hormones level and examining the histopathological changes.

Experimental

Fabrication and characterization of chitosan nanocomplex from selected plant materials

About 100 g (w/v) coarse powder from leaves of G. sylvestre and barks of C. zeylanicum, were filled in soxhlet apparatus for 8–10 hours separately for hydro-methanol (10 : 90) extraction. The extracts were dried completely and used for the synthesis of chitosan nanocomplex using sodium tripolyphosphate (TPP) as cross linking agent by adopting the ionic-gelation method. Under gentle magnetic stirring, the plant extracts (1.5% w/v) were mixed with TPP (0.5% w/v) separately and the solution was added drop wise using syringe into the chitosan solution containing 1% (w/v) chitosan and 2% (v/v) acetic acid. The synthesized nanoparticles were centrifuged at 8000 rpm for 20 min, sonicated, dried and were further used for characterization by FTIR, XRD, TEM analysis and DLS techniques.

In vivo study on estradiol valerate induced PCOS in female wistar rats

Animal care and acclimatization. Healthy female wistar rats weighing 90–120 g were selected for the present study and maintained in the animal house of Kovai Medical Centre College of Pharmacy, Coimbatore, Tamilnadu, India. Rats were housed in polypropylene cages lined with husk in standard environmental conditions (temperature 25 ± 2 °C, relative humidity 55 ± 10%) and constant light/dark cycle (12 h : 12 h). The rats were fed on a standard pellet diet (Amrut rat and mice feed, Sangli, India) and water ad libitum. The entire experiment was performed in accordance with the approval and clearance obtained from the Institutional Animal Ethics Committee, Periyar University, Salem – 636011, Tamil Nadu, India (Approval no. 1085/ac/07/PUIAEC/2012/17) by prescribed guidelines of Committee for the purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India.

Induction of PCOS by estradiol valerate in rat models. Fifty four animals displaying at least two consecutive normal 4 day estrous cycles were used in this study. Estradiol valerate (EV), an analogue of 17β estradiol was obtained from Himedia (Product Code: RM 4682-1G). The animals were divided into 8 experimental groups and one control group (n = 6 animals per group). A single dose of 2 mg kg⁻¹ b. wt in 0.2 ml olive oil (v/v) was administered intraperitoneally to the animals in the experimental group following the method of Brawer et al.³¹ The control animals were injected with equivalent amounts of vehicle only (0.2 ml corn oil).

Determination of vaginal smears. Daily vaginal smears were taken to check the estrous cyclicity in rats following the method of Jitendra et al.³² Briefly, a cotton bud immersed in normal saline was inserted gently in the vaginal opening of the female rats and a swab was obtained. The bud was then rolled on a clean slide to make a smear and allowed to dry. After adding few drops of methanol to fix the cells in the smear, Giemsa stain was added and covered in petridish. After 5 min, distilled water was added and further the slide was stained in dilute Giemsa. The stained slide was drained, washed, air dried and observed under microscope in 40× objectives. Cycles with duration of 4–5 days were considered regular. The smear was assessed by analysis under a microscope for the relative abundance of nucleated epithelial cells, cornified cells and leukocytes. The observation of cornified cells in the smears during a minimum period of 10 serial days was defined as persistent vaginal cornification (PVC) and considered to be an indication of follicular cystic development.³³

Animal groups. After the formation of polycystic ovaries, the animals in the nine experimental groups were treated with the synthesized biomolecule-loaded TPP–chitosan nanocomplex and plant extracts. The experimental groups were divided into group-I, normal control (untreated) rats; group-II, PCOS untreated rats; group-III, PCOS rats given metformin (28 mg kg⁻¹ bodyweight); group-IV, PCOS rats given Gymnema sylvestre leaf extract (150 mg kg⁻¹ bodyweight); group-V, PCOS rats given lower dose of GSCNPs (50 mg kg⁻¹ body weight); group-VI, PCOS rats given higher dose of GSCNPs (100 mg kg⁻¹ body weight); group-VII, PCOS rats given bark extracts of Cinnamon zeylanicum (150 mg kg⁻¹ body weight); group-VIII PCOS rats given lower dose of CZCNPs (50 mg kg⁻¹ body weight) and group-IX, PCOS rats given higher dose of CZCNPs (100 mg kg⁻¹ body weight). The treatment was carried out for 21 days, with oral administration of respective drugs.

Measurement of circulating levels of gonadotropins in the study groups. The effects of the synthesized GSCNPs, CZCNPs, GSLE, CZBE and standard drug metformin in the PCOS induced rats were estimated on the 21^st day of the treatment period after sacrificing the animals by decapitation. Blood was collected from all the groups by retro orbital puncture under light ether anesthesia for serum separation. Serum was separated by centrifugation at 3500 × g for 10 min at 25 °C. Serum luteinizing hormone (LH); Follicle Stimulating Hormone (FSH); prolactin (PRL); progesterone (PRG) and insulin (INS) levels were determined. A diagnostic kit of (MODULAR ANALYTICS E170, Elecys 2010 from Roche Diagnostics, Indianpolis, IN, USA) was used to measure the above said level of hormones bearing the ref. no. 11775863, 11732234, 03203093, 12145383 and 12017547 for FSH, LH, PRL, PRG and INS respectively.

Histopathological studies

After the collection of blood samples, the ovaries and uterus tissues from all the nine group of rats were subjected to histopathological studies. The ovaries and uterus were dissected out from the rats, cleaned of adherent connective fat tissue and fixed in 10% formaldehyde buffer (v/v) solution for at least 24 h. The sections were embedded in paraffin, cut in >7 μm thickness and then stained with hematoxylin and eosin for the histopathology study examined under bright field microscope.

Statistical analysis

The data obtained were statistically analyzed by one way ANOVA followed by Duncan's multiple range tests as a multiple comparison test. P < 0.05 values were considered. The results were expressed as mean ± S. E.

Results and discussion

Fabrication of biomolecules-loaded TPP–chitosan nanocomplex

This study describes an efficient protocol for the synthesis of biomolecule-loaded TPP–chitosan nanocomplex using methanol extracts of G. sylvestre and C. zeylanicum plants. The nanoparticles were fabricated using the ionic gelation protocol with sodium tripolyphosphate (TPP) as cross linking agent. After the formation of opalescent suspension, the samples were centrifuged, sonicated and used for further characterization. FTIR spectrum of biomolecule-loaded GSCNPs showed strong absorption peaks at 3423 cm⁻¹, 2924 cm⁻¹, 2360 cm⁻¹, 1638 cm⁻¹, 1539 cm⁻¹, 1385 cm⁻¹, 1074 cm⁻¹ and 883 cm⁻¹ while spectra of CZCNPs had absorption peaks at 3417 cm⁻¹, 2924 cm⁻¹, 2360 cm⁻¹, 1614 cm⁻¹, 1528 cm⁻¹, 1385 cm⁻¹, 1092 cm⁻¹, 883 cm⁻¹ and 530 cm⁻¹. The FTIR results revealed that the synthesized biomolecule-loaded chitosan nanoparticles were capped by the phytoconstituents such as phenols, flavonoids, saponins, alkaloids and aldehydes (Fig. 1A). The observed peaks were shifted from the higher frequencies to lower frequencies due to the increase in bond length and decrease in stretching frequency. The physical status of chitosan nanoparticles was determined by the X-ray diffractometer method confirmed the change in the chitosan nanoparticles packing structure due to the incorporation of bioactive molecules that were present in the methanol extracts of the plant samples (Fig. 1B). The average particle size of the chitosan and the synthesized nanoparticles were calculated by Debye–Scherrer equation. The results of XRD study revealed intense peaks at 2θ values of 18.21°, 25.03°, 28.19°, 35.12°, 43.00°, 46.47° for GSCNPs and 23.26°, 30.07°, 39.32°, 41.20°, 48.78°, 57.80° for CZCNPs. These peaks suggest that the diffraction width of the synthesized nanoparticles might be caused by the cross-linking reaction between chitosan and TPP molecules that indicates the crystalline structure. The size and morphology of the synthesized biomolecule-loaded chitosan nanoparticles predicted by TEM analysis are represented in Fig. 1C. The size of the CNPs obtained was ranged from 53.11 to 81.82 nm whereas the size of the synthesized nanocomplex was between 58–80 nm and 60–120 nm for GSCNPs and CZCNPs respectively. The stability and particles dispersity index (pdi) were determined by zeta potential and pdi values measurements adopting the phenomenon of DLS. The results of zeta particle size distribution peaks are illustrated in the ESI Fig. 1† and the details of particle density index are presented in the ESI Fig. 2.† The results indicated that the synthesized GSCNPs were found to be more stable (+3.25 mV) and homogeneous (0.474) in distribution when compared with CNPs and CZCNPs.

Fig. 1 (A) FTIR spectral analysis of chitosan nanoparticles, Gymnema sylvestre leaf extracts derived biomolecule-loaded chitosan nanoparticles (GSCZNPs) and Cinnamomum zeylanicum bark extracts derived biomolecule-loaded chitosan nanoparticles (CZCNPs). (B) X-ray diffraction peaks of chitosan nanoparticles, Gymnema sylvestre leaf extracts derived biomolecule-loaded chitosan nanoparticles (GSCZNPs) and Cinnamomum zeylanicum bark extracts derived biomolecule-loaded chitosan nanoparticles (CZCNPs). (C) Transmission electron microscopy images: (a) chitosan nanoparticles, (b) Gymnema sylvestre leaf extracts derived biomolecule-loaded chitosan nanoparticles (GSCZNPs) and (c) Cinnamomum zeylanicum bark extracts derived biomolecule-loaded chitosan nanoparticles (CZCNPs) (scale bar – 500×).

Efficacy of TPP–chitosan nanocomplex on EV induced PCOS in female wistar rats

The major goal of the present study was to evaluate the protective role of the supplementation of biomolecule-loaded TPP–chitosan nanoparticles derived from G. sylvestre leaf and C. zeylanicum bark methanol extracts on EV induced PCOS rats by analysing the biochemical and histopathological parameters in detail.

Effects of EV on the estrous cycle

The formation of polycystic ovaries was detected by the irregular estrous cycle in the experimental groups through the vaginal smears. Samples collected from different stages of the estrous cycle were analysed in all the group of the PCOS induced rat models to confirm the occurrence of PCOS (Fig. 2). The Fig. 2A shows the diestrus stage of the control group with the presence of prominent leukocytes. The PCOS untreated group displays the irregularity of the menstrual cycle mostly with the metestrus stage showing cornified cells (Fig. 2B). The metformin treated group in the Fig. 2C depicts the diestrus stage with the presence of leukocytes. The Fig. 2D indicated the metestrus stage with nucleated epithelial cells that was treated with GSLE. Predominant nucleated cells of the proestrus stage (Fig. 2E) were exhibited in the group of animals treated with lower doses of GSCNPs. In case of the group treated with higher doses of GSCNPs, dominant epithelial cells were observed (Fig. 2F). The Fig. 2G showed the diestrus phase with leukocytes in the group treated with CZBE. In the groups treated with lower and higher doses of CZCNPs, it was observed that epithelial cells were changed to metestrous phase (Fig. 2H) and cornified cells became estrous phase (Fig. 2I).

Fig. 2 Effect of estradiol valerate on various stages of rat estrous cycle. (A) Control (group 1) in diestrus stage. (B) PCO induced (group 2) in metestrus stage. (C) Metformin treated shows diestrus stage (group 3). (D) GSLE treated (group 4) shows metestrus stage. (E) GSCNPs treated-low dose shows proestrus (group 5). (F) GSCNPs treated-high dose shows estrus stage (group 6). (G) CZBE treated shows diestrus stage (group 7). (H) CZCNPS treated-low dose shows metestrus stage (group 8). (I) CZCNPS treated-high dose shows estrus stage (group 9).

Effect of the biosynthesized chitosan nanoparticles on gonadotropic hormone levels

In order to study the effect of the biomolecule coated chitosan nanoparticles on PCOS induced rats, the levels of FSH, LH, prolactin, progesterone and insulin were determined in serum samples collected from the different experimental groups after 21 days of treatment.

Determination of FSH level

The level of FSH in the control as well as the PCOS induced rats after treatment with plant extracts (GSLE, CZBE) and biomolecule-loaded chitosan nanoparticles (GSCNPs and CZCNPs) are depicted in the Fig. 3A. The results indicated that the level of FSH was significantly increased in PCOS induced rats with the administration of higher dose of GSCNPs (0.61 to 0.79 mIU ml⁻¹) followed by CZCNPs (0.61 to 0.76 mIU ml⁻¹) compared to the other treatments. The increased level of FSH was almost similar to that found with control and metformin (standard drug) treated group. It is noteworthy to mention that plant extracts had positive effect on FSH level in PCOS induced rats compared to the control and GSCNPs showed best results on FSH level in PCOS induced rats than CZCNPs.

Fig. 3 Determination of the level of (A) FSH, (B) LH, (C) PRL, (D) INS and (E) PRG in PCO induced rats after treatment with plant extracts and synthesized biomolecule-loaded chitosan nanoparticles. Values are statistically significant at P < 0.05 level by ANOVA test. ^aPCOS vs. treated, ^bsignificantly differ from PCOS, ^#non-significant vs. PCOS.

Analysis of LH level

The Fig. 3B illustrates the effect the biomolecule-loaded GSCNPs, CZCNPs and plant extracts on luteinizing hormone level in PCOS induced experimental and control rat groups. Results indicated that the level of LH was reduced from 0.65 to 0.57 mIU ml⁻¹ when the PCOS induced rats were treated with higher doses of GSCNPs compared with other experimental groups. A significant increase of LH hormone level was noticed in PCOS induced untreated group when compared with the control and metformin treated groups. In general, the experimental rat groups treated with the plant extracts showed almost similar LH level to control and nanocomplex treated groups exhibited on par with metformin (drug) treated PCOS induced experimental rat groups.

Estimation of prolactin level

The role of the biosynthesized chitosan nanocomplex on prolactin hormone level in PCOS induced experimental rats is depicted in Fig. 3C. The prolactin levels were found to be significantly decreased in PCOS induced experimental rats by the administration with metformin, GSCNPs and CZCNPs than PCOS untreated rats. It is interesting to mention that the prolactin levels were significantly decreased from 5.23 to 4.76 and 4.86 ng ml⁻¹ for GSCNPs and CZCNPs respectively, in PCOS induced rats when treated with higher doses of nanocomplex compared to the other groups.

Assessment of insulin level

The results of biomolecule-loaded nanocomplex, plant extracts and metformin treatment on regulation of serum insulin level in PCOS induced experimental rats are showed in Fig. 3D. The current study clearly revealed that the serum insulin level was significantly down regulated in PCOS induced experimental group by treated with the higher doses of biomolecule-loaded GSCNPs (from 4.4 to 2.8 μIU ml⁻¹) and CZCNPs (from 4.4 to 3.1 μIU ml⁻¹) than untreated PCOS group rats. It is noteworthy to mention that the serum insulin hormone level was significantly decreased in PCOS induced experimental groups due to the treatment of metformin (drug), nanocomplex and plant extracts compared with untreated PCOS rats.

Detection of progesterone level

The efficacy of the synthesized GSCNPs, CZCNPs and metformin in regulation of the progesterone levels in PCOS induced experimental rats is presented in the Fig. 3E. The elevated level of progesterone was noticed; when the PCOS induced rats were treated with biosynthesized chitosan nanoparticles compared to the PCOS untreated groups. Results clearly showed that the level of progesterone was increased from 4.16 to 8.00 ng ml⁻¹ in PCOS experimental rat group treated with higher dose of GSCNPs and it was found to be almost similar to metformin treated rats, while a decreased level of progesterone was recorded in untreated PCOS experimental group.

Efficacy of the biosynthesized chitosan nanoparticles on histopathological alterations

The effects of the biosynthesized chitosan nanoparticles on the macroscopic and microscopic morphological changes in ovary and uterus tissues from EV induced PCOS rats were examined by histopathological observations in detail after 21 days of treatment.

Histological analysis of ovaries

The microscopic examinations of the ovary in the control group showed the presence of normal matured follicles and development of corpus luteum. The follicles had thick granulosa layer and thin theca layer. Moreover with the presence of significant number of corpus luteum, ovulation induction is evident (Fig. 4a). Section of ovary from the PCOS untreated groups exhibited large number of cystic follicles accompanied by thin granulosa layer. The thickened thecal layer was found to be delineating, dying cells and the debris were collected in the antrum (Fig. 4b). The effect of EV induced PCOS resulted in reduced number of corpus luteum that significantly reduced ovulation rate. The Fig. 4c represented the normal follicular development when treated with the standard drug metformin. The effects of GSLE, GSCNPs treated with lower and higher doses on the morphology of ovaries from PCOS induced rats are presented in the Fig. 4d–f. Results indicated that the plant extract had positive effect on development of antral follicles with degenerating granulosa layer. It is interesting to note that GSCNPs treatment showed changing the PCOS to normal ovarian morphology with the formation of Graafian follicles and rapid development of follicles. The microscopic observations were also confirmed the presence of many defined follicles in the process of normalization after treatment of PCOS induced rats with chitosan nanocomplex. The development of corpora lutea in PCOS rats after the nanoparticle treatment suggested that the estrous cyclicity had restored back to normal due to the presence of bioactive molecules. The effect of CZBE and lower and higher doses of CZCNPs on ovarian morphological changes in PCOS rats are depicted in the Fig. 4g–i. It was observed that poly cysts were disappeared and the formation of normal follicles rate was found to be increased due to the treatment of PCOS induced rats with biomolecule coated nanoparticles. Results clearly showed that the CZCNPs also exhibited regaining of normal follicular development and formation of corpus luteum in PCOS induced rats, however, the recovery was slow when compared to the GSCNPs treatment.

Fig. 4 Histological section of the ovaries showing the follicular development in the normal and treated groups. (a) Section of ovary from the control group rats showing normal matured follicles and corpus luteum. (b) Section of ovary from the PCO induced untreated group showing the presence of cystic follicles. (c) Section of ovary from the metformin treated group showing the normal development of follicles and formation of corpus luteum. (d) Section of ovary from the group treated with GSLE exhibiting antral follicles with degenerating granulosa layer. (e) Section of ovary from the group treated with lower doses of GSCNPs exhibiting the formation of Graafian follicle. (f) Section of ovary from the group treated with higher doses of GSCNPs displaying normal follicular development. (g) Section of ovary from group treated with CZBE showing unremarkable changes in the follicular development. (h) Section of ovary from the group treated with lower doses of CZCNPs showing slower regeneration of follicular development. (i) Section of ovary from the group treated with higher doses of CZCNPs showing unclear organization of follicles and corpus luteum.

Histological analysis of uterus

The histological analysis of the uterus was examined to study the effect of the biomolecule coated chitosan nanoparticles on morphological variation occurred in the uterus of PCOS induced rats. The Fig. 5a of the control group showed normal matured uterus lining whereas the PCOS untreated group displayed disintegrated uterine walls (Fig. 5b). Normal uterus arrangement was also observed in the group treated with metformin (Fig. 5c). Results showed that the intact endometrial layer formation with thick stratified epithelium and better endometrial tissue rearrangements were noticed in the histological observations from the PCOS rat groups treated with GSLE and GSCNPs (Fig. 5d–f). It is interesting to note that the better endometrial cervical region (Fig. 5g), with loss of mucoidal layer in the endometrial lining (Fig. 5h) and cuboidal endometrial epithelium (Fig. 5i) were observed in the ovarian morphologies of PCOS rats treated with CZBE, and CZCNPs. The histopathological examination of the present study clearly reveals that the biomolecule-loaded chitosan nanoparticles treatment proved to be more effective in revert back to normal reproductive cycles in EV induced PCOS rats and this nanocomplex could be considered as good nanodrug for treatment of anovulation.

Fig. 5 Histological section of the uterus showing the endometrium development in the normal and treated groups. (a) Section of uterus from the control group rats showing normal matured uterus epithelial lining. (b) Section of uterus from the PCO induced untreated group showing the presence of disintegrated uterine cell walls. (c) Section of uterus from the metformin treated group showing the normal development of endometrial lining. (d) Section of uterus from the group treated with GSLE exhibiting intact endometrium layer. (e) Section of uterus from the group treated with lower doses of GSCNPs exhibiting the formation of thick stratified epithelium. (f) Section of uterus from the group treated with higher doses of GSCNPs displaying better endometrial arrangements. (g) Section of uterus from group treated with CZBE showing the endometrial cervical region. (h) Section of uterus from the group treated with lower doses of CZCNPs showing loss of mucoid layer in the endometrial lining. (i) Section of uterus from the group treated with higher doses of CZCNPs showing cuboidal inactive endometrial epithelium.

Polycystic ovary syndrome is considered as one of the most threatening gynecological disorders in young female population and ultimately causing serious infertility problems. In order to find out the suitable therapeutic potential, various experimental rat models have been developed to study the polycystic ovary syndrome recovery by treating with different form of herbal extracts as drugs in detail in the recent past. In spite of these studies, no effective therapeutic drug is identified to treat the PCOS so far. At present available few drugs and hormonal therapies used in the management of PCOS display various side effects.^34,35 However, to the best of our knowledge, no report is available on the protective role of nanoparticles based drug treatment in PCOS induced rats until now. In the present study, we investigated the protective role of biomolecule incorporated TPP–chitosan nanocomplex treatment based biochemical and histopathological alterations against PCOS induced rat model.

The current investigation describes the protective role of the biomolecule-loaded chitosan nanocomplex by ionic-gelation cross-linking method using the methanol extracts of G. sylvestre and C. zeylanicum plants against EV induced PCOS rat models by altering the gonadotropic hormone levels and histopathological changes. Fabrication of chitosan nanocomplex by cross-linking with sodium tripolyphosphate (TPP) through ionic gelation method has attracted much attention due to the encapsulation of bioactive molecules into chitosan nanoparticles and it is evident from the physical cross linking of chitosan with the multivalent anions derived from sodium tripolyphosphate in this study.³⁶ The particles are formed mainly through the electrostatic interaction between positively charged chitosan and negatively charged TPP molecules.³⁷ Recently, the drug loading efficiency of the most potent combination of staphylokinase and L. aspera plant extract were analysed using chitosan nanoparticles.³⁸

The synthesized nanoparticles were characterized by FTIR, XRD, TEM, zeta potential and polydispersity index analysis. The FT-IR spectroscopy investigated the interaction between chitosan–TPP nanoparticles and phytoconstituents from the plant extracts used. Results suggested that the peak shifts in the synthesized nanocomplex observed was due to the possible interaction between protonated amine or amide groups and negatively charged TPP cross linking agent. Similar results were also reported by Rejinold et al.³⁹ The successful formation of biomolecule coated chitosan nanoformulations was revealed by the XRD analysis. The shifts of the absorption peaks confirmed the crystalline nature of the synthesized chitosan and biomolecule-loaded chitosan nanoparticles. This is due to the potential interaction of chitosan with TPP molecules. AbdElhady et al.⁴⁰ also demonstrated a similar type of X-ray diffraction pattern for chitosan and chitosan–ZnO nanoparticles. TEM analysis revealed the smooth and spherical morphology of the synthesized nanoparticles obtained in the present study. When compared to chitosan nanoparticles, the synthesized GSCNPs and CZCNPs show increased in size and shape due to the entrapment of the active biomolecules from plant extracts on the chitosan nanoparticles. The colloidal stability and dispersion nature of the prepared nanoparticles were determined by the DLS techniques. Results confirmed that GSCNPs were found to be more stable and homogeneous in distribution due to the positive charges on the nanoparticles that were responsible for the cellular membrane components and the tight junctions triggering the paracellular permeation. The molecular weight and concentration of the chitosan are considered as the prime factor for the particle size of the nanoparticles. The present work was in parallel with the evidence of earlier studies.^41,42

As the experimental polycystic ovary syndrome (PCOS) in rodent model provide resemblance to human PCOS syndrome, changes in gonadotropin-releasing hormones (GnRH) in serum and development of multiple poly cysts, were induced by injecting any one of the hormones namely estradiol valerate (EV), letrozole, dihydrotestosterone (DHT) for inducting PCOS in rats.^34,35,43,44 The present study was focussed to induce polycystic ovaries with EV in normal healthy female wistar rats. After acclimatization under standard environmental conditions and diet of healthy female wistar rats, 54 animals that exhibited normal estrous cycle were used for the study. The animals were divided into nine groups including one control group and eight experimental groups. Apart from the control group (6 rats), the rest of the animals (48 rats) were treated with a single dose of EV for inducing PCOS. The effects of EV on the estrous cycle of the rat models were confirmed by the vaginal smear determinations. It is reported that PCOS results in the failure of cyclic reproductive mechanism and displays a number of metabolic abnormalities associated with hormonal imbalances in women.⁴⁵

In this study, the animal model with PCOS was developed using EV in rat which showed many biochemical (gonadotropin-releasing hormone – GnRH) and histopathological changes consisted with human PCOS. Therefore, the present study was focused to analyze the level of the gonadotropic hormones such as FSH, LH, prolactin, progesterone and insulin in the PCOS induced rat models by the administration with active biomolecule-loaded chitosan nanoparticles. The result indicated that there was increased level of LH in PCOS group while the levels of FSH and progesterone were decreased compared to the control group. FSH is responsible for the follicular growth and maturation of eggs in the ovaries. When the level of FSH decreases in EV induced PCOS rats, it leads to follicular growth inhibition that affects the release of eggs resulting in infertility. Thereafter, the immature follicles in ovaries develop into small cysts leading to PCOS. The treatment with GSCNPs and CZCNPs showed the increased level of FSH secretion in the treated PCOS rat groups compared to the untreated one. Similarly, Ouladsahebmadarek et al.⁴⁶ observed increased FSH level in the PCOS induced rats treated with omega-3 under diet. Pushpa and Kalavathy⁴⁷ also reported that the treatment with mehani was increased the FSH level in PCOS induced rats. Current results clearly showed that the LH level was significantly down regulated in PCOS induced rats by the treatment with biomolecule-loaded chitosan nanoparticles. Farideh et al.²³ demonstrated that the levels of LH and FSH were significantly decreased in Matricaria chamomilla extract treated rat group. It is reported that the phytochemicals were found to be effective in decreasing the LH level in PCOS induced rats.^47,48

Increased level of prolactin leads to increase production of androgen which again finally coincides with the symptoms of PCOS disorder. The present results indicated that the level of prolactin was significantly decreased in PCOS rats by the administration of biomolecule-loaded chitosan nanocomplex. The plasma progesterone levels decreased significantly in PCOS rats. Results strongly showed that biomolecule-coated chitosan nanoparticles lead to a significant increase in progesterone levels in PCOS induced rats. Similar results were also reported earlier by Pushpa and Kalavathy.⁴⁷ Jadhav et al.³⁵ described that increased level of progesterone was recorded in PCOS induced rats by the administration with high dose of Mimosa pudica extract. The elevated level of insulin significantly correlated with the insulin resistance in PCOS induced rats. The serum insulin level in PCOS rats was significantly decreased by the administration with higher dose of biomolecule-loaded chitosan nanoparticles. Pushpa and Kalavathy⁴⁷ also reported that the insulin level was down regulated by the treatment of PCOS rats with phytochemical formulations. The enzyme activity responsible for glucose uptake and insulin sensitivity has been enhanced with leaf extracts of G. sylvestre.⁴⁹ Most recently, Venkatachalam et al.⁵⁰ reported that the bioactive molecule-loaded chitosan nanoparticles prepared from G. sylvestre leaf extracts were found to be responsible for enhanced stimulation of insulin and restoration of metabolic activity in STZ-induced diabetic rats. Cinnamomum zeylanicum bioactive components (eugenol, cinnamaldehyde) were found to stimulate insulin release by enhancing activity of beta cells of pancreas to secrete elevated level of insulin.^51,52

The biochemical observations were strongly supported by the evidence of histopathological findings recorded by light microscopy. It has been explained that the histopathological examinations of PCOS induced rats had developed poly cysts in the ovary. In addition, the ovarian cortex showed the appearance of atretic follicles and the cysts were made by attenuated layer of granulosa cells and hyperplasia of thecal layer. Subsequently, the occurrence of massive disintegration of the atretic follicles is noticed and the number of corpora lutea are disappeared in the ovary showing anovulation and the frequency of estrous cycle is totally absent in PCOS induced rats.^34,35,53 Further, the elevated level of androgens may lead to disintegration of follicles by enhancing the rate of pyknotic granulosa cells and destroying oocytes.^34,35 Similar results were also noticed in the present experiment when the rats were subjected to PCOS induction with EV. The EV induced PCOS rats which had been treated by biomolecule-loaded chitosan nanoparticles showed restoration of normal estrous cycle described by histopathological investigation in the ovarian and uterine tissues. The histopathological findings in PCOS rats treated with biomolecule-loaded chitosan nanoparticles indicated the remarkable reorganization of ovarian tissue with the formation of normal follicle structure. According to the light microscopy observations, the presence of well-developed normal follicles was noticed and the follicles had normal granulosa with well-organized thecal layers and clear antrum with no cell debris. It is noteworthy to mention that the biomolecule-loaded chitosan nanoparticle treatment is not only responsible for corpora lutea formation but also involved in the estrous cycle back to normal in PCOS rats. The present result is in agreement with earlier report by Manneras et al.⁴³ who observed that the DHT induced PCOS rat model displayed large ovaries with atreticantral follicles, follicular cysts with thickened thecal layer and few corpora lutea. Generally, the increased level of LH in the body leads to over production of estrogen, androgen (male hormones), testosterone and DHEAS (dehydroepiandrosterone sulphate) levels that cause pathological appearances in the endometrial tissue and thickening of uterus which ultimately results in heavier/irregular periods.⁵⁴ Results revealed that the PCOS induced rat groups treated with GSCNPs and CZCNPs showed normalization of the follicular structures in the ovaries and uterus linings as well. It is very interesting to note that the poly cysts had mainly disappeared in the ovarian tissue and development of normal follicles had increased with better endometrial tissue rearrangements in uterus by the administration with biomolecule-coated chitosan nanoparticles. It has been reported that flower extracts of Matricaria chamomilla L. not only can induce recovery from PCOS rats to normal, but also can enhance the dominant follicles rate and endometrial tissue arrangements in the uterus.²³ Studies have reported that metformin when acting in ovarian thecal cells inhibits the production of androgens by reducing pituitary secretion of LH, leading to ovulation and regular menstrual cycles.⁵⁵ Similar histopathological results were also noticed with letrozole induced polycystic ovary rat model by treating with Symplocos racemosa plant extract.³⁷

The normalization of the estrous cycles was observed in the experimental PCOS induced rat groups when treated with synthesized biomolecule-coated nanoparticles using methanol extracts of G. sylvestre leaves and C. zeylanicum barks. It is presumed that this might be due to the presence of: (1) saponin component from the G. sylvestre leaf extract found in the GSCNPs and (2) polyphenolics (methyl-hydroxychalcone polymer), eugenol, cinnamaldehyde from C. zeylanicum bark extract incorporated into CZCNPs. The present result is in agreement with earlier findings reported by Santos et al.⁵⁶ who demonstrated when the female rats were exposed to plant steroidal saponin, rats enter into a state of permanent estrous and ultimately increased the duration of the estrous phase of their cycle. Present results clearly indicate that the conversion of PCOS induced estrous cycle back to normal following the treatment with nanoparticles could be achieved due to the presence of active biomolecule coated on chitosan nanocomplex which balancing sex hormone levels making into fertility condition to be retrieved. The FTIR data also strongly showed the presence of saponin, aldehyde and polyphenol which could be potential phytochemicals in regulating hyperglycaemic conditions and modulating steroidogenesis in PCOS induced rats. Similarly, Maharjan et al.²² also reported that phytocompounds such as phytosterol and polyphenol from Aloe vera involved in reversion of estrous cycle to normal in letrozole induced PCOS rats. Based on the results, it is hypothesized that the biomolecule-loaded chitosan nanocomplex may negatively regulate the activity of key enzymes such as 3β-hydroxysteroid dehydrogenase, and modulating the flux toward estradiol formation for normal follicle development in PCOS induced rats.

Conclusion

In conclusion, the bioactive molecule-loaded TPP cross-linked chitosan nanoparticles were successfully fabricated using methanol extracts of G. sylvestre leaves and C. zeylanicum barks via the ionic gelation technique and characterized by using FTIR, XRD, TEM and DLS. The synthesized nanoparticles were evaluated for their protective role against PCOS induced rat models by the assessment of gonadotropic hormones namely FSH, LH, PRL, PRG and INS level and histopathological examinations of ovary and uterus. The results confirmed that the PCOS experimental groups treated with GSCNPs and CZCNPs exhibited reverting pattern of estrous cycle back to normal compared to control. Interestingly, the biomolecule-loaded chitosan nanoparticles were found to be effective in decreasing the serum luteinizing hormone, prolactin and insulin level but increasing the level of follicle stimulating hormone and progesterone levels in PCOS rats than the control. The chitosan nanoparticles could also play a key role in maintaining balanced gonadotropic hormone levels and formation of corpora luteum and follicles in PCOS groups. Results reveal that the biomolecule-loaded TPP–chitosan nanocomplex was also comparable with metformin in PCOS treatment and leads to recovery of regular ovulation as well as fertility in PCOS induced rats. Further, the biomolecule-loaded chitosan nanoparticles could be used as potential nanomedicine to treat the PCOS in the future. To the best of our knowledge, this is the first detailed report on the effective role of biomolecule-loaded TPP cross-linked chitosan nanocomplex in the treatment of PCOS.

Conflict of interest

The authors have declared no conflict of interest.

Acknowledgements

The authors would like to acknowledge the management of Kovai Medical Centre College of Pharmacy, Coimbatore, Tamilnadu, India in proving the animal house for conducting the experiments. The authors are grateful to Mr G. Arihara Sivakumar, Assistant Professor, Department of Pharmacology, KMCH College of Pharmacy, Kovai Estate, Kalapatti Road, Coimbatore – 641 048 for his valuable suggestions and assistance during the experiment.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra07228c

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