A Nutraceutical approach to enhance Reproductive longevity and Ovarian health using Curcuma longa in wistar Rats

Damodara Gowda K M¹, Shailaja S Moodithaya¹*, Vishakh R Kedilaya², Nayanatara A K³

¹Dept of Physiology, K.S. Hegde Medical Academy, Nitte (Deemed to be University), Mangalore - 575018, Karnataka, India.

²Central Research Laboratory, K.S. Hegde Medical Academy, Nitte (Deemed to be University),

Mangalore - 575018, Karnataka, India.

³Department of Physiology, Kasturba Medical College, Mangalore, Manipal Academy of Higher Education, Manipal India.

*Corresponding Author E-mail: shailaja.moodithaya@nitte.edu.in

ABSTRACT:

Aging is associated with various physiological, pathological and psychosocial alterations. This study evaluates the benefits of Curcumin by assessing reproductive aging indices and ovarian health in Wistar rats. Laboratory bred adult rats selected for the experiment. After 12 months of follow-up, the animals were grouped into Normal control rats, Sham control group, Curcumin-1(100 mg/kg body weight), Curcumin-2(200 mg/kg body weight) and Curcumin-3 (400 mg/kg body weight). For the duration of six months Curcumin dosage was administered. The experimental parameters included estrous cycle and histological evaluation of ovarian follicles. Data were analyzed using one-way ANOVA. The percentage of primordial follicles was significantly more (p<0.001) in all the groups when compared with other types of follicles. Prolonged increase (p=0.0001) in the Diestrus phase in animals treated with different dosages of Curcumin. The current study concludes that Curcumin, an active component of Curcuma longa contributes to the anti-aging properties.

KEYWORDS: Curcumin, Anti-aging, Estrous cycle, Reproductive aging, Ovarian aging, Ovarian follicles.

INTRODUCTION:

Aging is a natural phenomenon. It has been associated with the decline in physiological, structural, and functional components in the associated organs¹. In females the health of the ovaries and the reproductive capability is the main facet for the determination of the ageing. Reproductive aging eventually leads to menopause². Ovarian aging occurs exceptionally early and rapid when compared to the other organs.

The hormonal benefits associated with the premenopausal state declines gradually as the age advances. This denotes that in postmenopausal life there is increased risk associated with cardiovascular, osteoporosis, and cognitive dysfunction directly effecting the quality of the life with advanced ageing. Age-related infertility is also commonly seen in females³. This may be due to the loss of ovarian reserve, which happens due to the decline in the quality and quantity of eggs. Preservation of the reproductive potential and the symptoms associated with the aging process is of prime importance. This is probably due to the reason that ovarian failure associated with aging cannot be reversed. Recent research has focused on improvement in the reproductive capacity with age using naturally available agents.

Anti-aging medicine plays an very important role in the therapeutic approach to problems associated with aging provoking healthy lifespan filled with youthfulness^4-7. Antiaging biomarkers mainly promotes the functional evaluation of body systems and helps in balancing physiological and pathological biomarkers^2,8-11. Nutrition plays a prime role in aging. The type of diet also influences the aging process. This might be probably associated through controlling the mechanisms associated with the biochemical process of aging^5,12. Numerous pharmaceutical components prompts healthy aging¹³. Literature survey documents the physiological benefits of these nutraceuticals against chronic disease ^14-17. Among nutraceuticals, Curcumin, commonly known as 'turmeric,' has gained interest due to its increased medicinal value against various diseases^18-20. Long-term dietary restriction negatively affects estrous cycles and reproductive output². Our previous study documented the role of Curcumin slowing the aging process considering inflammatory indices^21,22. However, studies investigating reproductive longevity and ovarian health in humans or mammalian models is very few. Therefore, the present research was aimed to explore the longevity benefits of Curcumin by assessing reproductive parameters focusing on the aging indices in female Wistar rats.

MATERIALS AND METHODS:

Ethics:

All the experiments were carried out following the National Institute of Health Guide for the Care, and Use of Laboratory Animals and associated guidelines and all the experimentation procedures were done after the prior approval from the Institutional Animal Ethics Committee.

Chemicals:

All the required kits and reagents were procured from Sigma– Aldrich (Merck), India.

Preparation of test sample:

Analytical grade curcumin powder was procured from Sigma-Aldrich. The required quantity of Curcumin was weighed and suspended in distilled water to get 100mg, 200mg, and 400mg of Curcumin per kg body weight in 2ml of the test solution.

Experimental Animals:

Laboratory bred adult two-month-old female albino Wistar rats were selected for the experimentation procedures. Rats were housed in separate polypropylene cages containing sterile paddy husk as bedding material with 12:12 light: dark cycle at room temperature 26±1°C. The animals were fed with standard rat feed (Gold Mohar. Hindustan Lever Limited, Mumbai) and water ad libitum. The animals were followed up to 12 months and were regularly examined throughout the study.

Experimental design:

Twelve months old experimental animals were divided into five groups. Each group contained of eight rats. The animals were grouped into

Normal control (NC):

These group of rats were fed with standard rat feed and water ad libitum

Sham control (SC):

The sham control group received distilled water along with standard rat feed and water ad libitum.

Curcumin was administrated orally, daily for six months in addition to standard rat feed and water. Based on the dosage administered the animals were further subdivided into

Curcumin-1- These animals were treated with curcumin dosage of 100mg/kg body weight

Curcumin-2- These animals were treated with curcumin dosage of 200mg/kg body weight

Curcumin-3-These animals were treated with curcumin dosage of 400mg/kg body weight

Estrous cycle evaluation:

Animals showing regularity in the normal cycle were separated and chosen for further studies. Daily vaginal smears were performed every morning between 8:00 AM and 9:00 AM. Vaginal secretion was collected with a plastic pipette filled with 2ml of normal saline (NaCl- 0.9%) by inserting the tip into the rat vagina. The vaginal fluid was placed on glass slides. A different glass slide was used for each animal. One drop was collected with a clean tip from each rat. Unstained material was observed under a light microscope, without using the condenser lens, with 10x and 40x objective lenses. The duration of the normal oestrous cycle in rats was 4–5 days. Three types of cells are recognized such as round and nucleated ones are the epithelial cells; irregular ones without a nucleus are the cornified cells, and the little round ones are the leukocytes. The proportion among them was used for the determination of the oestrous cycle stages. Proestrous is the short phase of the oestrous cycle that refers to the pre-ovulatory day. Oestrous was characterized by cornified epithelial cells, which were abundant and often non-nucleated with the granulated cytoplasm and irregular shaped. Meta-oestrous is a brief stage comprising of a large number of leucocytes and a small number of large, non-granular and non-nucleated cornified epithelial cells and Dioestrus was identified with many leucocytes.

After six months of intervention, the 18 months old animals were sacrificed, and ovary was obtained by bilateral oophorectomy.

Histological studies:

Ovaries were harvested by bilateral oophorectomy and cut into serial longitudinal sections. The follicles were counted at four parts distinct from each other by approximately 200 micrometres. The sections were stained with haematoxylin and eosin, the number of follicles (primordial, primary, preantral, antral) and corpora lutea were counted.

Statistical analysis:

All the data were expressed as mean±SD from 6 animals per group. Though, each group contained eight animals, the data from 6 animals were taken for analysis. The differences between the means of different experimental groups were compared for statistical significance by the Student's t-test, followed by Tukey's HSD test with the significance level set at p<0.05 using SPSS Version 16.

RESULTS:

Histological analysis of the ovary:

On comparison of the percentage of different types of follicles in the ovary in different experimental groups, a significant decline (p=0.001) in the number of primary, pre-antral, antral, and corpora lutea was observed when compared to primordial follicles in all the groups (Fig-1). Within-group comparison of different types of follicles in the ovary of different experimental groups showed a highly significant difference (p=0.0001) when compared to the primordial follicle with primary, preantral antral and corpora lutea of different experimental groups (Fig-2).

Cytological study to evaluate the phases of estrous cycle in wistar rats:

Four phases of the oestrous cycle were identified, depending upon the presence of cell types found in the smear (Fig-3). The analysis of the oestrus cycle showed that there is a significant decline in the proestrous phase (p=0.001), Oestrus phase (p=0.001), and Meta-oestrous phase (p=0.01) respectively in animals treated with 100mg, 200 mg and 400 mg of curcumin per kg bodyweight. Whereas, there was a significant increase (p=0.0001) in the Diestrus phase in animals treated with different dosages of Curcumin (Table-1).

Fig-1: Comparison between the percentage of different Types of follicles in the ovary of different experimental groups.

Fig-2: Comparison of effect of curcumin on different types of follicles in the ovary of animals within the different experimental groups.

Table-1: Comparison of effect of different dosages of curcumin on different phases of estrus cycle.

Estrous cycle stages	Normal control	Sham control	Curcumin-1	Curcumin-2	Curcumin-3
Proestrus	6.9±1.89	7.5±1.69 NS	4.3±2.64**	4.0±2.83**	3.3±3.01**
Estrous	8.5±0.83	7±2.32 NS	1.83±3.17***	1.83±3.17***	2.1±2.97***
Meta-estrous	8.80±1.32	8.83±2.79 NS	5.5±2.59*	5.0±2.51*	4.1±1.17*
Diestrus	6.6±1.03	5.83±2.44 NS	12.55±2.59 ***	12.83±2.69 ***	13.6±2.73 ***

***P<0.0001, **P<0.001, *P<0.01

Compared to Normal control, NS: Non-Significant

Fig-3: Effect of curcumin on cytological changes at different phases of estrous cycle in the ovary of different experimental groups. NEC: Nucleated Epithelial Cells, ANC: Anucleated Cornified Cells, L: Lymphocytes.

Fig-4: Effect of curcumin on the histology of the ovary of different experimental groups (H & E, x100). 4a. Primordial follicle, 4b. Primary follicle, 4c. Pre-antral follicle, 4d. Antral follicle, 4e. Corpus luteum.

Histological studies of the ovary:

Analysis of the percentage of different follicles in the ovary of different experimental groups showed a significant difference (p=0.001) between Normal Control and Sham Control with Curcumin-1, Curcumin-2, and Curcumin-3 for all the types of follicles (Fig-4). The total number of follicles (primordial, primary, preantral, antral) and corpora lutea were counted. Primordial follicle is partially/entirely encapsulated by squamous pre-granulosa cells (Fig-4a). Primary follicles showed an enlarged single layer of granulosa cells (Fig-4b). The oocyte was encapsulated in the preantral follicle by more than two layers of granulosa cells; no antrum formation (Fig-4c). The oocyte was encapsulated in the antral follicle by more than two layers of granulosa cells with antrum formation (Fig-4d). The corpus luteum exhibits the presence of both granulosa cells and thecal cells (Fig-4e). Analysis of the percentage of different types of follicles in the ovary of different experimental groups showed a significant difference (p=0.001) between Normal Control and Sham Control with Curcumin-1, Curcumin-2, and Curcumin-3 for all the types of follicles.

DISCUSSION:

Aging imposes various challenges and has been associated to various age-associated diseases decreasing the ability to survive. Increase in the level of the reactive oxygen species might be the main cause of the age related chronic and acute diseased conditions^23-26. This study is an approach to evaluate the anti-aging properties of Curcumin. Curcuma longa (turmeric) has been used in traditional medicinal system^27,28. The active metabolite of present in CUR is Tetrahydrocurcumin (THC). THC with the help of the reductase enzyme found in the intestinal epithelium helps in the metabolization of orally ingested curcumin. THC possesses diverse antioxidant activity. The role of tetrahydrocurcumin as an antioxidant role has been implicated in recovery from the injury to the real tissue in in mice apart from its anti-inflammatory responses²². Various studies document the protective role of curcumin. Comparison of immunological indices in different study groups indicated that animals treated with Curcumin showed a significant increase in A: G ratio and lymphocyte count compared to the control and sham control groups. However, a considerable reduction in c-reactive protein was observed in animals treated with Curcumin at a dosage of 400mg/kg body weight. In the present study the follicles with a one layer of squamous follicular cells was considered as primordial. Single layer of cuboidal follicular cells in single layer is considered as the primary follicle. Whereas, the secondary follicle is the one which contains more than one layer of follicular cells. Follicles with distinct antrum were considered as antral^29,30. Healthy follicles were categorized based on the absence or presence of oocyte and degeneration of the granular component’s³¹. Comparing the stages of follicles in various study groups showed that the percentage of primordial follicles was significantly higher in the treated groups. Thus, indicating the slowing down of the reproductive aging.

The rhythmic reproductive cycle in sexually mature female mammals is the estrous cycle. It is an index of the neuroendocrine activity. Di-estrous is immediately followed by the Proestrous phase of the next cycle. Disturbances in the oestrous period is considered as a reasonable index of the ovarian activity. It depends on various factors associated with the regulation of HPA axis. Any variations in the normal oestrus cycle might be due to the hormonal disturbances causing variations in the ovarian and endocrine function. Prolonged duration in the estrous cycle was taken as the varied cyclic phase^14,24. In the present study, Curcumin treated rats showed a decline in the duration of proestrous, oestrous, and metestrus phase. This confirms that Curcumin plays a vital role in fertility control in female rats.

In conclusion the current study concludes that Curcumin, an active component of Curcumin longa, exhibits the anti-aging properties as observed by selective reproductive aging indices. In addition to the evaluation of Curcumin's efficacy on reproductive aging, the assessment of other aging risk factors, such as oxidative, mental, physical, and metabolic (glycation) stress is essential to promote the health and improve quality of life. More studies are further essential to explore the mechanisms of anti-aging effect of Curcumin on reproductive longevity.

ACKNOWLEDGEMENT:

Authors kindly acknowledge the financial support given by Nitte (Deemed to be University), Mangalore, Karnataka, India.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 21.07.2020 Modified on 29.08.2020

Research J. Pharm. and Tech 2021; 14(10):5385-5390.

DOI: 10.52711/0974-360X.2021.00939