BIOTROPIKA Journal of Tropical Biology
https://biotropika.ub.ac.id/
Vol. 11 | No. 2 | 2023 | DOI: 10.21776/ub.biotropika.2023.011.02.05
THE EFFECT OF COMBINATION OF Marsilea crenata AND Moringa oleifera LEAVES ETHANOL EXTRACT ON FOLLICLES AND OOCYTES OF GOAT: IN VITRO STUDY
Septiawan Putranto1), Siska Nanda Widhaningrum1), Sri Rahayu1)*, Gatot Ciptadi2)
ABSTRACT
Marsilea crenata and Moringa oleifera are herbal plants widely used in various alternative medicines due to their antioxidants and estrogenic activity. The current study investigated the effect of a combination M. crenata and M. oleifera leaves ethanol extract on the growth of follicles and oocytes of goats in vitro. Twenty-four antral follicles of Ettawa Crossbred Goat (Capra aegagrus hircus) were randomly grouped into four groups: K (control, without extract treatment), D1 (21.6 μg/mL of M. crenata ethanol extract), D2 (21.6 μg/mL of M. oleifera leaves ethanol extract), and D3 (combination of M. crenata and M. oleifera leaves ethanol extract at the dose of 21.6 μg/mL). The follicle diameter was measured on days 0, 3, and 6 of culture, while the oocyte diameter was only examined on day 6. After being treated with the combination of M. crenata and M. oleifera leaves ethanol extract (D3), the diameter of the ovarian follicles increased significantly (p<0.05) from day-0 to day-6. D3 group also greatly enhanced estrogen levels in the follicular fluid of goats compared to a single extract. However, the diameter of the oocyte was not significantly increased after being treated with a single or combination of both extracts. Therefore, this study suggested that combination of M. crenata and M. oleifera leaves ethanol extract can be used as phytoestrogen to enhance follicular growth and estrogen levels.
Keywords: flavonoid, follicle, Marsilea crenata, Moringa oleifera leaves, oocyte
INTRODUCTION
The process of reproduction is essential to all living organisms [1]. The ovarian follicle contributes to follicular development or folliculogenesis [2]. Folliculogenesis is a physiological process requiring complex interactions between autocrine, paracrine, and endocrine systems for the growth and mature ovarian follicles. Steroidogenesis, angiogenesis, oocyte maturation, and follicular atresia can all be affected by this [3]. During folliculogenesis, cellular metabolism will generate reactive oxygen species (ROS). Even though aerobic cells require oxygen (O2), the normal metabolism of O2 will generate several free radicals [4]. In addition, the superoxide anion radical (O2-) interacts with reactive nitrogen species (RNS) by forming peroxynitrite (ONOO-) via the reaction of nitric oxide (NO-) and superoxide anion radical (O2-) [5].
Proteins, cellular lipids, and nucleic acids will react with elevated ROS levels and RNS, severely damaging cell structures and influencing oxidative stress. Oxidative stress triggers apoptosis in antral follicles, which affects steroidogenesis and folliculogenesis [6]. Furthermore, the function of endogenous antioxidants in normal physiological activities will minimize the rise of free radicals and retain the stabilization of redox conditions in cells [5]. During ovarian follicle growth, the size of the oocyte increases along with granulosa cell layers
and antrum development [7]. Oocyte quality can be evaluated by measuring oocyte diameter [8].
Water clover (Marsilea crenata) and moringa leaves (Moringa oleifera) are herbal plants that have been used as food in addition to an alternative treatment for osteoporosis, urinary tract infections, esophageal inflammation, and other circumstances [9, 10]. This is due to the flavonoid content in M.
crenata and M. oleifera leaves, which have antioxidant, anti-inflammatory, antihypertensive, anti-osteoporosis, anti-tumor, and estrogenic characteristics [11, 12]. M. crenata leaves contain a high flavonoid, especially isoflavones formononetin and biochanin A, which have estrogenic activity and are important in folliculogenesis [13]. Furthermore, the ethanol extract of M. oleifera also contained a high flavonoid, such as quercetin and kaempferol, which could enhance folliculogenesis in mice with endometriosis [14]. To the best of our knowledge, the combination of M. crenata and M. oleifera leaves has never been used for improving folliculogenesis. Thus, the present study was intended to investigate the effect of the combination M. crenata and M. oleifera leaf ethanol extract on the growth of ovarian follicles and oocytes in vitro.
METHODS
Extract preparation. M. crenata and M.
oleifera leaves were purchased from a local farmer
Submitted : January, 2 2023 Accepted : June, 20 2023
Authors affiliation:
1)Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Brawijaya, Indonesia
2)Faculty of Animal Husbandry, Universitas Brawijaya, Indonesia
Correspondence email:
How to cite:
Putranto, S, Widhaningrum SN, Rahayu S, Ciptadi G. 2023. The effect of combination of Marsilea crenata and Moringa oleifera leaves ethanol extract on follicles and oocytes of goat: in vitro study. Biotropika:
Journal of Tropical Biology 11 (2): 101- 105.
in Surabaya, Indonesia. All samples were then cleaned and allowed to dry at 25°C for 4-5 days.
The powdered sample was extracted with 70%
ethanol using a modified maceration method [15, 16]. The extraction process was done in the Biochemistry Laboratory, Faculty of Mathematics and Natural Sciences, Universitas Brawijaya, Malang. The macerated extract was filtered and then evaporated using a rotary evaporator until it formed a paste. The obtained extract was then refrigerated until used.
Isolation of ovarian follicle. The study used adult Etawa Crossbred Goats (Capra aegagrus hircus) aged more than one year. The ovarian follicles of an Etawa Goat were obtained from Slaughterhouse, Malang, East Java. The ovaries were cleaned using sterile physiological saline. The ovaries were brought to the Central Laboratory of Life Sciences (LSIH), Universitas Brawijaya, using a container consisting of 0.9% sterile physiological saline, penicillin and streptomycin solution (Meiji, Japan) at 37°C. Some fatty ovarian tissue was replaced and cleaned with sterile physiological saline [17].
In vitro culture. Twenty-four ovarian follicles (2.5-3.2 mm) were maintained in a culture medium. The culture medium consisted of TCM- 199 media, 10% heat-activated Fetal Bovine Serum (FBS), 1% Penicillin-Streptomycin, 4%
polyvinylpyrrolidone, 1 IU/mL human chorionic gonadotropin (HCG), and 0.1 IU/mL follicle- stimulating hormone (FSH). The culture plate was covered with sterile paraffin oil and placed in the incubator (5% CO2 at 37°C) [18]. The ovarian follicles were then treated with the following treatment: K (control, no extract treatment), D1 (21.6 μg/mL of M. crenata ethanol extract), D2 (21.6 μg/mL of M. oleifera leaves ethanol extract), and D3 (combination of M. crenata and M. oleifera ethanol extract at the dose of 21.6 μg/mL). The culture medium was changed daily. All extract treatment was exposed to ovarian follicles for six days.
Morphological examination of follicular growth. The morphology of ovarian follicle growth was evaluated on days 0, 3, and 6 of treatment using a stereo microscope (Nikon, Japan) with 20x magnification. ImageJ software was used to determine the follicular diameter [19]. The morphology of isolated follicles is shown in Figure 1.
Day 0 3 6
Figure 1. The morphology of isolated ovarian follicles. The observation was done using a stereo microscope at 20x magnification.
Evaluation of oocyte quality. The oocytes were isolated from antral follicles by the slicing method. Oocyte diameter was measured on day-6 of culture using an inverted microscope with 10x magnification [7].
Determination of the 17β-estradiol level. The slicing method was used to isolate follicular fluid (±3 μL) from the follicular antrum. Follicular fluid was added with PBS until it reached a volume of 100 μL. Samples were then centrifuged at 6000 g, 5 min, at 4°C. The supernatant was collected and stored at -20°C. The level of 17β-estradiol was analyzed using the protocol of Goat 17β-estradiol Enzyme-Linked Immunosorbent Assay (ELISA) kit (Catalog no: MBS265205, Bioassay Technology Laboratory).
Data analysis. Data were represented as mean
± standard deviation (SD). One Way ANOVA and Duncan test were used to analyze the difference between treatment groups using SPSS version 16 for Windows (p≤0.05).
RESULTS AND DISCUSSION
Ovarian follicle growth. The combination of M. crenata and M. oleifera extract (D3) exhibited the highest increase in ovarian follicles diameter from day-3 until day-6 (Table 1). The control group, D1 and D2, also showed an increase in follicles diameter only, but not as much as in the D3 group.
The involvement of FSH in granulosa cell growth is responsible for the rise in ovarian follicle diameter during folliculogenesis [20]. Follicles will form antrum, and then the follicle's diameter can increase. Furthermore, 17β-estradiol could promote the growth and differentiation of granulosa cells, either naturally occurring or produced through steroidogenesis [21]. There are two granulosa cells: mural granulosa cells (synthesize sex steroid hormones) and cumulus cells (enclose the oocyte and support oocyte maturation). Thus, a rise in granulosa cell number, antrum development, and oocyte diameter increases the diameter of the ovarian follicle [7].
The increase in follicle diameter after treatment with the combination of M. crenata and M. oleifera leaves ethanol extract is caused by an antioxidant compound (phenolic) binding with free radicals [22]. The high antioxidant activity could inhibit cell damage caused by increased free radicals, enabling antioxidants and prooxidants to maintain intracellular homeostasis [5]. Moreover, flavonoids could modulate antioxidant action by
Table 1. The follicles diameter in all group treatments
Groups Follicle diameter (µm)
Day-0 Day-3 Day-6
K 200,720.71±25,172.66 209,069.05±24,181.69 206,756.75±26,730.50 D1 192,504.68±28,255.85 208,400.91±21,772.20 189,729.75±20,163.10 D2 187,019.51±15,102.69 190,930.93±18,639.27 201,321.31±18218.24
D3 154,684.65±7,197.43 172,792.8±9,214.94 218,003±31,746.84
Note: K (control, no extract treatment), D1 (21.6 μg/mL of M. crenata ethanol extract), D2 (21.6 μg/mL of M. oleifera leaves ethanol extract), and D3 (combination of M. crenata and M. oleifera leaves ethanol extract at the dose of 21.6 μg/mL)
inhibiting cell damage [23]. Phytoestrogens also support follicle growth in ethanol extracts of M.
crenata and M. oleifera leaves. Thus, phytoestrogen binds to estrogen receptors (ERα and ERβ) with lower binding activity in the cytoplasm or nucleus through genomic and nongenomic actions [24]. This estrogenic complex has a role as a transcription factor [20, 21, 22], which triggers rapid cellular responses, including PI3K/AKT, PKC, and MAPK/ERK, cAMP/PKA, which then enhances transcriptional activity [25].
According to Rosales-Tores [21], estrogenic action enhances the granulosa cell's response to FSH. Due to the expression of numerous genes involved in folliculogenesis, this condition promotes enhanced proliferation and differentiation activity.
Oocyte diameter. The results showed that all group treatments did not significantly (p>0.05) increase the oocyte diameter (Table 2). According to Santini et al. [26], phytoestrogen compounds can stimulate oocyte activity by triggering endogenous estrogen production and growth factors from granulosa cells. Griffin et al. [27] revealed that an increase in follicular diameter strongly correlated with an increase in oocyte diameter. However, this study showed that an increase in follicle diameter did not correlate with oocyte diameter. The limited number of oocyte samples caused insignificant results in oocyte diameter because this study only used three oocytes in each group.
Table 2. The oocyte diameter in all group treatments
Groups Oocytes diameter (µm)
K 162.66 ± 4.93a
D1 156.66 ± 5.50a
D2 152.66 ± 12.09a
D3 156 ± 8.18a
Note: The same alphabet indicated an insignificant difference (p>0.05). K (control, no extract treatment), D1 (21.6 μg/mL of M. crenata ethanol extract), D2 (21.6 μg/mL of M. oleifera leaves ethanol extract), and D3 (combination of M. crenata and M. oleifera leaves ethanol extract at the dose of 21.6 μg/mL).
Estrogen levels in follicular fluid. The results showed that the estrogen levels in follicular fluid were markedly (p<0.05) increased after being treated with the combination of M. crenata and M.
oleifera extract (D3) compared to another group (Figure 2). D1 and D2 also showed increased estrogen levels, but not as much as in the D3 group.
This finding supported our previous results, which found that the D3 group could enhance the follicle diameter (Table 1).
The high increase in estrogen level in the follicular fluid might be caused by the phytoestrogen compound in a combination of M.
crenata and M. oleifera. The phytoestrogen compound, such as quercetin, could enhance the activity of the aromatase enzyme by increasing endogenous estrogen, progesterone, FSH, and luteinizing hormone/LH [28]. The use of phytoestrogen effectively enhances the estrogen level. However, the excessive use of phytoestrogen will cause reproductive dysfunction in long-term use [29].
Figure 2. The estrogen level in the follicular fluid after being treated with the combination of M.
crenata and M. oleifera extract. K (control, no extract treatment), D1 (21.6 μg/mL of M. crenata ethanol extract), D2 (21.6 μg/mL of M. oleifera leaves ethanol extract), and D3 (combination of M.
crenata and M. oleifera leaves ethanol extract at the dose of 21.6 μg/mL).
The combination of M. crenata and M. oleifera leaves ethanol extract significantly affects follicle
*
0 0.05 0.1 0.15 0.2 0.25
K D1 D2 D3
Estrogen level (pg/mL)
Treatment groups
diameter and estrogen level but does not affect oocyte diameter. The limited number of follicles and isolated oocytes in each group treatment might cause insignificant results on oocyte diameter.
Therefore, this study still needs more evaluation with the appropriate number of samples and further research to confirm the effect of a combination M.
crenata and M. oleifera on different stages of follicle development.
CONCLUSION
The combination of M. crenata and M. oleifera leaves ethanol extract significantly enhanced follicle diameter and estrogen level in the follicular fluid of goats compared to a single extract.
However, the diameter of the oocyte was not significantly increased after being treated with a single or combination of both extracts. Therefore, this study suggested that a combination of M.
crenata and M. oleifera leaves ethanol extract can be used as phytoestrogen to enhance follicular growth and estrogen levels.
ACKNOWLEDGMENT
The authors thank the staff of Central Laboratory of Biological Sciences for assisting this research.
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