The effect of Moringa leaves aqueous extract to Ovarian sodium dismutase and Apoptotic index in rats treated with depomedroxyprogesterone acetate

Ratna Dwi Jayanti¹, Ivon Diah Wittiarika¹, Rize Budi Amalia¹, Baksono Winardi²,

Sri Winarsih³, I Wayan Arsana Wiyasa⁴

¹School of Midwifery, Faculty of Medicine, Airlangga University, Surabaya, Indonesia.

²Department of Obstetrics and Gynecology, Dr. Soetomo General Hospital, Faculty of Medicine,

Airlangga University, Surabaya, Indonesia.

³Pharmacy Study Program, Faculty of Medicine, Brawijaya University, Malang, Indonesia.

⁴Department of Obstetrics and Gynecology, Syaiful Anwar General Hospital, Faculty of Medicine,

Brawijaya University, Malang, Indonesia.

*Corresponding Author E-mail: ratnadwijayantii@gmail.com

ABSTRACT:

Depomedroxyprogesterone acetate (DMPA) is a widely used progestin based contraception. The long term use of this contraception could be unpleasant including the disruption of ovarian hormonal production resulting the excessive oxidative stress. Oxidative stress affects the entire reproductive organ including uterus and ovaries even after the reproductive lifespan finished (menopause). Oxidative stress result as an imbalance between pro-oxidant and antioxidant level. Moringa oleifera leaves extract is known to have a high antioxidant compound such as flavonoid, fenolic, saponin, steroid, tannin, and terpenoid. This antioxidant activity has been displayed by the increase of antioxidant enzyme such as glutathione (GSH), sodium dismutase (SOD), catalase, and also significantly reduce the lipid peroxidation process. Previous studies have been explained the function of this antioxidant to reproductive organ. Despite of this, the SODand apoptotic rate of ovarian granulosa cells have not yet been explored in the aqueous extraction of Moringa oleifera leaves. Sodium dismutaseis the main antioxidant enzyme with the central role in stress oxidative elimination. Meanwhile the apoptoticrate in the ovarian granulosa cells reflects the damage in the ovarian tissuethus reduce reproduction performance. In this report, we investigated the effect of Moringa oleifera leaves aqueous extract on SOD antioxidant using spectrophotometry SOD assay kit in DMPA exposed rats model. In addition, the apoptotic index in the granulosa ovarian cell was also assessed. We showed thatadministration of moringa oleifera leaves aqueous extract with the dose dependent manner would significantly increase the SOD level in the DMPA exposed rats model. Moreover, the increased of this antioxidant dose was dramatically reduced the apoptotic index in the ovarian granulosa cell. This result confirmed the high effect of moringa oleifera leaves aqueous extract as the potential plant to reduce oxidative stress with the huge impact on the reproductive organ.

KEYWORDS: Moringa oleifera, Ovarian sodium dismutase, Apoptotic index, depomedroxyprogesterone acetate

INTRODUCTION: 

The high dose of MDPA as a contraceptive method has been accepted in the world as the most promising and effective contraceptive method with a relative low failure¹. Unfortunately, the high dose and long term use of progesterone based contraceptive such as in DMPA affect the disruption of estrogen level in the body resulting in the low antioxidant level within ovaries². This condition affects the increase of the oxidative stress level and could be dangerous to the follicles³.

Centuries of research in finding antioxidant compound has been becoming the centre of attention. Moringa oleifera leaves extract has been postulated to be a potent antioxidant to prevent the stress oxidative. The leaves have a positive compound including vitamins, phenolic acids, flavonoids, isothiocyanates, tannins and saponins and act as a potent antioxidant⁴. The protective effect of this antioxidant could be in the various role such as cancer, inflammatory diseases, cystic fibrosis, ischemia, aging, rheumatoid arthritis, neurodegenerative diseases, and diabetes⁵.

Previously, it has been shown that Moringa oleifera leaves extract has the protective effect to the reproductive organ. For instance, Zheng and colleagues, has been proved that Moringa oleifera leaves extract  can increased litter size and antioxidant ability, reduced the rate of sperm abnormality and the expression of Bax⁶. Unfortunately, it was unknown whether the aquous extract leaves of Moringa oleifera has the protective effect especially in the ovarian follicle toxicity. The aquous technique is very crucial to explore since the large community apply the various part this plant on water based processed food.

METHODS:

Animal and ethics statement:

All of the procedure has been conducted under University of Brawijaya animal ethics committee 222B/EC/KEPK/08/217. The animal model was female Rattus norvegicus weighed 100-150g aged 10-11w that was acclimatized for 1w and maintained in ad libitum supply for standard feed and water in 27-28⁰C temperature on 12/12 photo period. Moringa leavesaquousextract were administered at day-1 to day-28 and DMPA was injected intramuscular at days 1, 8, 15, 22 of menstrual cycle.

The total of 30 rats that were divided into 5 groups: negative control group (distilled water 0,2ml/week), positive control group (receiving DMPA 2,7 mg/rat/week), P1 (DMPA 2,7mg/rat/week + Moringa leaves extract 100mg/kg body weight/day), P2 (DMPA 2,7mg/rat/week + Moringa leaves extract 150mg/kg body weight/day), P3 (DMPA 2,7mg/rat/week+ Moringa leaves extract 200mg/kg body weight/day). DMPA was given intramuscularly and Moringa leaves was given via lavage once a day.

All mice were euthanized at day-28 after Moringa leaves extract administration and both of the ovaries were collected. The right ovary was stored at Phosphate buffer saline (PBS) and the left ovary was stored at 10% formalin buffer.

Moringa leaves extraction:

Moringa leaves were collected from the local area in Indonesia. One hundred and fifty grams of moringa leaves powder were soaked in 1,5L water with temperature of 70⁰C for 1h, stirred and filtered with whatman filter paper (number 3). The extract then was evaporated using rotary evaporator with 45⁰C then stored in 4⁰C freeze until use.

Superoxide dismutase assessment:

The freshpost mortem ovarian tissue washomogenized and centrifuged at 1500g in 4⁰C temperature, stirred at 80⁰C freezer until use. Two hundred μlradical detector of Superoxide Dismutase Assay Kit Item No. 706002 (Cayman Chemical) was prepared in 96 wells plate, and 10μl of sample was put in each wells. The reaction was initiated by adding 20μl of diluted xanthine oxidase. The plate was incubated for 20m in RT. The absorbance was read with the plate reader at 440-460nm.

Apoptosis index measurement in the ovaries:

Five μm of the tissue section was deparaffinization in 60⁰C for 1h followed by incubation with xylene for 10m. Rehydration was done with the degrade concentration of ethanol and washing process using double distilled water. Apoptotic cells were assessed using In Situ Cell Death Detection Kit (Roche). The sample then incubated with proteinase K working solution for 15-30 minutes in 21-37⁰C followed by washing step with PBS.  The slides then were incubated with micro nuclease coccal for 10m in 15-25⁰C to induce the DNA fragmentation before the labelling. Fifty ml converter POD were added to the sample and incubated in 37⁰C temperature. After washing with PBS, slides were ready to be assessed after the mounting. Semi-quantitative analysis was performedand the samples were examined by 40x objective to measure the apoptotic index by dividing the cells that displayed the brownish colour with the total cell multiplied by 100 (source) in the 10 different areas of interest to obtain the percentage of apoptotic index using Image J (Fiji) software⁷. The score was compared using the Statistical Package for Social Science (SPSS) 16.0 software program by one-way ANOVA test. A p<0.05 was considered as a statistically significant value.

RESULT:

Superoxide dismutase assessment on ovarian lesion affected by medroxyprogesterone acetate exposure treated with moringa leaves extract.

We performed the assessment to identify the SOD level in DMPA exposed rats model using spectrophotometry SOD assay kit. The graph below showed the groups of treatment and the mean±standard deviation (SD) of SOD. The increased dose of moringa leaves aquousextract would significantly increase the antioxidant SOD in DMPA exposed rats model.

Figure 1: The absorbance of superoxide dismutase in rats ovarium. The bars showed the value from normal rats, DMPA rats model, DMPA + Moringa oleifera leaves extract 100mgDMPA + Moringa leaves extract 150 mg,DMPA + Moringa leaves extract 200 mg respectively. The dose dependent of moringa leaves aquous extract could significantly increase the SOD in rats treated with DMPA (p<0.05)

A                                                             B

C                                                              D

E

Figure 2. A.Ovary from negative controlrats model B. Ovary from DMPA exposed rats model. C. DMPA + Moringa oleifera leaves extract 100mg, (JC) D. DMPA + Moringa leaves extract 150 mg, E. DMPA + Moringa leaves extract 200 mg . Orange arrows shows the apoptotic body. In Situ Cell Death assay. 40x magnifications

Figure 3: Semi-quantitative analysis of apoptotic index in DMPA exposed rats model. The apoptotic index was quantified by divided the brownish color apoptotic cells divided by the total cells multiple with 100% using Image J (Fiji) software. There was significantly decreased of apoptotic index with the increase dose of moringa leaves aquousextract compared to control positive rats (p<0.05)

DISCUSSION:

Depot-medroxyprogesterone acetate is a progestin injectable hormonal contraception, has been widely used as the hormonal contraceptive worldwide and also has the impact in reducing antioxidant level preventing causing oxidative stress^8,9. Previous studies have been proved that a high dose of progestin intake are associated with hipotalamus-pituitary-ovarium axis impairment¹⁰. The external injection of a high dose of progestin affect the gonadotropin releasing hormone (GnRH) and lutheinizing hormone (LH) level which in turn reduce the esterogen release by ovarium¹⁰. As if, esterogen has been known for the role in the oxidative stress and low level of estrogen in the maternal circulation affect oxidative stress¹¹.

Previous studies have been explained Moringa oleifera leaves extract as the antioxidant plant based. The antioxidant compound of moringa oleifera leaves extract including total phenolic content (TPC), total flavonoid contents (TFC), free radical scavenging activity (DPPH), and ferric reducing antioxidant power (FRAP)¹². Sreelatha and colleagues have been proved that moringa oleifera leaves extract has a potent antioxidant activity against free radical by preventing oxidative damage to the majority of the biomolecules against oxidative damage¹³. In addition, moringa oleifera leaves extract has a protective effect in the reproductive organ such as sperm motility, count, and testosterone level¹⁴.

In this study, we sought to determine whether Moringa oleifera leavesaquous extract could increase the SOD level, as the most potent antioxidant, in dose dependent manner. In addition, the apoptosis rate of the follicle was also displayed in order evaluate the strength of the antioxidant in preventing stress oxidative. With the age matched control, we proof that MDPA administration without moringa oleifera leaves extract exposure inhibit the SOD production and increase the apoptosis rate.

Spectrophotometry assessment of SOD in the DMPA-exposure mice ovarian showed the increase of SOD level along with the increase of moringa oleifera leavesaquous extract administration. The molecular mechanism of this phenomenon is unknown, but previous report showed that the SOD level can increase by either the production of SOD or the antioxidant component in the compound inhibit the oxidant thus affect SOD improvement. Although the mechanism of this is not clear, it is possible that they might play an important role in inhibiting the oxidant, since the high compound of bioactive components, including vitamins, phenolic acids, flavonoids, isothiocyanates, tannins and saponins was found in their leaves ⁴. Further, the high amount of antioxidant can break the chain reaction and protect the cell, thus save the SOD level to prevent the stress oxidation¹⁵.

In DMPA exposed rats model ovary, the apoptotic granulosa cells raised. In addition, the increased dose of moringa oleifera leaves aquousextract would reduce the apoptotic bodies dramatically. It could be explained by the ability of their antioxidant compound in preventing cell death via receptor-mediated apoptotic pathway as well as cell death via the mitochondrial pathway¹⁶. In addition, reactive oxygen species are mediators required for the apoptotic program or inhibit a survival pathway¹⁶.

CONCLUSION:

Our result strongly indicated that the administration of moringa oleifera leaves aquousextract have a protective effect of DMPA effect exposure.

ACKNOWLEDGEMENT:

The authors thank to Brawijaya University for the support and help during the research and Airlangga university for the funding.

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Received on 30.09.2021            Modified on 08.05.2022

Accepted on 02.12.2022           © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(5):2103-2106.

DOI: 10.52711/0974-360X.2023.00345