Abstract
Background and Objective:
The aging process in women is still a problem, especially in developing countries that will become developed countries, including Indonesia. Here, we investigated the action of Michelia champaca L. extract administration on antioxidant modulation to inhibit hippocampal apoptosis in middle-aged female rats.
Materials and Methods:
Thirty-two female Wistar rats were divided into four groups (n = 8 each group): the middle-aged rats without any treatment (control group) and three M. champaca L. extract groups (treated at doses of 100, 200, or 300 mg/kg b. w). The superoxide dismutase (SOD) levels and Bax and Bcl-2 expressions in the hippocampal region were analyzed using the technique of enzyme-linked immunosorbent assay.
Results:
The second and third doses of M. champaca L. extract significantly increased the SOD hippocampal levels compared with the control (P < 0.05). This extract also decreased Bax expression (at the second and third doses) and significantly increased Bcl-2 expression (at the highest dose) than that of the control (P < 0.05).
Conclusions:
The ethanol extract of M. champaca L. could modulate SOD and regulate apoptotic-related proteins in middle-aged female rats. Thus, the extract of M. champaca L. can be an alternative to prevent the degeneration of hippocampus due to the aging process.
Keywords: Aging, antioxidant, apoptosis, oxidative stress
INTRODUCTION
Old age is described as being older than 365 years, however, it can alternatively be considered being older than 60 or 70 years. The aging population reflects a country’s well-being. By 2050, 22% of the global population will be over 60 years old.[1] Life expectancy in the United States is anticipated to rise from 63 to 83 years. In 2010, the senior population in Indonesia was around 5%, and this figure is expected to rise to 11% by 2035.[2] The proportion of elderly women will rise from 5% in 2019 to 12% in 2050.[3,4] This population is growing year by year and has its own set of issues, such as chronic diseases, polypharmacy, medication side effects, and health-care utilization.[5,6,7,8,9]
Compounds generated during oxygen metabolism and ATP generation are known as reactive oxygen compounds. When the body’s synthesis of reactive oxygen molecules exceeds its antioxidant capacity, oxidative stress occurs, resulting in oxidative damage.[10] TheOxidative damage includes the oxidation of enzyme thiol groups as well as changes in the structure and integrity of biomolecules.[11] These outcomes may jeopardize the attainment of healthy aging. Apoptosis and necrosis are the mechanisms by which oxidative stress causes oxidative damage.[12,13,14] The accumulation of oxidative stress as a result of aging is a mechanism for cognitive deterioration in the elderly. The hippocampus’s functional and structural integrities are critical in learning and remembering. The presence of oxidative stress in the hippocampus has been linked to age-related cognitive impairment.[15,16,17]
Endogenous antioxidant superoxide dismutase (SOD) catalyzes the conversion of superoxide radicals to hydrogen peroxide.[18] SOD mutant mice had oxidative stress in all organs and plasma, as well as a shorter life span (up to 30%).[19,20,21,22] Apoptosis is triggered by oxidative stress through a variety of pathways, including mitochondrial damage, disruption of calcium homeostasis, and DNA damage. Reactive oxygen molecules that cause the oxidation of mitochondrial lipids and proteins cause membrane degradation as well as the release of cytochrome-C and calcium.[23]
Michelia champaca L. flower is the mascot of Aceh Province, Indonesia. This plant grows abundantly in Aceh, although supposedly it originated from Taiwan, Malaysia, and South China.[24] Previous studies have proven the active compound of M. champaca.[25] This plant has antifungal and antimicrobial,[26] antifertility,[27] antidiabetic,[28] anticancer,[29] and antileishmaniasis properties.[30] Thus far, studies on the benefits of M. champaca L. extract as an antioxidant to reduce hippocampal apoptosis in middle-aged female rats have not been carried out yet. Therefore, this study is aimed at evaluating the pharmacological action of plants in inhibiting oxidative stress and apoptosis in the aging process.
MATERIALS AND METHODS
Subjects
Thirty-two, 18-month-old, female Wistar rats at an average weight of 350–450 g were divided into four groups (n = 8 per group), including untreated group (control); middle-aged rats treated with M. champaca L. extract (100 mg/kg of body weight; MC100); middle-aged rats treated with extract (200 mg/kg of body weight; MC200); and middle-aged rats treated with extract (300 mg/kg of body weight; MC300). The extracts were given for 15 days orally with the probe. These doses were modified from a previous study.[27]
Extraction
M. champaca L. flowers were picked up on August 2016 from Langsa City, East Aceh, Indonesia. The flowers were cut into small pieces and then dried and ground into powder. The powders were then extracted with 96% ethanol using the Soxhlet method. The extraction process was carried out as described in the previous studies.[28,31]
Isolation of hippocampus
After the treatment was completed, the rats were sectioned to get the hippocampus. The rats were anesthetized with ketamine at the dose of 80 mg/kg and then the head cavity was opened to take the brain. The hippocampi were divided into two sections, homogenized and centrifuged, one of which was used for SOD level analysis and another one for Bax and Bcl-2 analysis.[32]
Analysis of superoxide dismutase level
SOD levels in the hippocampal region were analyzed by enzyme-linked immunosorbent assay technique (ELISA). The ELISA kit used here was Rat SOD ELISA Kit bought from MyBioSource, catalog MBS722675 (San Diego, CA, USA). Analysis procedures were carried out according to the procedures as stated in the kit.
Bax expression analysis
Bax expressions were analyzed using ELISA Kit (Rat-BCL-2-associated X protein) from Elabscience, catalog E-EL-R0098 (Houston, Texas, USA). The analysis steps were done in accordance with the procedures as stated in the kit.
Bcl-2 expression analysis
Bcl-2 expressions in the hippocampal region were analyzed using ELISA technique. The ELISA kit used was the Apoptosis Regulator, Bcl-2 ELISA Kit. This kit was bought from MyBioSource, catalog MBS28817313 (San Diego, CA, USA). Detailed analysis steps were done in accordance with the instructions as stated in the kit.
Ethics
The Faculty of Nursing, University of North Sumatera, Medan, North Sumatera, Indonesia, granted research ethical approval for this study (Number 944/VI/SP/2016).
Statistical analysis
The data are presented as the means ± standard deviation, and the differences between the treatment groups were analyzed by one-way analysis of variance and LSD test. The analysis was performed using SPSS 23.0 statistical package for Windows program (IBM, New York, United States). The probability value (P < 0.05) is significantly different.
RESULTS
Figure 1 shows SOD levels in the hippocampal region in the various treatment groups. SOD levels increased significantly in the MC200 or MC300 group compared with the MA group.
Figure 1.
Superoxide dismutase levels in the hippocampus in all treatment groups. Note: aThe P < 0.05 is compared with the control group; bP < 0.05 is compared with the middle-aged female rat group treated with Michelia champaca L. extract of the first dose; cP < 0.05 is compared with the middle-aged female rats treated with Michelia champaca L. extract of the second dose; Control: Middle-aged female rats as control; MC100: Middle-aged female rats treated with Michelia champaca L. extract of the first dose; MC200: Middle-aged female rats treated with Michelia champaca L. extract of the second dose; MC300: Middle-aged female rats treated with Michelia champaca L. extract of the third dose
Bax expressions in the hippocampal region are presented in Figure 2. Bax expression of the hippocampal region was significantly lower in the MC200 or MC300 group than the controls (P < 0.05). There are no differences in Bax expression between these two doses (P < 0.05).
Figure 2.
Hippocampal Bax expression in all experimental groups. Note: aThe P < 0.05 is compared with the control group; bP < 0.05 is compared with the middle-aged female rat group treated with Michelia champaca L. extract of the first dose; cP < 0.05 is compared with the middle-aged female rats treated with Michelia champaca L. extract of the second dose; Control: middle-aged female rats as control; MC100: middle-aged female rats treated with Michelia champaca L. extract of the first dose; MC200: middle-aged female rats treated with Michelia champaca L. extract of the second dose; MC300: middle-aged female rats treated with Michelia champaca L. extract of the third dose
Figure 3 shows the Bcl-2 expression of the hippocampal region in the various treatment groups. There was a significant increase in Bcl-2 expression in the MC300 group compared with the control group and the lower dose (P < 0.05).
Figure 3.
Hippocampal Bcl-2 expression in all experimental groups. Note: aThe P < 0.05 is compared with the control group; bP < 0.05 is compared with the middle-aged female rat group treated with Michelia champaca L. extract of the first dose; cP < 0.05 is compared with the middle-aged female rats treated with Michelia champaca L. extract of the second dose; Control: middle-aged female rats as control; MC100: middle-aged female rats treated with Michelia champaca L. extract of the first dose; MC200: middle-aged female rats treated with Michelia champaca L. extract of the second dose; MC300: middle-aged female rats treated with Michelia champaca L. extract of the third dose
DISCUSSION
Some previous studies stated that ethanol extract of M. champaca L. contains carbohydrates, flavonoids, alkaloids, and tannins. This plant also contains sesquiterpene lactones including cycloguaianolide, michampane, michampanolide, 8-acetoxyparthenolide, and magnograndiolide.[33] Previous studies have proven that SOD levels decreased significantly in postmenopause than premenopause.[34] In this study, there is a significant increase in SOD levels in the administration of M. champaca L. extract of both higher doses than the control. This finding indicates that the extract of M. champaca L. acts as an antioxidant through the mechanism of SOD antioxidant upregulation or scavenging of superoxide radicals so that the endogenous SOD enzyme level remains high. This finding extends the earlier findings, stating that M. champaca L. can scavenge free radicals,[35] even it contains SOD.[36]
Bcl-2 family is a protein that controls apoptosis, which is divided into anti-apoptosis and pro-apoptosis.[37] The extract in the second and third doses could significantly suppress the Bax expression in the hippocampal region. At the highest dose, the decrease in Bax expression was followed by a significant increase in Bcl-2 expression. This finding shows that the extract of M. champaca L. has the characteristic of apoptosis protein modulator by regulating anti-apoptotic and pro-apoptotic proteins in the hippocampal region of menopausal rats. We suggest that the anti-apoptosis mechanism was underlined by antioxidant mechanisms. Severe oxidative stress will trigger apoptosis,[38] and it has been proven that M. champaca L. is an antioxidant.[35,36] (−)-Anonaine is an active substance of M. champaca L. that can regulate apoptosis.[39]
Thus, it is concluded that M. champaca L. extract can modulate SOD and apoptosis regulating proteins in hippocampus of middle-aged rats. Thus, this herbal can be an alternative to prevent the degeneration of hippocampus due to the aging process.
CONCLUSION
Thus, it is concluded that M. champaca L. extract can modulate SOD and apoptosis regulating proteins in hippocampus of middle-aged rats. Thus, this herbal can be an alternative to prevent the degeneration of hippocampus due to the aging process.
Ethics approval and consent to participate
This study was approved (Number 944/VI/SP/2016) by the Ethics Committee from the Faculty of Nursing, University of North Sumatera, Medan, North Sumatera, Indonesia.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
Acknowledgments
We acknowledged all technicians in the Physiology Laboratory, Faculty of Medicine, Universitas Brawijaya, Malang, East Java, Indonesia, for helping in experiment.
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