The study investigated the protective and curative effects of unripe palm kernel seeds on monosodium glutamate-induced uterine fibroid in rats. The rats were in eight experimental groups according to the time of administration of the unripe palm kernel seeds: the first four groups investigated protective effects of UPK for 28 days and the last four groups investigated the curative effects of UPK after treatment with MSG for 28 days. MSG was administered to the animals by oral gavage at a dose of 200 mg/kg bd wt and UPK was administered as feed at a dose of 1.14 mg/g bd wt during the period of study. Histopathology studies of the uterus in addition to serum total protein, total cholesterol, estrogen and progesterone levels were determined. Significant increased estrogen and progesterone levels were observed in MSG-treated animals. UPK prevented and also led to the reversal of these biomarkers in this study. MSG also resulted in endometrial epithelium distortion and lamina propria fibrosis with UPK ameliorating the distortion observed in the uterus. These findings suggest that UPK contains bioactive constituents that are useful in preventing and reversing MSG-induced uterine hyperplasia.
Fibroid Estrogen Palm Kernel1. Introduction
African oil palm (Elaeis guineensis), native to west and southwest Africa, is mainly grown for its industrial production of oil. The fruit produces two types of oil: the palm oil from the mesocarp and the kernel oil from its kernel [1] [2]. The various parts of the tree have been reported to possess several medicinal uses [3].
Uterine fibroids are common non-cancerous or benign tumors that develop in or around the uterus, often affecting women in reproductive age [4]. Several genetic, biological and hormonal factors have been shown to contribute to the development and growth of fibroid, with uterine fibroid often most prevalent in black women [5]. Ample evidence supports hormones, estrogen and progesterone, as important factors in the etiology of uterine fibroids, with high level of estrogen reported to be the most common cause of fibroid [6] [7] [8].
Monosodium glutamate, a sodium salt of glutamic acid, is used in the food industry as a flavor enhancer and a popular food additive [9] [10]. Studies have shown that monosodium glutamate (MSG) induces uterine fibroid in rats by increasing the levels of total protein, cholesterol and estradiol [11] [12] [13].
Current treatment of uterine fibroids involves evasive and non-evasive methods like myomectomy, hysterectomy, hormone replacement therapy and uterine artery embolization [14] [15]. Given the risks, side effects associated with these methods and health care costs, there is an increased demand to find alternative and cheap means for the treatment and management of numerous disease conditions. Herbal practitioners have often recommended daily doses of unripe African oil palm (Elaeis guineensis) kernel seeds over a period of time, and this has been reported to be successful in reducing fibroids. However, there is unavailability of scientific data on the mechanism of this action and thus, this research was aimed at discovering the mechanism(s) by which unripe palm kernel seed is used in managing and/or treating uterine fibroid in female Wistar rats.
2. Materials and Methods2.1. Experimental Animals
Forty female albino rats of Wistar strain with weights ranging from 90 - 110 g were purchased from the Department of Biochemistry, University of Benin. The animals were allowed to acclimatize for 14 days under standard laboratory conditions (12-hour light/dark cycle, standard conditions of temperature and humidity, and good hygiene practices) during which they were given chow and clean drinking water. The rats used in this study showed regular estrous cycle length (4 to 5 days) with these cycles assessed by observing the vaginal smear in the morning according to the procedure described by Solomon et al. [16].
2.2. Oil Palm Kernel
Unripe oil palm kernel (UPK) fruits were purchased from Nigeria Institute for Oil Palm Research (NIFOR), Edo State. Fruit samples were authenticated and identified at the Department of Plant Biology and Biotechnology, University of Benin, and a specimen with voucher number UBH-E444, deposited. The unripe seed nuts were cracked open with nut crackers and chaffs separated from the seeds, after which they were refrigerated to avoid desiccation and fermentation.
2.3. Monosodium Glutamate
A stock solution of monosodium glutamate (Sigma Aldrich Chemical Co., St Louis, USA) was prepared according to methods described by Olowofolahan et al. [17] by dissolving 200 mg of the salt in 200 mL distilled water. The stock MSG was kept under room temperature for the total period of administration.
2.4. Ethical Approval
Experimental protocols were carried out according to the approval and guidelines given by the University of Benin Ethical Committee. These rules, similar to international guidelines on animal handling, conformed to the acceptable guidelines on the ethical use of animals in research.
2.5. Experimental Design
After the period of acclimatization, when the rats reached an average weight of 150 g, the rats were divided into two groups: protective and curative; with each group containing four sub-groups of five rats each.
Protective studies:
Group A rats received neither MSG nor UPK seeds during the period of study (this served as the control group). Group B rats received MSG for 28 days. Group C rats received UPK seeds for 28 days. Rats in group D received MSG and UPK seeds for 28 days.
Curative studies:
Group E rats received neither MSG nor UPK seeds for 42 days, the period of study (this was the control group). Group F rats received no MSG, but were given UPK seeds from day 28 to day 42.
Group G rats received MSG for 28 days and thereafter stopped, with normal rat chow continued till day 42. Group H rats received MSG for 28 days, with UPK seeds started thereafter till day 42.
Monosodium glutamate was administered daily as a single oral dose, by oral gavage, at 200 mg/kg body weight of animals. The unripe palm kernel seeds were also administered based on the weight of the individual rat. Seeds were crushed to obtain smaller pieces which were sizeable for administration to experimental animals. Rats were given 1.14 mg UPK seed/g rat body weight as food, with the amount given calculated from the corresponding amount prescribed by the herbal practitioners to an average female human (in weight) during the period of treatment, which is 25 seeds daily/60kg weight of average female.
2.6. Sample Collection and Preparation
At the end of the period of treatment, the animals were sacrificed by chloroform anesthesia. Blood was collected by cardiac puncture into EDTA sterilized sample containers. Samples were centrifuged (3000 rpm, 20 min) and serum collected for biochemical analyses. The uterus samples were harvested and preserved using 10% neutral buffered formalin placed in pre-labelled universal containers for histology.
2.7. Biochemical Assay and Histology Studies
Total protein and total cholesterol were determined spectrophotometrically as described in the Randox kit (CH200 and TP4001 respectively, Randox Laboratories Ltd., Crumlin, UK). Estrogen and progesterone levels were measured by modifications of Competitive Enzyme Immunoassay as described by Meyer et al. [18]. Using all standard operating procedures, uterus samples were dissected and placed in labelled tissue cassettes with thickness of tissues not exceeding 3 - 5 mm. Tissues were processed using the Leica TP2010 automatic tissue processor (Leica Microsystems, Germany) for 18 hours passing the tissues through fixation (using 10% neutral buffered formalin), dehydration (using ascending grades of isopropyl alcohol), cleared using xylene and finally impregnated by molten paraffin wax. Paraffin wax-embedded tissues were subsequently processed into ultra-thin sections of 5 microns using a thermo-scientific semi-automated rotary microtome, dried overnight and subjected to hematoxylin and eosin staining. Stained slides were mounted on DPX and subsequently viewed under the microscope (Leica DM 500 microscope, Germany), and photomicrographs taken.
2.8. Statistical Analysis
Results are expressed as Mean ± SEM. Statistical differences were derived using one-way analysis of variance (ANOVA) with Turkey’s post hoc test. A p-value < 0.05 was considered statistically significant. Bar graphs were obtained using Graph Pad Prism software 8.01.
3. Results3.1. Protective Effects of Unripe Palm Kernel Seeds on Monosodium Glutamate-Induced Uterine Fibroid
The effect of unripe palm kernel seeds on preventing MSG-induced changes in the serum levels of protein, cholesterol, estrogen and progesterone is shown in Figure 1. Treatment with MSG significantly led to an increase in serum cholesterol and estrogen levels; 123.20 mg/dL and 106 pg/mL respectively, when compared to rats in the control group (78.48 mg/dL and 43.00 pg/mL).
Figure 2 shows the photomicrographs of the uteri of rats in the protective study group. Rats given only unripe palm kernel seeds showed prominent endometrial glands (long arrow) embedded in the lamina propria (short arrow) and visible close up of simple columnar epithelium and lamina propria, which corresponded to the photomicrograph of the control group. Monosodium glutamate administration resulted in uteri with endometrial epithelium surrounding
the lumen having distorted elongated epithelial cells. There was also evidence of distortion and hyperkeratosis of chromatin. Co-administration of MSG and UPK resulted in lamina propria with little fibrosis compared to the rats which received only MSG, thus suggesting some form of mitigation of the effect of MSG by the UPK seeds.
3.2. Curative Effect of Unripe Palm Kernel Seeds after Monosodium Glutamate-Induced Uterine Fibroid
The effect of unripe palm kernel seeds on ameliorating MSG-induced changes in the serum levels of protein, cholesterol, estrogen and progesterone is shown in Figure 3. Rats treated with MSG only for 28 days had significantly high levels of estrogen (119.00 pg/mL) and progesterone (1.2 ng/mL) compared to other treatment groups. Subsequent administration of unripe palm kernel (UPK) seeds for 14 days after treatment with MSG to female rats reversed the levels of these hormones by reducing levels of estrogen (36.50 pg/mL) and progesterone (0.90 ng/mL) to levels, even comparable to the control group.
The photomicrographs of the uterus of rats in the curative treatment design are presented in Figure 4. Rats which received MSG only had atrophied endometrial epithelium and distortion in the lamina propria, which are early signs of benign tumor growth. Adding UPK seeds to the diet of the female rats reduced the distortion observed in the epithelial cells and lamina propria.
4. Discussion
The potential benefits of unripe oil palm kernel seeds in the prevention and
treatment of uterine fibroids was investigated in this study. Ovarian steroid hormones have been shown to be important biomarkers associated with the growth and development of uterine fibroid. While estrogen has been considered the major factor, there is growing evidence suggesting that progesterone and its receptors, play a key role in the growth and development of uterine fibroid [19].
Monosodium glutamate led to an increase in the levels of estrogen and progesterone, which corroborates reports of [17] [20] [21] [22] who observed similar increase in these biochemical markers. Monosodium glutamate increases estrogen levels by activating aromatase, an enzyme that catalyzes estrogen (estradiol) synthesis by converting testosterone to β-estradiol and subsequent aromatisation of β-estradiol [12]. Monosodium glutamate also led to an increase in serum total cholesterol levels which agrees with previous studies [12] [17] [20] [23]. Cholesterol is an important precursor in the biosynthesis of several steroid hormones which are powerful signal molecules regulating a host of functions in living organisms [24]. The elevated levels of cholesterol in rats treated with monosodium glutamate only may aid in explaining the observed elevated serum estrogen and progesterone levels. Histopathology studies of the uterus revealed distorted or atrophied endometrial epithelium and evidence of fibrosis in the lamina propria of rats treated with monosodium glutamate, which is similar to reports by [20].
Co-treatment with monosodium glutamate and unripe palm kernel seeds led to a decrease in estrogen levels. This agrees with findings of Oyebode et al. [20] who studied the protective effects of alpha stone decoction on monosodium glutamate-induced uterine fibroid. It also agrees with other studies by Obochi et al. [12] and Olowofolahan et al. [17], who investigated the effects of garlic extracts and extracts of Drymaria cordota respectively, on MSG-induced uterine fibroid. One of the proposed mechanisms of action of unripe palm kernel seeds against uterine fibroid growth and development can be attributed to the inhibition of aromatase, thus preventing the synthesis of estrogen. The protective effect of unripe oil palm kernel seeds was also confirmed by a decrease in serum progesterone levels in the co-treated rats as observed in previous study [19]. Reis et al. [25] showed that estradiol and progesterone are important in the transformation of myometrial cells into leiomyoma cells with progesterone required for the complete development and proliferation of leiomyoma cells. As estrogen and progesterone are key hormones in the pathogenesis of leiomyoma formation [26], decrease in the serum levels of these hormones associated with unripe palm kernel seeds may explain the use of this treatment method by herbal practitioners. The ability of unripe palm kernel seeds to reverse induced fibroids was also investigated by administering the seeds after treatment with monosodium glutamate. Unripe palm kernel seeds reversed the elevated estrogen and progesterone levels caused by monosodium glutamate.
Unripe palm kernel seeds co-treatment also resulted in decreased the cholesterol levels as observed in similar studies which involved several other ameliorative natural products [12] [17] [20]. The decrease in serum cholesterol levels may be another mechanism of action by which the unripe palm kernel seeds acts in protecting against uterine fibroid development. Photomicrographs of the uteri of the female rats which were co-treated with unripe palm kernel seeds, and the rats which received unripe palm kernel seeds showed little evidence of fibrosis in the lamina propria, suggesting a protection against the development of leiomyoma and reduction in its growth.
This study provides scientific explanation to the claims by herbal practitioners who recommend daily doses of unripe palm kernel seeds in the management and treatment of fibroids. Unripe palm kernel seeds reduced the estrogen and decrease in progesterone levels, with a concomitant decrease in cholesterol levels, all of which have been reported to be the mechanism by which other natural products affect uterine fibroids.
5. Conclusion
In conclusion, these findings suggest that unripe palm kernel provides a natural, readily available and cheap alternative in the management and treatment of fibroids. Unripe palm kernel seeds may contain bioactive constituents useful in the management of fibroids. Unripe palm kernel reduces serum hormone levels which play roles in the etiology of uterine fibroid. Further work would be important in determining the potential effects or toxicity associated with daily consumption of unripe palm kernel seeds over an extended period of time.
Acknowledgement
The authors acknowledge the Department of Anatomy, University of Benin Teaching Hospital (UBTH) for the help with histology studies. The authors would also like to express gratitude to MeRB Africa, Benin city, for providing laboratory space for biochemical analysis.
Declaration of Interest Statement
The authors declare that there is no conflict of interest.
Cite this paper
Agbadua, O.G., Idusogie, L.E., Chukwuebuka, A.S., Nnamdi, C.S. and Sylvester, S. (2020) Evaluating the Protective and Ameliorative Potential of Unripe Palm Kernel Seeds on Monosodium Glutamate-Induced Uterine Fibroids. Open Access Library Journal, 7: e6461. https://doi.org/10.4236/oalib.1106461
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