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Introduction
Recently, medicinal plants are widely used in traditional treatments to relieve severity of many diseases. The use of alternative medicine tends to increase in the modern societies because of their antioxidant capacity and less toxicity. Although medicinal plants have many advantages in treatments, long-term use may affect physiological systems, including male reproductive system.
Momordica cochinchinensis Spreng (MC) is botanically classified in the Cucurbitaceae family and a tropical plant found in China, Myanmar, Laos, Cambodia, Vietnam, Malaysia, Bangladesh and Philippines including Thailand1. Previous studies showed that this plant has various phytochemical components2-9. Pharmacological properties of MC consist of antioxidant activities7, anti-inflammatory effect10, immunomodulatory activity11, anti-tumor properties12, and anti-cancer activity13,14. Additionally, aril part of MC was documented to have anti-cancer activity15 and anti-hyperglycemia16,17. In Thailand, this plant is most popular in consumption and research to be developed as skin care products and food supplements. Since many reports showed the toxic sensitivity of various plants on male reproductive system19,20, such effects of aril MC have never been studied. Therefore, this study aimed to investigate the toxic effects of aril MC on reproductive parameters in adult male mice.
Methods
Plant collection and extraction
M. cochinchinensis (MC) Spreng was cultured at Kuchinarai district, Kalasin province, Thailand from January to May, 2014. The plants were authenticated for its actual species by Prof. Dr. Pranom Chantaranothai, Department of Biology, Faculty of Science, Khon Kaen University, Thailand and kept in the Herbarium (#Apichakan Sampannang 01 [KKU]), Khon Kaen University, Thailand. The fresh-ripe MC arils (8000 g) were mixed with distilled water in the ratio (1:1) and then filtered by the nylon cloth. The aril MC filtrate was dried using Spray dryer. Percentage (%) yield of MC aril extract was 10.50.
Animals and treatment regime
Twenty-one ICR male mice, aged 6-8 weeks, were purchased from National Laboratory Animal Center (NLAC), Mahidol University, Salaya, Nakhon Pathom, Thailand. Mice were acclimatized at Northeast Laboratory Animal Center (NELAC), Khon Kaen University, Thailand. This study was approved by the Animal Ethics Committee of NELAC, Khon Kaen University, Thailand, based on the Ethics of Animal Experimentation of the National Research Council of Thailand (ref. No. 0514.1.12.2/35 with record No. AEKKU-NELAC 29/2557). Mice were randomly divided into three groups (control, vehicle, and MC treated groups). In control group, mice were not treated with DW or MC extract. Mice in vehicle group were administered with only DW. In MC treated group, animals were orally received with aril MC 1000 mg/kg BW for 35 consecutive days (one spermatogenesis cycle). At the end of experiment, all animals were euthanized by cervical dislocation. Blood samples by cardiac puncture were centrifuged by microcentrifuge (Microfuge 22R Centrifuge), 800 rpm at 4°C for 5 minutes to collect serum. The serum was delivered to the radio-chemical unit of Srinagarind hospital, Department of Radiology, Faculty of Medicine, Khon Kaen university, Thailand for measurement the levels of testosterone hormones by using radioimmuno assay.
Epididymal sperm concentration
Sperm concentration analysis was performed as previously described by Iamsaard et al19. Briefly, sperm fluid was collected from epididymis and vas deferens. Its fluid was dipped and re-suspended in 1 ml of phosphate buffer saline (PBS). Then re-suspended sperm was centrifuged (3000 rpm, 2 min) to wash and separate the mature sperm pellet from supernatant. To analyze the sperm concentration, the sperm pellets were re-suspended with fresh 1 ml PBS before dilution. The diluted sperm (1:20 dilution) were triplicately counted in each animal using a Neubauer's counting chamber.
Histopathological examinations of the testes and epididymes
Testes and caudal epididymes were fixed with 10% formalin and embedded with paraffin and sectioned by microtome about 5 µm thicknesses. The tissue sections were deparaffinized and stained by hematoxylin and eosin (H&E) before histological observations under light microscope.
Statistical analysis
All quantitative results were expressed as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) and Duncan test were used SPSS program (version 16) software to examine the significance of differences among sets of data. The level of statistical significance was p<0.05.
Results
Aril MC extract did not affect final body weight, absolute and relative weights of testis, seminal vesicle, and epididymis plus vas deferens as compared to control and vehicle groups (Table 1). Additionally, sperm concentration and testosterone levels in MC treated group were not changed as compared to the control as shown in Table 1 (p>0.05).
Table 1 Effects of aril MC on body weight, reproductive organ weights, sperm concentration, and testosterone levels
|
Parameters |
Control |
Vehicle |
MC treated |
|
Initial body weight (g) |
32.8 ± 1.5 |
33.9 ± 1.3 |
33.8 ± 1.2 |
|
Final body weight (g) |
42.6 ± 1.6 |
41.8 ± 2.3 |
41.9 ± 2.0 |
|
Absolute weight of testis (x10-1g) |
2.4 ± 0.4 |
2.8 ± 0.3 |
2.7 ± 0.3 |
|
Relative weight of testis (x10-3g) |
6.7 ± 0.9 |
6.7 ± 0.8 |
6.6 ± 0.8 |
|
Absolute weight of seminal vesicle (x10-1g) |
3.4 ± 0.4 |
3.5 ± 0.6 |
3.5 ± 0.6 |
|
Relative weight of seminal vesicle (x10-3g) |
7.9 ± 1.0 |
8.3 ± 1.4 |
8.5 ± 1.2 |
|
Absolute weight of epididymis plus vas deferens (x10-2g) |
7.4 ± 0.8 |
7.7 ± 0.6 |
7.7 ± 0.4 |
|
Relative weight of epididymis plus vas deferens (x10-3g) |
1.7 ± 0.2 |
1.9 ± 0.2 |
1.8 ± 0.1 |
|
Sperm concentration (x10-6cell/ml) |
64.4 ± 8.4 |
60.3 ± 4.3 |
62.0 ± 9.1 |
|
Testosterone levels (ng/ml) |
0.7 ± 0.3 |
0.9 ± 0.3 |
0.7 ± 0.2 |
* Significant difference (p<0.05). Data are represented as mean ± S.D. (n = 7 mice each group).
In Figure 1, the results showed that gross morphologies of testis, seminal vesicle and epididymis plus vas deferens were not obviously altered as compared to that of control and vehicle groups, which they related to the results of absolute and relative weights of male reproductive organ as shown in Table 1.
Figure 1 Representa
tive gross morphologies of testis, seminal vesicle, and epididymis plus vas deferens compared among the control, vehicle, and MC treated groups.
In testicular histological observations, seminiferous tubules in MC treated group were found to have a normal arrangement of spermatogenic and Sertoli cells with no histopathological lesions (Fig. 2C). Similar to control and vehicle groups (Fig. 2A & B), interstitial compartment in MC treated group was not altered (Fig. 2C). Furthermore, the aril MC extract also did not damage the epididymal-epithelial cells and density of sperm mass within epididymal lumen (Fig. 2F) as compared to that of control and vehicle groups (Fig. 2D & E).
Figure 2 Showing representative histologies of testis and caudal epididymis among control (A & D), vehicle (B & E), and MC treated (C & F) groups, respectively.
Discussion
Since many studies have previously reported the pharmacological properties of aril Momordica cochinchinensis (MC) Spreng in both animal models and cell cultures15-17, this study has demonstrated for the first time about the non-toxicity effect on male reproductive system. The results showed that aril MC extract was not toxic to the weights of body and reproductive organs, sperm concentration, testosterone levels, and histological structures of testis and epididymis (shown in Table 1, Fig. 1 & 2) in adult male mice. Our results may be similar to that of previous studies which also found that the aril MC extract did not affect morphological vital organs and behavioral changes16-18. Although the dose of MC extract (1000 mg/kg BW) used in this study was higher than that of previous investigations (200 and 400 mg/kg BW)16,17, it did not adversely affect male reproductive parameters. It is possible that dose of the MC extract was lower than its LD50 documented in an acute toxicity study (2000 mg/kg BW)18. Recent findings indicate that a dose of aril MC extract used in this study is not toxic to male reproductive parameters in mice. In addition, it supports the safe dose of use for other pharmacological properties demonstrated previously7, 10-18. However, to investigate the chronic toxic effects on male reproductive system, the longer-experimental study of aril MC administration should be performed in further study to observe the side effects of this plant in prolonged consumptions.
Conclusion
Aril-MC extract did not affect the changes of testosterone levels, body weight, reproductive organ structures, and sperm quality in adult male mice.
Acknowledgments
We would like to gratefully thank the invitation research grants (No. IN58123) from Faculty of Medicine, Khon Kaen University for financial support this project.
References
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