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OVARIUM HISTOPATHOLOGIC AND ENDOMETRIAL THICKNESS AFTER TREATMENT OF KEPEL FRUIT EXTRACT IN AN EXPERIMENTAL MICE
MODEL
Israhnanto Isradji1*, Iwang Yusuf1, Suparmi1, Dina Fatmawati1
1 Medical Faculty; Sultan Agung Islamic University,
Jl. Raya Kaligawe KM. 4 PO Box 1054/SM Semarang 50112, Indonesia
* Corresponding author, email: [email protected]
Abstract
Kepel (Stelechocarpus burahol) fruit used to prevent a pregnancy by princess palace, but there has been no scientific evidence. This study was conducted to determine the effects of kepel fruit extracts on ovarian histology and endometrial thickness. Ten female Balb/C mice were used in this study and randomly divided into two groups: group 1 as control (n = 5) was treated water and group 2 (n = 5) was treated with kepel fruit extract on dose 65 mg/kg BB BB for 14 days. All animals were sacrificed by decapitation to obtain ovaries for histological examination and uterus for endometrial thickness. Independent Sample T-Test analysis was used and statistical significance was defined as p < 0.05. Histopathologic evaluation of ovaries showed that ovaries from the control group have the mean number of primary, secondary, and de Graaf’s follicles were 11.40 ± 5.18, 12.60 ± 4.67, 6.60 ± 1.14, respectively. On the group 2, the mean number of primary, secondary, and de Graaf’s follicles were 20.25 ± 9.18, 20.25 ± 6.75, 7.50 ± 2.08, respectively. There was no difference in each folicles number variables between groups (p > 0.05). The endometrial thicknes in the kepel fruit extract group (695.96 ± 252.70 µm) was higher than control group 582.70 ± 120.52 µm), but there was no significantly difference (p=0.401). The results of this experimental mice model suggest that the treatment of kepel fruit extract on dose 65 mg/kg BBaffect ovarian histology and endometrial thickness, but not significant.
Keywords: Kepel fruit extracts, primary follicle, de Graaf’s follicle, secondary follicle, ovarian histology, endometrial thickness
INTRODUCTION
Kepel Fruit (Stelechocarpus burahol) belongs to the family of Annonaceae. Ripe fruits are eaten fresh, which its flavor to that of the mango. Kepel fruit has traditionally been known in Java to have value as an oral deodorant, antihyperuricemic, and to have the function of preventing kidney inflammation. This fruit also believed to be used by Javanese Princesses as contraception. Warningsih (1995) reported that the pulp fruit extracts of plant possess antimplatation effect. Heriyanto and Garsetiasih (2005 ) reported that alkoloid isolated from Kepel fruit to have abortifacient properties, indicating their potential to be used as an antifertility treatment in animals. Darusman (2010) have identified phytochemicals, such flavonoids, tannins, and saponins However, the phytochemical analysis of the ethanolic extracts of Kepel pulp fruit and its effect on the pregnancy prevention based on ovarium histopathologic and endometrial thickness has not been studied yet.
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Ovarium and endometrium play a fundamental role in the female reproduction (Frandson, 1996). Approximately 40 % of the infertility were caused by ovulation disorders due to abnormalities in hypothalamus and pituitary (Samsulhadi and Hendarto , 2009), feedback mechanism abnormalities, and ovulation failure (Djuanna et al., 2005).The mechamism of antifertility agent are suppressed the ovulation suppression, prevention of sperm migration through cervical mucus, and creating unfavorable endometrium for implantation (Cunningham et al ., 2012) . Progesterone stimulates the glandular cells that have evolved under the influence of estrogen so that the endometrium reaches a maximum thickness as a result of the secretions accumulation and oedem in the stroma (Junqueira , 2007) .
Antifertility activity are showed by interfering with the system at pre -ovulatory and pre-implantation stages. This Antifertility compounds were linked to steroids, so directly react to the ovaries, or indirectly inhibits the gonadotropins secretion (Cambie and Brewis, 1996). Siddiqi et al .( 2008) has been evaluated the antifertility activity phytoestrogens from kepel fruit extract. Genistein and daidzein are known a phytoestrogen compound, because it’s estrogenic activity. Estrogen can be used for contraception by inhibiting ovulation.
The common contraseption methods which are partially effective and also associated with undesirable side-effects, including obesity, hyperpigmentation ( melasma), hypertension, and infertility. Hence natural new contraceptive materials are required to be identified that can act as antiimplantation. The aim of the present study was to to determine the effects of kepel fruit extracts on ovarian histology and endometrial thickness.
MATERIAL AND METHODS Plant Material
Kepel Fruit were collected from banaran Coffee Plantation Ambarawa, Central Java.
Preparation of the ethanolic extract from kepel pulp fruit
The pulp kepel was dried in the shade and ground to powder. One kilogram of powdered pulp fruit was extracted in 96% ethanol for 28 h in soxhlet flask. The filtrate obtained was concentrated under reduced pressure then filtered then evaporated to dryness at 65°C, and exposed to a stream of nitrogen gas (N2) until completely dried.
The dried sample was stored at 4 °C, until the later analysis.
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Animal experimentSexually mature female albino mice (Balb/C strain; body weight, 20.37±1.40 g) bred in the university animal house and maintained on a normal laboratory diet (LPPT Gadjah Mada Universitu) were used. Animal experiments were conducted in accordance with the institutional ethical committee guidelines of committee for the purpose of control and supervision of experiments on animals (Ethical Unit Medical Faculty Sultan Agung Islamic University), Semarang, Indonesia. Animals were housed in polypropylene cages in a room that was maintained between 28 and 32°C with 12:12-h light/dark cycle. Food and water were given ad libitum. Food consumption was similar in all groups. Animals were randomly divided into two groups of six each: 1.
Control, and ethanolic extract of kepel fruit treated. Kepel fruit ethanolic extract treated were dissolved in distilled water and given orally by gastric intubation. Control animals were given the vehicle. The dose of the Kepel fruit ethanolic extract) was fixed to be 65 mg/kg body weight a day after. The duration of the experiment was 14 days. At the end of the experimental period, the animals were fasted overnight and sacrificed.
Histopathological Studies
Ovarium and uterus of each mice were fi xed in Bouin’s fluid passed through ascending series of ethanol and then through xylene and embedded in paraffin wax.
Tissues were sectioned at 5 µm and stained with hematoxylin and eosin. Ovarian histology were observed based on the structure and number of primary follicles, secondary follicles, follicle and corpus luteum de Graaf for each ovarium, and then averaged Calculations performed on 6 follicles visual field at histology preparations right and left ovary with 100 × magnification. The endometrium thickness were measured from the surface of endometrium functional layer to the basal layer of the endometrium using a microscope equipped with a 100x magnification with a micrometer connected to the OptiLab camera.
Statistical analysis
The SPSS 16.0 statistical program was employed, and the results were evaluated.using analysis of variance utilizing the F test. The results are presented as mean value±SD for the control and treated animals. Difference among the mean for the groups were assessed using independent T Test to determine which mean values were significantly different at p <0.05.
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RESULTS AND DISCUSSIONKepel (Stelechocarpus burahol) Fruit Extraction
Kepel fruit (1 kg) after going through the stages of extraction resulting 5 g thick brown extract (Figure 1). Kepel fruit extract yield was 0.5%, per 100 g of Kepel fresh fruit produce 0.5 g of crude extract. Extraction is a process that selectively take solute contained in a mixture with the help of solvent.
(a) (b)
Figure 1. (A) Samples Kepel fruit; (b) Extract Kepel fruit
The test results showed that the phytochemical content of ethanol extract of Kepel fruit contains saponins, flavonoids, alkaloids and tannins (Table 1). Its phytochemical have antiimplantation and antifertility effect. Flavonoids in general have a similar structure and function as estrogen (Zand, 2000). Consists of triterpenoid saponins and steroidal saponins elements. This result was consistent with Sunardi (2010) that Kepel fruit contains alkaloids, flavonoids, tannins, triterpenoids and steroids. Kepel fruit pulp contains saponins, flavonoids and polyphenols (Widaningsing, 2013). Limitations of this research is a method of testing the content of saponins, alkaloids and tannins were still qualitative by Thin Layer Chromatography (TLC) so do not know the percentage of the ingredient.
Table 1. Phytochemical content of fruit extracts test Kepel
No. Parameter Method Result
1. Saponin KLT Positive
2. Total Flavonoid Spectrofotometry 7,38%
3. Alkaloid KLT 266,95 ppm
4. Tanin KLT Positive
Kepel Fruit Extract Effect on Mice Reproductive Females
The knowledge about Kepel fruit extracts effects on the female reproductive can later be used as the basis of acute toxicity Kepel fruit extracts dose. Kepel fruit extract at a dose of 65 mg / kg for 14 days in feamle mice strains Balb / C led to an increase in the number of primary follicles, secondary follicles and follicle de Graaf compared with
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the control group (Table 2) , although it showed no statistically significant difference (p
> 0.05). Achdiat ( 2003) says that phytoestrogens required in very large quantities to obtain sufficient effect as estrogen. The substrate binds to the estrogen receptors of new estrogenic effects occur. With high levels of phytoestrogens and recurrent circulation can cause potential effects. This is due to the estrogen receptor is blocked by phytoestrogens and can not be occupied by estrogen. The binding of phytoestrogens on estrogen receptor , will cause weak estrogenic activity (Tsourounis, 2004) .
Table 2.The number of ovarian follicles
Follicle type The mean number of ovarian follicles on the right and left
Control Kepel
Primary follicles 5.60±2.70 10.12±4.59
Secondary follicles 6.30±2.33 10.12±3.37
de Graaf Folicles 4.80±2.46 3.75±1.04
Corpus Luteum 2.00±1.58 2.12±2.27
One way ANOVA test results showed no differences were significant at each of the ovarian follicle after treatment Kepel fruit extract (p> 0.05) (Figure 2). The increase in the number of follicles caused by the active ingredient in Kepel fruit extract of triterpenoid saponins, namely asiatikosida and madekossida were able to repair damaged cells and collagen fibers forming, the active ingredients are also able to repair the granulosa cells of the ovary (Suhaemi, 2007). The increase in the number of follicles suspected because of the content of flavonoid in Kepel fruits (Darusman, 2010). Flavonoids act as an antioxidant (Zand, 2000) which can prevent oxidation and protect cells from damage, so folliculogenesis in the ovary can be improved (Zilliken, 2009).
(a) (b)
Figure 2. Ovarian Microscopic Histopathologic image on magnification 100x by HE staining: (a) Kepel group (b) the control group. (1) primary follicles, (2) secondary follicles, (3) de Graaf follicle
The endometrial thickness in Kepel extract treated mice was 695.96 ± 252.70 µm, whereas the control group was 582.70 ± 120.52 µm. Although the endometrium of
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treaed mice was thicker than control mice, but there were no significantly difference (p>
0.05). Histopathological picture of the endometrium in the treatment group, the basal layer of the blood vessels more obvious and more winding uterine glands and distended (Figure 3), it is presumably due to the effects of Kepel fruit extract containing alkaloids and flavonoids.
(a) (b)
Figure 3. Histopathologic endometrium uterine in 100x magnification with HE staining ( a) . Kepel group ; ( b ) Control group. white line shows the location of the measurement .
Compounds in the form of aglycone isoflavones genistein and daidzein are known a phytoestrogen, because it has some estrogen activity. This result is consistent with Agustini et al., ( 2007) reported that the content of alkaloids, flavonoids, and saponins in the ethanol extract of Trigonella foenum - graecum L. can increase uterine wet weight and uterine growth (diameter and thickness of the endometrium of the uterus). Guyton and Hall (2007) led to the proliferation of estrogen on the uterus.
Phytoestrogens are a natural estrogen compounds capable of binding to estrogen receptors α and β (Morito et al. , 2002) , generally phytoestrogens worked as Selective Estrogen Receptor Modulators (SERMs) .
Limitations in this study were not know the equalization cycle during any phase of the most influential Kepel fruit extract on mice endometrial thickness . Doses of the extract in this study only uses empirical dose and dose variation is not done , so the possibility of the dose given less effect on the ovarium and endometrium.
CONCLUSION
The treatment of kepel fruit extract on dose 65 mg/kg BB affect ovarian histology and endometrial thickness, but not significant.
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ACKNOWLEDMENTThis study was supported by Research Funding from Sultan Agung Islamic University based on Contract Number: 01/P-KEL/UPR-FK/VII/2012, 27 July 2012.
Thanks to Dya Kusumawati and Rahmawati Indah Sribuhati for helping experimental activity.
REFERENCES
Achdiat CM., 2003, Fitoestrogen Untuk Wanita Menopause, http://www.situs.
kesrepro.info/aging/jul/2003/ag01.html, Diakses 25 Februari 2014.
Agustini, K., Wiryowidagdo, S., Kusmana, D., 2007, Pengaruh Ekstrak Biji Klabet (Trigonella foenum-graecum L.) terhadap Perkembangan Uterus Tikus Putih Betina Galur Wistar Prepubertal, Jurnal Sains dan Teknologi Indonesia 9(1), 8- 16.
Cambie, R.C., dan Brewis, A.A, 1996, Anty Fertility Plants of The Pacific, C. Siro, Australia.
Cuningham, F. G., 2012, Obstetri Williams, EGC, Jakarta.
Darusman HS., 2010, Aktivitas farmakologis tanaman kepel (Stelechocarpus burahol (BL) Hookf & Thoms.) sebagai deodoran topikal dan oral [tesis], Bogor, Prog Pascasarjana, Institut Pertanian Bogor.
Darusman, Rahminiwati, Sadiah, Batubara, Darusman, dan Mitsunaga, 2012, Indonesian Kepel fruit (Stelechocarpus burahol) as oral deodorant, Research Journal of Medical Plant, 6(2), 180-188.
Djuanna, Arifuddin , A Husni Tanra,Syarifuddin Wahid, Biran Affandi.2005. Pengaruh Teknik “Double Circle Stitching” dan atau Pemasangan Tourniquet terhadap Jumlah Perdarahan Uterus pada Tindakan Miomektomi Saat Seksio Sesar., J Med Nus,24(2).
Frandson, R.D, 1996, Anatomi dan Fisiologi Ternak, Alih Bahasa Oleh Srigandono, B Dan Praseno, K, UGM Press, Yogyakarta, hal 680-689.
Guyton, A. C., dan Hall, J. E., 2007, Buku Ajar Fisiologi Kedokteran, EGC, Jakarta.
Heriyanto, N.M. dan R. Garsetiasih, 2005, Kajian Ekologi Pohon Burahol (Stelechocarpus burahol) di Taman Nasional Meru Betiri, Jawa Timur, Buletin Plasma Nutfah ,11 (2), 65-73.
Junqueira, L. C., 2007, Histologi Dasar : teks & atlas, Edisi 10, EGC, Jakarta.
Morito, K., Aomori, T., Hirose, T., Kinjo, J., Hasegawa, J., Ogawa, S., Inoue, S., Muramatsu, M., dan Masamune, Y. 2002. Interaction of Phytoestrogens with Estrogen Receptors α dan β (II). Biol. Pharm. Bull. 25(1):48-52.
Samsulhadi, Hendarto H., 2009, Aplikasi Klinis Induksi Ovulasi dan Stimulasi Ovarium Buku Panduan Praktis bagi Klinis, Sagung seto, Jakarta.
Shiddiqi, T., Y. Rindiastuti, dan N. A. Sri W. 2008. Potensi In Vitro Zat Sitotoksik Anti Kanker Daun Tanaman Kepel (Stelechocarpus buharol) terhadap Carcinoma Colorectal. Karya Tulis Ilmiah. Fakultas Kedokteran Universitas Sebelas Maret Surakarta.
Suheimi, K., 2007, Fisiologi Folikulogenesis dan Ovulasi, Dalam makalah pada symposium pertemuan ilmiah.
Sunardi, C., 2010, Structure of Steroids in Stelechocarpus burahol Hook F. & Thomson Stem Bark, The Journal of Indonesian Medicinal Plants, 3(2).
Tsourounis, C. 2004, Clinical effects of fitoestrogens, Clinical Obstetrict and Genycology, 44 (4): 836-42.
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Warningsih,1995,Uji fitokimia dan efek antiimplantasi ekstrak etanol bunga Hibiscus rosa sinensis Linn., buah Piper nigrum Linn.,dan buah Stelechocarpus burahol hook.f.& Th, Abstrak, Penelitian Tanaman Obat di beberapa Perguruan Tinggi di Indonesia X, Pusat Penelitian dan Pengembangan Farmasi, Badan Penelitian dan Pengembangan Kesehatan Departemen Kesehatan RI, Jakarta.
Widaningsing, N. A., 2013, Kepel, dari Deodoran Alami Sampai Kontrasepsi. Dalam : http://ppvt.setjen.deptan.go.id/ppvtpp/halkomentar-587-4.html. (02 Mei 2013).
Zand, R.S.R., David, J.A.J., Eleftherios, P.D., 2000, Steroid Hormone Activity of Flavonoids and Related compounds, Breast Cancer Research and Treatment, 62, 35-49.
Zilliken, F.I., 2009, Production of Novel Isoflavons. Material Meeting, BMBF,Bonn, Germany, http://www.naturalwoman.com/phyto.htm, Diakses tanggal 4 Februari 2014
