| dc.description.abstract | Daun gedi populer dikonsumsi sebagai sayuran oleh masyarakat di Indonesia bagian Timur dan dipercaya turun-temurun dapat meningkatkan produksi ASI. Penelitian ini bertujuan mengkaji komponen gizi dan senyawa aktif fitokimia dalam ekstrak daun gedi dan khasiatnya sebagai laktagogum pada tikus menyusui. Tahap pertama: identifikasi, budi daya, ekstraksi daun gedi. Tahap kedua: evaluasi kandungan gizi daun gedi; komponen fitokimia, aktivitas antioksidan, senyawa fitokimia dugaan dalam ekstrak daun gedi; uji in silico senyawa fitokimia dalam ekstrak daun gedi. Tahap ketiga: uji in vivo toksisitas akut dan khasiat laktagogum. Analisis hormon estrogen (E), prolaktin (PRL), cAMP, TNF-a menggunakan uji serologi ELISA. Analisis ekspresi gen menggunakan sistem Real Time-PCR. Analisis statistik menggunakan program SPSS versi 21.0 for Windows. Perbedaan antar kelompok untuk (1) konsumsi susu, bobot badan (BB) induk tikus, diameter alveoli, hormon E dan PRL, cAMP, TNF-a menggunakan uji Kruskal Wallis, Post Hoc Mann Whitney yang di-adjust Bonferroni; (2) BB anak tikus menggunakan uji ANCOVA, Post Hoc Bonferroni; (3) bobot kelenjar mammae, ekspresi gen, jumlah alveoli, menggunakan uji one way ANOVA, Post Hoc Bonferroni (untuk bobot kelenjar mammae dan ekspresi gen) dan LSD (untuk jumlah alveoli).
Hasil analisis menunjukkan bubuk daun gedi memiliki kandungan karbohidrat (51,11%), serat kasar (10,25%), vitamin K (1.110,31 µg/100 g), magnesium (699,98 mg/100 g), sodium (89,59 mg/100 g), dan kalsium (2.670,10 mg/100 g) tertinggi dan berbeda signifikan dengan bubuk daun torbangun dan daun katuk, yang umum digunakan sebagai laktagogum di Indonesia (p < 0,05).
Daun gedi diekstrak menggunakan pelarut air, etanol 70%, etanol absolut, etil asetat, n-heksana. Fenolik, flavonoid, tanin, steroid terdeteksi dalam semua ekstrak. Kuersetin tidak terdeteksi dalam ekstrak etil asetat. Hasil analisis menunjukkan semua ekstrak tergolong antioksidan sangat kuat, dengan aktivitas penghambatan pembentukan malondialdehida pada IC50: ekstrak etanol absolut (6,77 ppm) > n-heksana (7,67 ppm) > etil asetat (9,10 ppm) > etanol 70% (9,12 ppm) > air (9,76 ppm). Kapasitas antioksidan tertinggi pada ekstrak etanol absolut dan n-heksana diprediksi karena kandungan kuersetin (0,246 mg/g) dan steroid (201,948 mg/g) tertinggi dibandingkan ekstrak lainnya, secara berurutan.
Interpretasi metabolit menggunakan UPLC-QToF-MS/MS memprediksi 88 dan 93 senyawa dugaan dalam ekstrak etanol absolut dan n-heksana, secara berurutan. Kuersetin tidak terdeteksi dalam ekstrak etanol absolut karena analisis untargeted, steroid total terdeteksi 18,38% dalam ekstrak n-heksana. Uji in silico memprediksi senyawa steroid dugaan dalam ekstrak n-heksana, 2-hidroksiestradiol (LD50 = 2000 mg/kg), berpotensi sebagai fitoestrogen. Uji in vivo toksisitas akut menunjukkan ekstrak n-heksana “tidak toksik” (LD50 > 5000 mg/kg BB mencit).
Uji in vivo khasiat laktagogum menggunakan desain rancangan acak lengkap. Induk tikus dikelompokkan ke dalam lima kelompok: kontrol normal (KN), kontrol positif (KP; Domperidon), ekstrak n-heksana (mg/kg BB tikus): 50 (G50), 100 (G100), 200 (G200). Perlakuan diberikan sejak hari ke-3 (H3) hingga H17 pascamelahirkan. Produksi (konsumsi) susu pada H17: G200 > G50 > G100 > KN > KP. Konsumsi susu di kelompok ekstrak pada H17 lebih tinggi dibandingkan H4, dan terjadi sebaliknya di kelompok kontrol.
Penambahan BB anak tikus di kelompok ekstrak lebih tinggi dibandingkan KN, perbedaan nyata terjadi antara G100, G200 dengan KN (p < 0,05). Penambahan BB anak tikus: G100 > KP > G200 > G50 > KN, begitu juga ekspresi gen reseptor prolaktin (PRLR) di kelenjar mammae. Ekspresi gen reseptor estrogen alfa (ERa) di kelenjar mammae: G100 > G50 > G200 > KP > KN. Fitoestrogen dugaan, terutama di G100, berpotensi berikatan efektif dengan ERa intraseluler di kelenjar mammae dan menstimulasi ekspresi gen PRLR.
Penurunan BB induk tikus di kelompok ekstrak lebih tinggi dibandingkan KN, perbedaan nyata terjadi antara G50 dengan KN (p < 0,05). Di kelenjar pituitari anterior, ekspresi gen PRL: G50 > G200 > G100 > KP > KN, begitu juga penurunan BB induk tikus; ekspresi gen ERa: G100 > G200 > KP > KN > G50; ekspresi gen D2R: G200 < KN < G50 < KP < G100. Pada G50, G200, fitoestrogen dugaan berpotensi kuat berikatan efektif dengan mERa, menghambat kerja reseptor dopamin 2 (D2R), terjadi peningkatan cAMP, dan respon seluler yang cepat untuk menstimulasi ekspresi gen PRL. Pada G100, fitoestrogen dugaan berpotensi kuat berikatan efektif dengan ERa intraseluler dan menstimulasi ekspresi gen PRL. Namun, peningkatan cAMP serum: G100 > G200 > G50, terdapat potensi jalur lain dalam interaksi fitoestrogen dugaan pada G100, ekspresi gen mER, ekspresi gen D2R. Hormon PRL memasuki dinding sel laktosit di kelenjar mammae, berikatan dengan PRLR sehingga produksi susu meningkat, terjadi perbaikan pertumbuhan anak tikus, ditandai dengan penambahan BB anak tikus.
Bobot relatif kelenjar mammae di kelompok ekstrak lebih tinggi dibandingkan KN, diprediksi karena peningkatan produksi susu dan sisa susu di kelenjar mammae. Bobot relatif kelenjar mammae: G50 > G100 > G200 > KN, begitu juga penurunan TNF-a serum. Terdapat aktivitas antioksidan fitoestrogen dugaan dalam ekstrak untuk memperbaiki stres psikososial sehingga terjadi peningkatan produksi susu.
Tidak terdapat perbedaan nyata pada jumlah alveoli kelenjar mammae antar kelompok. Deskripsi histologi kelenjar mammae menunjukkan kelompok ekstrak memiliki cairan susu lebih banyak di dalam lumen alveoli aktif. Perbesaran alveoli: G100 > KP > KN > G200 > G50, karena terisi dengan susu, mempengaruhi produksi dan sekresi susu. Kelenjar mammae memproduksi susu selama terjadi sekresi susu karena isapan anak tikus. Di lain pihak, sekresi susu yang tidak optimal juga dapat menyebabkan akumulasi susu di kelenjar mamme sehingga menimbulkan faktor lokal (FIL) yang dapat menghambat produksi susu.
Pada H18, peningkatan kadar E serum hanya terjadi di G100 (0,02 (0,1) ng/ml). Kadar PRL hanya terdeteksi di G100 (0,04 ng/ml) dan KP (6,651 ng/ml). Penelitian ini menunjukkan potensi aktivitas ekstrak n-heksana daun gedi sebagai laktagogum, bergantung pada konsentrasi ekstrak yang diberikan, terutama di konsentrasi 100 mg/kg bobot badan tikus. | |
| dc.description.abstract | Gedi leaves are popularly consumed as a vegetable by Eastern Indonesia people and are believed to increase breast milk production for generations. This study aimed to examine nutritional components and active phytochemical compounds in gedi leaves extracts and its efficacy as a galactagogue in lactating rats model. Stage one: identification, cultivation, extraction of gedi leaves. Stage two: evaluation of nutritional content of gedi leaves; phytochemical components, antioxidant activity, predicted phytochemical compounds in gedi leaves extracts; in silico test of phytochemical compounds in gedi leaves extracts. Stage three: in vivo test of acute toxicity and efficacy as a galactagogue. Estrogen (E), prolactin (PRL), cAMP, TNF-a were analysed using ELISA serology test. Gene expression was analysed using Real Time PCR system. Statistical analysis was done using SPSS version 21.0 for Windows. Differences between groups for (1) milk consumption, mother rats body weight (BW), alveoli diameter, E and PRL, cAMP, TNF-a were analysed using Kruskal Wallis test, Post Hoc Mann Whitney adjusted Bonferroni; (2) rat pups BW were analysed using ANCOVA test, Post Hoc Bonferroni; (3) mammary gland weight and gene expression, mother rats alveoli number, were analysed using one way ANOVA test, Post Hoc Bonferroni and LSD, respectively.
The analysis showed that gedi leaves powder had the highest carbohydrate (51.11%), crude fiber (10.25%), vitamin K (1,110.31 µg/100 g), magnesium (699.98 mg/100 g), sodium (89.59 mg/100 g), and calcium (2,670.10 mg/100 g) and significantly different with torbangun and katuk leaf powder, which are commonly used as galactagogues in Indonesia (p < 0.05).
Gedi leaves were extracted with water, 70% ethanol, absolute ethanol, ethyl acetate, and n-hexane. Phenolic, flavonoid, tannin, steroid were detected in all extracts. Quercetin was not detected in ethyl acetate extract. The analysis showed that all extracts were classified as very strong antioxidants, with malondialdehyde formation inhibitory activity at IC50: absolute ethanol extract (6.77 ppm) > n-hexane (7.67 ppm) > ethyl acetate (9.10 ppm) > 70% ethanol (9.12 ppm) > water (9.76 ppm). The highest antioxidant capacity in absolute ethanol and n-hexane extracts were predicted because of highest quercetin (0.246 mg/g) and steroids (201.948 mg/g) content, respectively.
Metabolites interpretation using UPLC-QToF-MS/MS predicted 88 and 93 compounds in absolute ethanol and n-hexane extracts, respectively. Quercetin was not detected in absolute ethanol extract due to untargeted analysis, total steroids detected were 18.38% in n-hexane extract. In silico test predicted the steroid compound in n-hexane extract, 2-hydroxyestradiol (LD50 = 2000 mg/kg), had a potency as a phytoestrogen. In vivo acute toxicity test showed that n-hexane extract was classified as "nontoxic" (LD50 > 5000 mg/kg mouse BW).
In vivo efficacy test as a galactagogue was done using completely randomized design. Mother rats were allocated into five groups: normal control (KN), positive control (KP; Domperidone), n-hexane extract (mg/kg rat BW): 50 (G50), 100 (G100), 200 (G200). The treatment was given from day 3 (D3) to D17 postpartum. Milk production (consumption) on D17: G200 > G50 > G100 > KN > KP. Milk consumption in extract groups on D17 were higher than D4, and the opposite happened in control groups.
The increase of rat pups BW: G100 > KP > G200 > G50 > KN, with significant difference between G100, G200 and KN (p < 0.05). The same sequence happened in prolactin receptor (PRLR) gene expression in mammary gland. Estrogen receptor alpha (ERa) gene expression in mammary gland: G100 > G50 > G200 > KP > KN, with significant difference between G100 and KP, KN (p < 0,05). Predicted phytoestrogen, especially in G100, had the potential to effectively bound to intracellular ERa in mammary gland and stimulated PRLR gene expression.
The decrease of mother rats BW in extract groups were higher than KN, with significant difference between G50 and KN (p < 0.05). Gene expression in anterior pituitary gland, PRL gene: G50 > G200 > G100 > KP > KN, as well as the decrease of mother rats BW; ERa gene expression: G100 > G200 > KP > KN > G50; D2R gene: G200 < KN < G50 <. KP < G100. In G50, G200, predicted phytoestrogen had the potential to effectively bound to mERa, inhibiting dopamine 2 receptor (D2R) work, increasing cAMP, and gave a rapid cellular response to stimulate PRL gene expression. In G100, predicted phytoestrogen had the potential to effectively bound to intracellular ERa in mammary gland and stimulated PRL gene expression. However the increase of cAMP serum: G100 > G200 > G50. Other potential pathway may be involved in the interaction between predicted phytoestrogen, mERa, and D2R gene expression in G100. The PRL entered lactocyte cell wall in mammary gland alveoli, bound to PRLR so that milk production increased, with an improvement in the growth of rat pups, indicated from the increase of rat pups BW.
The mammary glands relative weight in extract groups were higher than KN, predicted due to the increase of milk production and milk left in mammary glands. The mammary glands relative weight: G50 > G100 > G200 > KN, as well as the decrease of TNF-a serum. Antioxidant activity of predicted phytoestrogen improved psychosocial stress so that milk production could increase.
No significant difference in mammary gland alveoli number between groups. Histological description showed that extract groups had more milk secretion in the lumen of active alveoli. Alveoli distention: G100 > KP > KN > G200 > G50, happened because they were filled with milk, affected the milk production and secretion. Mammary gland produced milk as milk secreted due to continuous suckling. Meanwhile, suboptimal milk secretion could also cause milk accumulation in mammary gland, causing local factors (FIL) which could inhibit milk production due to milk left in mammary gland.
At H18, the increase of estrogen serum only happened in G100 (0.02 (0.1) ng/ml). The highest ERa gene expression was also in G100. Prolactin (PRL) serum was only detected in G100 (0.04 ng/ml) and KP (6,651 ng/ml). This study showed potency of n-hexane extract of gedi leaves as a galactagogue, depending on the concentration given, especially at 100 mg/kg rat BW. | |
