Pluchea indica (P. indica) and Sauropus androgynus (S. androgynus) are medicinal plants empirically used as an herbal galactogogue, but their mechanism of action has not been elucidated at the molecular level in the form of a mixed extract. This research aimed to standardize and investigate the combined effects of P. indica leaf extract (EPI) and S. androgynus leaf extract (ESA) on the expressions of prolactin (PRL), PRL receptor (PRLR), oxytocin (OXT), and OXT receptor (OXTR) genes in pituitary and mammary glands of lactating rats. The extracts were prepared by maceration in 70% ethanol. Standardization was done by examining non-specific parameters and determining total flavonoids using spectrophotometric analysis. Twenty-four lactating rats were divided into six groups. Group I: CMC 0.5% as control, II: EPI 500 mg/kgBW, III: ESA 125 mg/kgBW, and IV–VI were given combination formula EPI-ESA 1: 125+31.25, EPI-ESA 2: 250+62.5, and EPI-ESA 3: 500+125 mg/kgBW (orally, once a day) from the 2nd–15th day after parturition. On the 16th day, experimental animals were euthanized for tissue sampling, and gene expression was measured using real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The study of the non-specific parameters revealed water content, total ash, and acid-insoluble ash met criteria based on the Indonesian Herbal Pharmacopoeia. Evaluation of total flavonoids yielded 7.33% ± 0.00% and 3.84% ± 0.01% w/w for EPI and ESA, respectively. Test results showed balanced induction of all target genes was achieved when subjects received EPI-ESA 2. We concluded that EPI-ESA can potentially support lactogenesis by stimulating the expressions of PRL, PRLR, OXT, and OXTR genes.
Wandansari ED, Syarif RA, Septyaningtrias DE, Purwono S, Sholikhah EN. Standardized mixture of Pluchea indica and Sauropus androgynus extract stimulates the gene expression associated with lactogenesis in rats. J Appl Pharm Sci. 2024. Online First. http://doi.org/10.7324/JAPS.2024.164075
1. Sánchez C, Franco L, Regal P, Lamas A, Cepeda A, Fente C. Breast milk: a source of functional compounds with potential application in nutrition and therapy. Nutrients 2021;13(3):1–34. doi: https://doi.org/10.3390/nu13031026
2. Scherbaum V, Srour ML. The role of breastfeeding in the prevention of childhood malnutrition. World Rev Nutr Diet. 2016;115:82–97. doi: https://doi.org/10.1159/000442075
3. Vitalis D, Witten C, Pérez-Escamilla R. Gearing up to improve exclusive breastfeeding practices in South Africa. PLoS One 2022;17(3):1–12. doi: https://doi.org/10.1371/journal.pone.0265012
4. Victora CG, Bahl R, Barros AJD, França GVA, Horton S, Krasevec J, et al. Breastfeeding in the 21st century: epidemiology, mechanisms, and lifelong effect. Lancet 2016; 387(10017):475–90. doi: https://doi.org/10.1016/S0140-6736(15)01024-7
5. Rollins NC, Bhandari N, Hajeebhoy N, Horton S, Lutter CK, Martines JC, et al. Why invest, and what it will take to improve breastfeeding practices? Lancet 2016; 387(10071):491–504. doi: https://doi.org/10.1016/S0140-6736(15)01044-2
6. Kennett JE, Mckee DT. Oxytocin: an emerging regulator of prolactin secretion in the female rat. J Neuroendocrinol. 2012;24(3):403–12. doi: https://doi.org/10.1111/j.1365-2826.2011.02263.x
7. Grzeskowiak LE, Wlodek ME, Geddes DT. What evidence do we have for pharmaceutical galactagogues in the treatment of lactation insufficiency?—a narrative review. Nutrients 2019;11(5):1–21. doi: https://doi.org/10.3390/nu11050974
8. Golan Y, Assaraf YG. Genetic and physiological factors affecting human milk production and composition. Nutrients 2020;12(5):1–20. doi: https://doi.org/10.3390/nu12051500
9. Matare CR, Craig HC, Martin SL, Kayanda RA, Chapleau GM, Kerr RB, et al. Barriers and opportunities for improved exclusive breast-feeding practices in Tanzania : household trials with mothers and fathers. Food Nutr Bull. 2019;40(3):308–25. doi: https://doi.org/10.1177/0379572119841961
10. Zapantis A, Steinberg JG, Schilit L. Use of herbals as galactagogues. J Pharm Pract. 2012;25(2):222–31. doi: https://doi.org/10.1177/0897190011431636
11. Tabares FP, Jaramillo JVB, Ruiz-Cortés ZT. Pharmacological overview of galactogogues. Vet Med Int. 2014;2014:1–20. doi: https://doi.org/10.1155/2014/602894
12. Gimpl G, Fahrenholz F. The oxytocin receptor system: Structure, function, and regulation. Physiol Rev. 2001;81(2):629–83. doi: https://doi.org/10.1152/physrev.2001.81.2.629
13. Ministry of Health of the Republic of Indonesia. Indonesian Herbal Pharmacopeia. 2nd ed. Jakarta, Indonesia: Ministry of Health of the Republic of Indonesia; 2017.
14. Ruan J, Li Z, Yan J, Huang P, Yu H, Han L, et al. Bioactive constituents from the aerial parts of pluchea indica less. Molecules 2018;23(9):1–11. doi: https://doi.org/10.3390/molecules23092104
15. Syarif RA, Anggorowati N, Munawaroh M, Wahyuningsih MSH. Ethanolic extract of pluchea indica less leaf increases serum growth hormone in lactating rats. Trad Med J. 2021;26(2):111–6. doi: https://doi.org/10.22146/mot.62060
16. Syarif RA, Anggorowati N, Munawaroh M, Adyaksa DNM, Wahyuningsih MSH. Lactogenic activity of ethanolic extract of Pluchea indica Less leaf in lactating rats. J Herbmed Pharmacol. 2023;12(3):380–7. doi: https://doi.org/10.34172/jhp.2023.41
17. Zhang B dou, Cheng J xin, Zhang C feng, Bai Y dan, Liu W yuan, Li W, et al. Sauropus androgynus L. Merr.-A phytochemical, pharmacological and toxicological review. J Ethnopharmacol. 2020;257:1–13. doi: https://doi.org/10.1016/j.jep.2020.112778
18. Soka S, Alam H, Boenjamin N, Agustina TW, Suhartono MT. Effect of Sauropus androgynus leaf extracts on the expression of prolactin and oxytocin genes in lactating BALB/C Mice. J Nutri Nutrigenom 2010;3(1):31–6. doi: https://doi.org/10.1159/000319710
19. Zhong J, Liang Y, Chen Y, Zhang J, Zou X, Deng J, et al. Study and experimental validation of the functional components and mechanisms of Hemerocallis citrina Baroni in the treatment of lactation deficiency. Foods 2021;10(8):1–14. doi: https://doi.org/10.3390/foods10081863
20. Tarko A, Štochmal’Ová A, Jedli?ková K, Hrabovszká S, Vachanová A, Halim Harrath A, et al. Effects of benzene, quercetin, and their combination on porcine ovarian cell proliferation, apoptosis, and hormone release. Arch Anim Breed. 2019;62(1):345–51. doi: https://doi.org/10.5194/aab-62-345-2019
21. Huang X, Ai C, Xiao J, Xiang C. Guideline for the extraction, isolation, purification, and structural characterization of polysaccharides from natural resources. EFood 2022; 3(e37):1–16. doi: https://doi.org/10.1002/efd2.37\
22. Sulaiman CT, Balachandran I. Total phenolics and total flavonoids in selected Indian medicinal plants. Indian J Pharm Sci 2012;74(3):258–60. doi: https://doi.org/10.4103/0250-474X.106069
23. Garber JC, Barbee RW, Bielitzki JT, Clayton LA, Donovan JC, Hendriksen CFM, et al. Guide for the care and use of laboratory animals. 8th ed. Washington, DC: The National Academies Press; 2011.
24. Mustofa, Yuliani FS, Purwono S, Sadewa AH, Damayanti E, Heriyanto DS. Polyherbal formula (ASILACT®) induces Milk production in lactating rats through upregulation of α-Lactalbumin and aquaporin expression. BMC Complement Med Ther. 2020;20(1):1–8. doi: https://doi.org/10.1186/s12906-020-03152-7
25. Charan J, Kantharia N. How to calculate sample size in animal studies? J Pharmacol Pharmacother. 2013;4(4):303–6. doi: https://doi.org/10.4103/0976-500X.119726
26. Chou TC. Preclinical versus clinical drug combination studies. Leuk Lymphoma 2008;49(11):2059–80. doi: https://doi.org/10.1080/10428190802353591
27. Hosseinzadeh H, Tafaghodi M, Mosavi MJ, Taghiabadi E. Effect of aqueous and ethanolic extracts of Nigella sativa seeds on milk production in rats. JAMS J Acupunct Meridian Stud. 2013;6(1):18–23. doi: https://doi.org/10.1016/j.jams.2012.07.019
28. Honvo-Houéto E, Truchet S. Indirect immunofluorescence on frozen sections of mouse mammary gland. J Vis Exp. 2015; 106:1–24. doi: https://doi.org/10.3791/53179
29. Cao D, Ma X, Zhang WJ, Xie Z. Dissection and coronal slice preparation of developing mouse pituitary gland. J Vis Exp. 2017; 129:1–5. doi: https://doi.org/10.3791/56356
30. Unger C, Lokmer N, Lehmann D, Axmann IM. Detection of phenol contamination in RNA samples and its impact on qRT-PCR results. Anal Biochem. 2019; 571:49–52. doi: https://doi.org/10.1016/j.ab.2019.02.002
31. Ochoa A, Montes de Oca P, Rivera JC, Dueñas Z, Nava G, Martínez de la Escalera G, et al. Expression of prolactin gene and secretion of prolactin by rat retinal capillary endothelial cells. Investig Ophthalmol Vis Sci. 2001;42(7):1639–45.
32. Clapp C, Torner L, Gutiérrez-Ospina G, Alcántara E, López-Gómez FJ, Nagano M, et al. The prolactin gene is expressed in the hypothalamic-neurohypophyseal system and the protein is processed into a 14-kDa fragment with activity like 16-kDa prolactin. Proc Natl Acad Sci USA. 1994;91(22):10384–8. doi: https://doi.org/10.1073/pnas.91.22.10384
33. Xi D, Kusano K, Gainer H. Quantitative analysis of oxytocin and vasopressin messenger ribonucleic acids in single magnocellular neurons isolated from supraoptic nucleus of rat hypothalamus. Endocrinology 1999;140(10):4677–82. doi: https://doi.org/10.1210/endo.140.10.7054
34. Liu CX, Takahashi S, Murata T, Hashimoto K, Agatsuma T, Matsukawa S, et al. Changes in oxytocin receptor mRNA in the rat uterus measured by competitive reverse transcription-polymerase chain reaction. J Endocrinol. 1996;150(3):479–86. doi: https://doi.org/10.1677/joe.0.1500479
35. Popovics P, Rekasi Z, Stewart AJ, Kovacs M. Regulation of pituitary inhibin/activin subunits and follistatin gene expression by GnRH in female rats. J Endocrinol. 2011;210(1):71–9. doi: https://doi.org/10.1530/JOE-10-0485
36. Svingen T, Letting H, Hadrup N, Hass U, Vinggaard AM. Selection of reference genes for quantitative RT-PCR (RT-qPCR) analysis of rat tissues under physiological and toxicological conditions. PeerJ 2015;3:1–15. doi: https://doi.org/10.7717/peerj.855
37. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method. Methods 2001;25(4):402–8. doi: https://doi.org/10.1006/meth.2001.1262
38. Lee S, Lee DK. What is the proper way to apply the multiple comparison test? Korean J Anesthesiol. 2018;71(5):353–60. doi: https://doi.org/10.4097/kja.d.18.00242
39. Patil AS. Plant Secondary Metabolites: Isolation, Characterization and Biological Properties. Delhi, India: Studera Press; 2020.
40. Zhang QW, Lin LG, Ye WC. Techniques for extraction and isolation of natural products : a comprehensive review. Chin Med. 2018;13(20):1–26. doi: https://doi.org/10.1186/s13020-018-0177-x
41. World Health Organization. Quality control methods for medicinal plant materials. Geneva, Switzerland: World Health Organization; 1998.
42. Rao Y, Xiang B. Determination of total ash and acid-insoluble ash of Chinese herbal medicine Prunellae Spica by near infrared spectroscopy. Yakugaku Zasshi 2009;129(7):881–886. doi: https://doi.org/10.1248/yakushi.129.881
43. Caesar LK, Cech NB. Synergy and antagonism in natural product extracts: when 1 + 1 does not equal 2. Nat Prod Rep. 2019;36(6):869–88. doi: https://doi.org/10.1039/c9np00011a
44. Xi X, Wang J, Qin Y, You Y, Huang W, Zhan J. The Biphasic effect of flavonoids on oxidative stress and cell proliferation in breast cancer cells. Antioxidants 2022;11(4):1–22. doi: https://doi.org/10.3390/antiox11040622
45. Liu X, Bushnell DA, Kornberg RD. RNA Polymerase II transcription: structure and mechanism. Biochim Biophys Acta. 2012;1829(1):2–6. doi: 10.1016/j.bbagrm.2012.09.003
46. Mercadante AA, Dimri M, Mohiuddin SS. Biochemistry, replication and transcription internet. Treasure Island, FL: StatPearls Publishing; 2022 cited 2022 Dec 28.. Available from: https://www.ncbi.nlm.nih.gov/books/NBK540152/
47. Alberts B, Heald R, Johnson A, Morgan D, Raff M, Roberts K, et al. Molecular biology of the cell. 7th ed. New York, NY: W. W. Norton & Company; 2022.
48. Kiyama R. Estrogenic flavonoids and their molecular mechanisms of action. J Nutr Biochem. 2023;114:1–50. doi: https://doi.org/10.1016/j.jnutbio.2022.109250
49. Brisken C, Ataca D. Endocrine hormones and local signals during the development of the mouse mammary gland. Wiley Interdiscip Rev Dev Biol. 2015;4(3):181–95. doi: https://doi.org/10.1002/wdev.172
50. Carroll JS. Mechanisms of oestrogen receptor (ER) gene regulation in breast cancer. Eur J Endocrinol. 2016;175(1):41–9. doi: https://doi.org/10.1530/EJE-16-0124
51. Kiyama R. Estrogenic terpenes and terpenoids: pathways, functions and applications. Eur J Pharmacol. 2017;815:405–15. doi: https://doi.org/10.1016/j.ejphar.2017.09.049
52. Björnström L, Sjöberg M. Mechanisms of estrogen receptor signaling: convergence of genomic and nongenomic actions on target genes. Mol Endocrinol. 2005;19(4):833–42. doi: https://doi.org/10.1210/me.2004-0486
53. Freeman ME, Kanyicska B, Lerant A, Nagy G. Prolactin: structure, function, and regulation of secretion. Physiol Rev. 2000;80(4):1523–631. doi: https://doi.org/10.1152/physrev.2000.80.4.1523
54. Ye Q, Zhang QY, Zheng CJ, Wang Y, Qin LP. Casticin, a flavonoid isolated from Vitex rotundifolia, inhibits prolactin release in vivo and in vitro. Acta Pharmacol Sin. 2010;31(12):1564–8. doi: https://doi.org/10.1038/aps.2010.178
55. Zárate S, Seilicovich A. Estrogen receptors and signaling pathways in lactotropes and somatotropes. Neuroendocrinology 2010;92:215–23. doi: https://doi.org/10.1159/000321683
56. Featherstone K, White MRH, Davis JRE. The prolactin gene: a paradigm of tissue-specific gene regulation with complex temporal transcription dynamics. J Neuroendocrinol. 2012;24(7):977–90. doi: https://doi.org/10.1111/j.1365-2826.2012.02310.x
57. Kavarthapu R, Dufau ML. Essential role of endogenous prolactin and CDK7 in estrogen induced upregulation of the prolactin receptor in breast cancer cells. Oncotarget 2017;8(16):27353–63. doi: https://doi.org/10.18632/oncotarget.16040
58. McFarland-Mancini M, Hugo E, Loftus J, Ben-Jonathan N. Induction of prolactin expression and release in human preadipocytes by cAMP activating ligands. Biochem Biophys Res Commun. 2006;344(1):9–16. doi: https://doi.org/10.1016/j.bbrc.2006.03.168
59. Kuiper GGJM, Lemmen JG, Carlsson B, Corton JC, Safe SH, Van der Saag PT, et al. Interaction of estrogenic chemicals and phytoestrogens with estrogen receptor β. Endocrinology 1998;139(10):4252–63. doi: https://doi.org/10.1210/endo.139.10.6216
60. Shughrue PJ, Dellovade TL, Merchenthaler I. Estrogen modulates oxytocin gene expression in regions of the rat supraoptic and paraventricular nuclei that contain estrogen receptor-β. Prog Brain Res. 2002;139:15–29. doi: https://doi.org/10.1016/S0079-6123(02)39004-6
61. Acevedo-Rodriguez A, Mani SK, Handa RJ. Oxytocin and estrogen receptor β in the brain: an overview. Front Endocrinol. 2015;6:1–7. doi: https://doi.org/10.3389/fendo.2015.00160
62. Pauciullo A, Ogah DM, Iannaccone M, Erhardt G, Di Stasio L, Cosenza G. Genetic characterization of the oxytocin-neurophysin I gene (OXT) and its regulatory regions analysis in domestic Old and New World camelids. PLoS One 2018;13(4):1–17. doi: https://doi.org/10.1371/journal.pone.0195407
63. Drummond AE, Fuller PJ. The importance of ERβ signalling in the ovary. J Endocrinol. 2010;205:15–23. doi: https://doi.org/10.1677/JOE-09-0379
64. Haddad JJ. Antioxidant and prooxidant mechanisms in the regulation of redox(y)-sensitive transcription factors. Cell Signal. 2002; 14:879–97. doi: https://doi.org/10.1016/S0898-6568(02)00053-0
Year
Month