INTRODUCTION:

Breast cancer is a leading health concern among women due to its high mortality and morbidity rate. It is considered to be a multifarious disease and is considered as the second largest cancer¹. Mutations in breast cancer genes BRCA1, BRCA2, environmental factors, the female hormones estrogen, progesterone, prolactin are linked to breast cancer etiology² Increased exposure and fluctuations in estrogen occur due to exogenous menopausal hormone therapy and usage of oral contraceptives.

The risk factors that contribute in both the onset and progression of mammary carcinoma were broadly classified in to two categories, as Endogenous factors Exogenous factors such as family history, smoking, radiation, chemical carcinogens and radium. Both these risk factors are inseparable³. As per the breast cancer statistics, every three minutes a woman is diagnosed and every 13 minutes a death case gets reported. One in eight women could be diagnosed and one in thirty will die from breast cancer during their lifetime. The involvement of steroid hormones namely estradiol or progesterone has been extensively studied Estrogen plays a role in causing certain cancers. several different factors play a role in turning healthy cells cancerous. When these factors are present, estrogen can act as a spark. The hormone causes cancer cells to multiply and spread. Estrogen is primarily an ovarian hormone that regulates cellular development and differentiation. The major target of this hormone includes mammary gland, uterus, and urogenital tract of both males and females^4-7 Estrogen brings about proliferation of both the normal and neoplastic breast epithelium through activation of estrogen receptors (ER)-α and ER-β, regulate the expression of genes that contribute to tumor progression by both classical and non-classical activating mechanisms.^8,9

The major mechanisms estrogen intervenes its carcinogenic effects such as cellular proliferation through their receptor-mediated hormonal activity, the direct genotoxic effects by increasing mutation rates through a cytochrome P450-mediated metabolic activation and induction of aneuploidy¹⁰ The signiﬁcant amounts of ER-α receptors being there in breast cancer tissue at the time of diagnosis is considered as a marker of hormone dependence¹¹.This form the basis, for the treatment with the selective estrogen receptor modulator (SERM) drugs like tamoxifen, raloxifen and specific antiestrogens such as fulvestrant is considered as the ﬁrst-line remedy for metastatic mammary carcinoma¹¹ .In recent years, breast cancer control and preventive strategies have proven to be the most effective measures in reducing mortality rates and improving patient prognosis¹². Breast cancer prevention researches need molecularly targeted approach for success treatment and less toxic drugs. Alternative or Herbal medicine is widely practiced in India. Many active compounds and their semi-synthetic products or derivatives obtained from different medicinal plants, their parts have been evaluated for their usefulness and tolerability in breast cancer treatment^13-14Few biological response modifiers, herbs derivatives, are known to deter cancer cell growth by altering the activity of specific enzymes and hormones, while other known herbs reduce the lethal side effects and the complications arising due to chemotherapy and radiotherapy¹⁵. Vitex agnus castus or Vitex nirgundi commonly called as chasteberry or emmenagogue herb known to effectively ameliorate the symptoms of pre-menstrual syndrome, dysmenorrhea, amenorrhea, menstrual headaches and cramps through its modulatory effects on female endocrine system. It also blocks the production of prolactin from pituitary, shortening luteal phase and thus antagonize hormonal imbalance (low progesterone synthesis in luteal phase). Moreover, Vitex agnus castus induce cell death (tumoricidal) in ovarian, cervical, breast, gastric, colon, lung cancer cells. Though signiﬁcant amounts of ER-α receptors in breast cancer tissue is considered as a risk marker and many treatment modalities have been tried. However, not a single study has been reported in the literature about the role of vitex agnus and level of estrogen Apart from the plant extract we have used the anti-prolactin and anti-growth hormone therapy too in single and in combination dose. Therefore, this study is aimed to find out the role of vitex agnus and anti - prolactin and anti-growth hormone drugs on serum estrogen level in tumorigenic induced rats.

MATERIALS AND METHODS:

Materials:

In-house bred healthy female Sprague Dawley (SD) rats (40 to 60 days old) of weight 80-100 gm were selected for the study. All rats were fed with standard rat food and water ad libitum. Institutional Animal Ethical Committee (IAEC) approval was obtained before the conduct of the study (IAEC/KMC/28/02/14)) and care was taken to handle the rats in humane manner. Necessary precautions were taken to use only minimum number of required animals to generate significant data, diligently following all the ethical guidelines.

Experimental group.

Experimental overview:

Inbred female SD rats were selected and randomly allotted to different groups. The rats were induced with MNU for mammary tumor formation. MNU was weighed with respect to the standard dose of 50mg/kg body weight (BW) to that of the calculated body weight of the recruited rats. Acidified saline (0.9% w/v) whose pH was adjusted to 4 by the addition of 3% glacial acetic acid was used as vehicle. MNU was dissolved in the acidified vehicle to obtain a final formulation of 10ml/kg. A single intraperitoneal dose was administered to 40-50 days old female SD rats^16,17 The rats were observed for first palpable tumour appearance. treatment plans to check the occurrence and extent of mammary carcinogenesis and progression. The rats were treated prior to induction of tumor and post tumor with Vitex seed extract. Once the experimental procedure was completed, rats were anesthetized individually in a glass desiccator containing saturated ether vapours and blood was collected under aseptic conditions through intra-cardiac puncture. The blood sample was then centrifuged at 5000rpm for 10 minutes in a cooling centrifuge. The serum was collected in sterile 1.5mL aliquots and stored at -20^o C for further analysis.

Estimation of Estrogen:

Method: Sandwich enzyme linked immune-sorbent assay technology. (Shanghai Yehua Biological Technology Co., Ltd. China. Catalog No: BYEK2676)

Assay Procedure: A volume of 50μL standards, serum samples and HRP conjugated detection antibody were added to the appropriate wells subsequently, mixed and incubated with the pre-coated anti- E antibody bound to the wells for 60 minutes at 37^oC. The unbound conjugated antibody was washed away 1x with wash buffer (five washes). After complete drying, chromogen A and B were added and incubated for 10minutes to visualize HRP enzymatic reaction. TMB was catalyzed by HRP to produce a blue color product that changed into yellow after the addition of acidic stop solution. The intensity of yellow color was proportional to the amount of estrogen bound in the plate which was read at 450 nm and the concentration was calculated.

Precision: Intra-Assay: CV<10% and Inter-Assay: CV<12% Sensitivity: 0.252ng/

RESULT:

Estimation of Serum Estrogen Levels of the Groups

The study results showed significantly (p<0.001) elevated estrogen levels in tumour induced and hyperprolactinemia compared to control group and Estrogen levels were significantly (p<0.001) increased in tumour induced group than hyperprolactemia (HPRL) group (III). The level of estrogen was lowered in cabergoline, and octreotide group compared to HPRL and Induction group In pre and post Vitex treated groups showed decrease estrogen levels (p<0.01) when equated with MNU induced and HPrl groups. Pre-treatment with Vitex extract presented significant (p<0.05) reduction in estrogen levels when matched with Vitex post treated group. A significant decrease (P<0.05) was observed between the combined cabergoline and octreotide group and the pretreated herbal group.

Figure 1: Effect of vitex agnus on serum estrogen level

*p<0.001 Vs Control, # p<0.001 Vs MNU Induced, $ P<0.001 Vs HPrl

Fig 2. Comparison of different treatment strategies on serum estrogen level

Values expressed as Mean±SD *p<0.001 Vs Control, # p<0.001 Vs MNU Induced, $ P<0.001 Vs HPrl, €p<0.001 VI Vs (IV andV), † p<0.05 VI Vs VII

Table 1: Animal Group

Groups	Treatment
1. Control	Normal saline
2. N-Methyl-N-nitrosourea (MNU)	50µg/kg
3. Sulpiride	20mg /kg
4. Cabergoline + MNU	0.5mg/kg +50µg/kg
5. MNU+ cabbergoline	50µg/kg+0.5mg/kg
6. Octreotide + MNU	50µg/kg+ 50µg/kg
7. MNU+ octreiotide	50µg/kg+ 50µg/kg
8. cabergoline+ octreotide + MNU	0.5mg/kg + 50μgm/kg+50µg/kg
9. Octreotide + MNU	50µg/kg+ 50µg/kg
10. Pretreated with vitex Agnus	200mg/kg+50µg/kg
Post treated with vitex Agnus	50µg/kg +200mg/kg

Table 2. Effect of vitex agnus, cabergoline and octreotide on serum estrogen level in MNU induced SD rats

Groups	Estrogen level (ng/L)
Control group I	9.1± 0.72#$
MNU induced group II	27.03±0.95*$
Hyperprolactemia (Group III)	19.23±0.92*#
Cabergoline +MNU (group IV)	14.06±0.83*#$€
MNU+ cabergoline (group IVA)	18.96±0.76*#$
Octreotide +MNU group V	15.07±1.14*#$€
MNU+ octreotide (group VA)	20.25±0.70*#$
Cabergoline + octreotide +MNU (group VI)	12.69±1.69 *#$
Pretreatment with vitex agnus (group VII)	14.75±0.74*#$†
Post treatment with vitex agnus (group VII A)	16.66±0.62 *#$

Values expressed as Mean ± SD *p<0.001 Vs Control, # p<0.001 Vs MNU Induced, $ P<0.001 Vs HPrl, €p<0.001 VI Vs (IV andV), † p<0.05 VI Vs VII

DISCUSSION:

Mammary cancer is among the prevalent malignancies can have effect on both men and women worldwide. Breast cancer is complex and multifactorial¹⁸. Numerous risk factors such as age, hormones involved in menstrual cycle namely estrogen and progesterone, and sedentary lifestyle can lead to obesity and various life threatening diseases So early diagnosis is the cornerstone for the treatment of breast cancer. The protocol for diagnosis includes screening tests such as mammogram, breast ultrasound and diagnostic tests include biopsy. All these procedures have their own merits and demerits. Early detection of breast cancer can be improved by having a greater knowledge of the extent of the disease and causes behind the delay in the diagnosis. About two out of three breast cancers are hormone receptor positive, they can be estrogen receptor (ER) positive or progesterone receptor (PR) positive¹⁹. Recently the role of prolactin and growth hormone alone or in conjunction with estrogen and progesterone in mammary tumorigenesis are main areas of research^20-21. The role of ovarian hormones in the development of mammary gland is well known Estrogen is primarily an ovarian hormone that modulates cellular growth and differentiation. Its major targets are the mammary gland, uterus, bone, cardiovascular system, brain and urogenital tract of both males and females^22-23 The effects of estrogen on the growth, differentiation and function of the mammary gland have long been well known. It plays a major role in promotion and proliferation of both the normal and neoplastic breast epithelium through activation of estrogen receptors (ER)-α and ER-β12^24-26. They regulate the expression of genes that contribute to tumor progression by both classical (e.g., binding to specific response elements in a gene promoter) and non-classical (e.g., activating growth factor pathways in the membrane) mechanisms^27,28.

Traditional herbs and plant derived products has been used from ancient days to treat various diseases including cancer^29-31. Vitex agnus is one such herb which is used as a folk medicine for relieving pre-menstrual syndrome problems from many years. Clinical studies done on Vitex showed its effect in latent hyperprolactinemia cases, providing a basis of its anti-prolactin activity^32-33. However, there is not wide information about the relation between the estrogen and vitex agnus. The present study is planned to find out the role of Vitex agnus on estrogen level in mammary carcinogenesis and progression. Clinical research and scientific findings show that hormones play a major role in the mammary carcinogenesis. It is mainly these hormones which fluctuate to create a suitable environment for the cancer cell to grow, divide and propagate. In breast cancer estrogen happens to play the main role in mammary cancer progression leading to the deadly malignancy. The action of the hormones might be through, and with proto-oncogenes and growth factors to affect breast cell proliferation and breast cancer etiology. Previous experiments on animal models reports that estrogen causes interlobular ductal cell division in the luteal phase. Most breast carcinomas originate from terminal duct lobular unit cells³⁴.

In the present study the tumour induced group (II) showed significantly highest level of estrogen as compared to all other groups. HPRL group (III) showed significantly high estrogen levels when compared to all other. The cabergoline, octreotide therapy group showed an increase in estrogen level compared to control, however lesser than the HRPL and tumor induction group. The pre and post treatment with Vitex showed significant reduction in estrogen levels compared to tumour induced (II) and HPRL (III) groups. Cabergoline is a prolactin lowering hormone, works by stimulating dopamine receptors in the brain. This leads to a decrease in the secretion of prolactin from the pituitary gland. Octreotide Somatostatin analog and mimics natural somatostatin, a hormone that inhibits the release of several other hormones and substances. By binding to somatostatin receptors, it reduces the secretion of growth hormone, insulin, glucagon, and various gastrointestinal hormone. In this study the administration of cabergoline and octreotide singly and conjointly prior to initiating tumor induction resulted in decrease level of estrogen compared to HRPL and Tumor induction groups. However, In unison brought about a better significant outcome. Cabergoline and estrogen are two distinct substances with different medical uses and usually cabergoline is a prolactin lowering drug there is limited evidence suggesting a direct interaction between cabergoline and estrogen. However, both medications can influence hormone levels and potentially impact conditions related to hormonal balance therefore, the exact mechanism of interaction between these to be elucidated. Similarly with octreotide too there is no well-documented direct interaction between octreotide and estrogen the possible mechanism. While octreotide primarily affects growth hormone secretion, its inhibition of various hormones could potentially influence estrogen levels and activity indirectly. The reduction in the estrogen levels in herbal extract treated group compared to the other groups can be attributed to its effect on estrogen receptor and hence following decrease in the levels. This finding is similar as seen in other studies³⁵. Vitex has been traditionally used for various women's health issues such as menstrual irregularities and symptoms of menopause. Vitex is believed to influence hormonal balance by affecting the pituitary gland and indirectly impacting estrogen and progesterone levels. Vitex agnus-castus is thought to exert its effects through dopaminergic mechanisms, specifically by stimulating dopamine receptors in the brain. It may also modulate the release of prolactin from the pituitary gland, which can indirectly influence estrogen and progesterone levels.

CONCLUSION:

This study also showed pre-treatment with Vitex extract improved oxidative state of the mammary gland. Though the level did not reach to the normal level, vitex agnus could decrease the level of estrogen in hypoprolactemia and tumor induced groups and could maintenance of the near normal. Therefore, this study indicates a possible therapeutic potential of Vitex fruit/seeds extract to be used as a traditional treatment for mammary carcinogenesis

CONFLICT OF INTEREST:

The authors declare that there are no conflicts of interest.

REFERENCES:

1. Nidhi Hariramani, Sivaraman Jayanthi. A Systematic Review of Intrinsic Factors and its Influence in Breast Cancer. Research J. Pharm. and Tech. 2018; 11(8): 3543-3546. doi: 10.5958/0974-360X.2018.00654.6

2. van Erkelens A, Derks L, Sie AS, Egbers L, Woldringh G, Prins JB, Manders P, Hoogerbrugge N. Lifestyle Risk Factors for Breast Cancer in BRCA1/2-Mutation Carriers Around Childbearing Age. J Genet Couns. 2017; 26(4): 785–791.

3. Dossus L, Benusiglio PR. Lobular breast cancer: incidence and genetic and nongenetic risk factors. Breast Cancer Res. 2015; 17: 37.

4. Workman P, Aboagye EO, Balkwill F, Balmain A, Bruder G, Chaplin DJ, et al. Guidelines for the welfare and use of animals in cancer research. Br J Cancer. 2010; 102: 1555–1577.

5. Clark JH, Shrader WT, O’Malley BW. Mechanisms of action of steroid hormones. In: Wilson JD, Foster DW, editors. Textbook of Endocrinology. New York: Saunders; 1992. p. 35–90.

6. Turner RT, Riggs BL, Spelsberg TC. Skeletal effects of estrogens. Endocr Rev. 1994; 15: 275–300.

7. Farhat MY, Lavigne MC, Ramwell PW. The vascular protective effects of estrogen. FASEB J. 1996; 10: 615–624.

8. Beato M, Herrlich P, Schutz G. Steroid hormone receptors: many actors in search of a plot. Cell. 1995; 83: 851–857.

9. Roger P, Sahla ME, Makela S, Gustafsson JA, Baldet P, Rochefort H. Decreased expression of estrogen receptor beta protein in proliferative preinvasive mammary tumors. Cancer Res. 2001; 61: 2537-2541.

10. Osborne CK, Bardou V, Hopp TA, Chamness GC, Hilsenbeck SG, Fuqua SAW, et al. Role of the Estrogen Receptor Coactivator AIB1 (SRC-3) and HER-2/neu in Tamoxifen Resistance in Breast Cancer. J Natl Cancer Inst. 2003; 95(5): 353–361.

11. Asaad RA, Abdullah SS. Breast Cancer Subtypes (BCSs) Classification according to Hormone Receptor Status: Identification of patients at High Risk in Jableh-Syria. Res J Pharm Technol. 2018;11(8):3703-3710.

12. Russo JI, Russo HR. The role of estrogen in the initiation of breast cancer. J Steroid Biochem Mol Biol. 2006; 102(1–5): 89-96.

13. Dhania Novitasari, Riris Istighfari Jenie, Febri Wulandari, Rohmad Yudi Utomo, Dyaningtyas Dewi Pamungkas Putri, Jun-ya Kato, Edy Meiyanto. Curcumin-like structure (CCA-1.1) induces permanent mitotic arrest (Senescence) on Triple-negative breast cancer (TNBC) cells, 4T1. Res J Pharm Technol. 2021; 14(8): 4375-2. doi: 10.52711/0974-360X.2021.00760

14. Hanaa H. Ahmed, Hadeer A. Aglan, Ghada H. Elsayed, Hebatallah G. Hafez, Emad F. Eskander. Quercetin Offers Chemopreventive Potential against Breast Cancer by Targeting a Network of Signalling Pathways. doi: 10.52711/0974-360X.2021.00499

15. Muhammad Isrul, Risky Juliansyah, Ahmad Saleh, Wa Ode Yuliastri, Jastria Pusmarani, Himaniarwati, Wa Ode Wahyuni Maulidina. Phytochemical Analysis, Standardization and Cytotoxic Activity of Curcuma aureginosa Extract in Human Breast Cancer (MCF-7) Cell Line. Research J. Pharm. and Tech. 2019; 12(4): 1967-1973. doi: 10.5958/0974-360X.2019.00329.9

16. Ohyama K, Akaike T, Hirobe C, Yamakawa T. Cytotoxicity and apoptotic inducibility of Vitex agnus-castus fruit extract in cultured human normal and cancer cells and effect on growth. Biol Pharm Bull. 2003; 26(1): 10–18.

17. McCormick DL, Adamowski CB, Fiks A, et al. Lifetime Dose Response Relationships for Mammary Tumor Induction by a Single Administration of N-Methyl-N-nitrosourea. Cancer Res. 1981; 41: 1690–1694.

18. Airo T, Yen-chang L, Hisanori M, Tomo S, Uehara N, Takashi Y, Katsuhiko Y. Animal Models of N-Methyl-N-nitrosourea-induced Mammary Cancer and Retinal Degeneration with Special Emphasis on Therapeutic Trials. Int J Exp Clin Pathophysiol Drug Res. 2011; 25(1): 11-22.

19. Punna Venkateshwarlu, Mehul M. Patel. A Review: Method Development Validation and Degradation Studies of some Anticancer Drugs. Res J Pharm Technol. 2021; 14(10): 5443-8. doi: 10.52711/0974-360X.2021.00949

20. Clevenger CV, Furth PA, Hankinson SE, Schuler LA. The role of prolactin in mammary carcinoma. Endocr Rev. 2003; 24: 1–27.

21. Manni A, Boucher AE, Demers LM, Harvey HA, Lipton A, Simmonds MA, Bartholomew M. Endocrine effects of combined somatostatin analog and bromocriptine therapy in women with advanced breast cancer. Breast Cancer Res Treat. 1989; 14: 289–298.

22. Anu Singh, Purnima Bharati, Vinita Singh, Ratna Chowdhary. An Immunohistochemical Study to assess the Expression of Estrogen and Progesterone Receptor in Endometrial hyperplasia and Endometrial carcinoma compared to normal Endometrium: A Prospective Pathological Study. . Res J Pharm Technol. 2024; 17(2): 491-5. doi: 10.52711/0974-360X.2024.00076

23. Remal Abdulaziz Asaad. Hormone Receptor Status and its Relation to C-Reactive Protein and other Prognostic factors in Breast Cancer in Jableh- Syria. Research J. Pharm. and Tech. 2017; 10(9): 3003-3010. doi: 10.5958/0974-360X.2017.00532.7

24. Farhat MY, Lavigne MC, Ramwell PW. The vascular protective effects of estrogen. FASEB J. 1996;10:615–624.

25. Beato M, Herrlich P, Schutz G. Steroid hormone receptors: many actors in search of a plot. Cell. 1995;83:851–857.

26. Roger P, Sahla ME, Makela S, Gustafsson JA, Baldet P, Rochefort H. Decreased expression of estrogen receptor beta protein in proliferative preinvasive mammary tumors. Cancer Res. 2001; 61: 2537–2542.

27. Osborne CK, Bardou V, Hopp TA, Chamness GC, Hilsenbeck SG, Fuqua SAW, et al. Role of the Estrogen Receptor Coactivator AIB1 (SRC-3) and HER-2/neu in Tamoxifen Resistance in Breast Cancer. J Natl Cancer Inst. 2003; 95(5): 353–361.

28. Syam M, Ahmad B, Siddig I, Adel S. In vitro ultramorphological assessment of apoptosis on CEMss induced by linoleic acid-rich fraction from Typhonium flagelliforme tuber. Evid Based Complement Alternat Med. 2011; 12.

29. Bharti Ahirwar, Dheeraj Ahirwar. In vivo and in vitro investigation of cytotoxic and antitumor activities of polyphenolic leaf extract of Hibiscus sabdariffa against, breast cancer cell lines. Research J. Pharm. and Tech. 2019; 13(2): 615-620. doi: 10.5958/0974-360X.2020.00116.X

30. N. Habeela Jainab,, M. K. Mohan Maruga Raja. In Silico Molecular Docking Studies on the Chemical Constituents of Clerodendrum phlomidis for its Cytotoxic Potential against Breast Cancer Markers. Research J. Pharm. and Tech. 2018; 11(4): 1612-1618. doi: 10.5958/0974-360X.2018.00300.1

31. Raghad J. Fayyad, Alaa Naseer Mohammed Ali, Noor T. Hamdan. The Specific Anti-cancerous Mechanisms Suggesting Spirulina Alga as a Promising breast Cancer Fighter. Research Journal of Pharmacy and Technology. 2021; 14(10): 5599-2. doi: 10.52711/0974-360X.2021.00974

32. Merz PG, Gorkow C, Schrodter A, Rietbrock S, Sieder C, Loew D, Dericks-Tan JS, Taubert HD. The effects of a special Agnus castus extract (BP1095E1) on prolactin secretion in healthy male subjects. Exp Clin Endocrinol Diabetes. 1996; 104: 447–453.

33. Pike MC, Spicer DV, Dahmoush L, Press MF. Estrogens, progestogens, normal breast cell proliferation, and breast cancer risk. Epidemiol Rev. 1993; 15(1): 17–35.

34. Carmichael AR. Can Vitex agnus-castus be used for the treatment of mastalgia? What is the current evidence? Evid Based Complement Alternat Med. 2008; 5(3): 247–250.

35. Bounous G, Gervais F, Amer V, Batist G, Gold P. The influence of dietary whey protein on tissue glutathione and the diseases of aging. Clin Invest Med. 1989; 12: 343–349.

Received on 08.02.2025 Revised on 17.06.2025

Accepted on 25.08.2025 Published on 13.01.2026

Available online from January 17, 2026

Research J. Pharmacy and Technology. 2026;19(1):377-381.

DOI: 10.52711/0974-360X.2026.00055

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.