Phytochemical analysis and anti-cancer effect of a polyherbal extract for mammary cancer by in vitro study

Introduction

Breast cancer is frequently diagnosed in women worldwide, leading to the second most common cause of death in women. Currently, breast cancer represents a serious public health problem in developing countries (Ullah, 2019) [27] . The incidence of mammary tumours in dogs is three times greater than humans. Mammary tumour occurrence is more common in intact females comprising up to 50 to 70 percent of all tumours (Moe, 2001) [19] . Many treatments directed against breast cancer are now available, some examples include; chemotherapy that can be used alone or in combination with surgery, radiotherapy, hormone therapy and targeted therapy (Tinoco et al., 2013) [26] . However, these therapies do not absolutely reverse metastatic breast cancer and are laden with many other drawbacks including: chemotherapeutic drug toxicity on healthy cells, endometrial cancer risk and cardiotoxicity (Kumar, et al., 2014) [13] . Phytochemicals act through multiple pathways that are involved in cancer progression, including cell cycle control, apoptosis, angiogenesis, and metastasis. Thus, their multimodal actions offer synergy with conventional therapeutic modalities, potentially improving therapeutic outcomes and reduces side effects (Aljabali et al., 2025) [2] . Annona muricata Linn (common name "soursop") is a plant widely distributed in tropical regions. It is known to possess biological properties such as sedative, bactericidal, antiparasitic, anti-inflammatory, astringent, haemostatic, antispasmodic, anti-ulcer, antidiarrhoeal, antitussive, hypotensive, antipyretic, galactogogue and anticancer (Maignien and Corosol, 2005) [17] . Administration of an ethanolic fraction of neem leaf (A. indica) inhibits progression of chemical carcinogen induced mammary tumourigenesis in rat models. Ethanolic fraction of neem leaf treatment was also highly effective in reducing mammary tumour burden and in suppressing mammary tumour progression even after the cessation of treatment (Arumugam et al., 2014) [3] . Aegle marmelos leaf extract is found to be very effective against various tumour cell lines including breast cancer cell line MCF-7 (Michigan [15,9] . The extract of soursop leaf (A. muricata) is very active in inhibiting the growth of breast cancer cell line MCF-7, showing higher activity using MTT assay (Hasan et al., 2020) [7] . When Aegle marmelos leaves extract extract treated MCF-7 cells was assessed by MTT assay using Dulbecco's Modified Eagle Medium. It inhibited the proliferation of MCF-7 cells, changed the cytoplasmic morphology, reduced the invasion and migration ability of MCF-7 cells and exhibited pro-apoptotic effects (Kumar et al., 2023) [14] . The extracts and phytochemicals compounds of Nigella sativa extract containing its active component such as thymoquinone (TQ), carvacrol, and trans-anethole have been studied extensively for therapeutic potential against many diseases especially cancer.

Preparation of polyherbal extract

Fresh leaves of Annona muricata, Azadirachta indica and Aegle marmelos were collected and cleaned with water. These were dried in shade for 20-30 days. All the three leaves and seeds of Nigella sativa were individually crushed into powder using mixture grinder. The ethanolic extract of individual herbals were prepared by macerating the respective herbal powder in 70% ethanol for five days with intermittent shaking in the Department of Veterinary pathology, Veterinary College and Research Institute, Orathanadu. It was filtered using Whatman No.1 filter paper. The extracts were kept in rotary evaporator at 50 ℃ for three hours at the Department of Veterinary Pharmacology and Toxicology, Veterinary College and Research Institute, Orathanadu and dried to obtain a blackish-green semisolid substance. The dried extract was weighed and stored at-20℃ in an air tight container until further use. The individual plant extracts were mixed at the ratio of 1:1:1:1.

Phytochemical analysis

Qualitative phytochemical analysis to confirm the presence or absence of different phytochemical constituents like tannins, phenols, alkaloids, flavonoids, terpernoids, saponins, sterols, carbohydrates and proteins in the ethanolic extract of A. muricata, A. indica and A. marmelos leaves and seeds of N. sativa individually as well as in polyherbal formulation were done following standard procedures. Quantitative phytochemical analysis of selective phytochemicals was analysed by using spectrophotometer. Total flavonoid content in polyherbal extract was determined according to the method of Zhishen et al. (1998). Tannin in polyherbal extract was estimated as per the procedures of The Ayurvedic pharmacopoeia of India 2008. Total phenolic content in polyherbal extract was determined as per Gupta et al. (2003). Saponin content in polyherbal extract was determined using the method of Obadoni and Ochuko (2001). The terpenoid content in polyherbal extract was determined using method of Indumathi et al. (2014).

In vitro study with MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay

Cell viability assay using MCF-7 cell line was carried out using 10 per cent fetal bovine serum supplemented Dulbecco's Modified Eagle media. The cells were seeded in a 96-well flat-bottom microtiter plate at a density of 10 4 cells/well and allowed to adhere for 24 hours at 37 °C in a CO 2 incubator. After 24 hours of incubation, culture medium was replaced with a fresh medium. Stock solution of polyherbal extract was prepared by mixing with Dimethyl sulfoxide. The cells were then treated with various concentrations (25, 50, 100, 200 and 400 µg/ml) of the polyherbal extract with three replicates for 24 hours at 37 °C in a CO 2 incubator. After 24 hours of incubation, culture medium was replaced with a fresh medium. Subsequently, 10 μL of MTT working solution (5 mg/mL in phosphate buffer saline solution) was added to each well and the plate was incubated for 4 hours at 37 °C in a CO 2 incubator. The medium was then aspirated, and the formed formazan crystals were solubilized by adding 50 μL of DMSO per well for 30 min at 37 °C in a CO 2 incubator. The plate was cover it with aluminum foil and mildly shaken for 10-15 minutes. Finally, the absorbance was read on a BIORAD iMax microtitre plate reader at 490 nm. The inhibition concentration IC 50 was calculated based on the OD value.

Absorbance of treatment % Cell viability = ×100 Absorbance of control

Results

Qualitative analysis

The results of qualitative analysis of the ethanolic extract of A. muricata, A. indica, A. marmelos and N. sativa as well as polyherbal extract are given in the Table 1. The leaves of A. muricata and A. indica were positive for tannins, phenols, flavanoids, terpernoids, saponins, sterols, and carbohydrates except alkaloids and proteins. The leaves of A. marmelos and seeds of N. sativa were positive for tannins, phenols, alkaloids, flavanoids, terpernoids, sterols, carbohydrates and proteins except saponins. The polyherbal formulation were positive for all the constituents like tannins, phenols, alkaloids, flavanoids, terpernoids, saponins, sterols, carbohydrates, and proteins.

Quantitative analysis

The quantitative analysis of selective phytochemicals for polyherbal formulation are given in the Table 2. The concentration of phytochemicals were flavonoids, saponins, phenols, terpernoids, tannins and alkaloids were 13.65, 06.22, 03.89, 02.49, 01.05 and 00.40 (% w/w) in decreasing order respectively.

In vitro study with 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay Cytotoxic effect of polyherbal extract in different concentration of 25, 50, 100, 200 and 400 µg/ml on MCF-7 cells was measured by MTT assay after 24 h of incubation. Cell viability reduced from 100% to 95, 87, 85, 73, 65% in response to dose dependent higher concentration of polyherbal extract from 25, 50, 100, 200 and 400 µg/ml respectively. The IC 50 value of polyherbal extract was estimated as 358.374 µg/ml (Fig. 1). The morphological analysis of polyherbal extract treated MCF-7 cells was observed in inverted phase contrast microscope. The MCF-7 cells treated with polyherbal extract 25, 50, 100, 200 and 400 µg/ml compared with untreated (control) cells are given in the Fig. 2. The significant morphological changes were apoptosis which were characterized by reduced cell density, rounded up cells, loss of contact with neighboring cells and cell shrinkage and were observed in a dose dependent manner.

Discussion

Qualitative phytochemical analysis

The leaves of A. muricata and A. indica were positive for tannins, phenols, flavanoids, terpernoids, saponins, sterols and carbohydrates. These findings were in accordance with earlier reports of Daud et al. (2016) [4] who reported the presence of alkaloids, terpenoids, flavonoids, coumarins, steroids, fatty acids, phlobatannins, phenolics, tannins, and saponins in A. muricata leaves extract. The results were in agreement with Ravi et al. (2015) [23] who reported the phytochemical screening of ethanol extract of A. indica leaves and twigs revealed the presence of alkaloids, flavonoids, saponins, tannins, polyphenols and steroids. The leaves of A. marmelos and seeds of N. sativa were positive for tannins, phenols, alkaloids, flavanoids, terpernoids, sterols, carbohydrates. The results are in strong agreement with Veer and Singh (2019) [29] who reported the phytochemical screening of A. marmelos dried leaves revealed the presence of alkaloids, carbohydrates, flavonoids, phenols, saponins, steroids, and tannins. These results were similar to Umadevi et al. (2012) [28] who reported the presence of carbohydrates, alkaloids, flavonoids, tannins, terpenoids, saponins, quinones, glycosides, coumarins in the ethanolic leaf extract of A. marmelos. These results also were in agreement with Ahmad et al. (2014) [1] who reported that the phytochemical screening of methanolic extract of N. sativa seeds revealed the presence of carbohydrates, phenolic compounds, flavonoids, alkaloids, proteins, saponins, lipids, sterols and tannins. Qualitative phytochemical screening of combined polyherbal extracts from A. muricata leaves, A. indica leaves, A. marmelos leaves and N. sativa seeds extract confirmed the presence of a wide range of secondary metabolites including tannins, phenols, alkaloids, flavanoids, terpernoids, saponins, sterols, carbohydrates, and proteins in ethanolic extracts. The detection of these bioactive compounds strongly supports the therapeutic potential of the polyherbal extract, as each plant component is already documented to contain similar phytochemical classes individually. These results align with published phytochemical reports for each species (Daud et [4,23,28,1] .

Quantitative phytochemical analysis

The quantitative analysis of selective phytochemicals of polyherbal extract in the current study contained higher concentration of flavonoids (13.65%) followed by saponin (6.2%). The results were in strongly agreement with Khanal (2021) [11] who reported the quantitative estimation of A. indica contained highest flavonoids percentage (13.8%) in leaves. Salsabila et al. (2021) [24] reported 2.3% of flavonoids in A. muricata leaves extract. It strongly supports the polyherbal extract as an anticancer agent. They also reported that flavonoid compounds are known for their antioxidant activity due to their compound structure which has many phenolic residues bound to the generic carbon ring framework. Phytochemicals are bioactive compounds produced naturally by plants and have great potential in human health and disease. These compounds possess antiproliferative, anti-oxidant, and immunomodulatory properties. The flavonoids are polyphenolic compounds that are ubiquitous in nature and are categorized, according to chemical structure, into flavonols, flavones, flavanones, isoflavones, catechins, anthocyanidins and chalcones. Numerous in vitro and animal model data suggest that flavonoids modulate important cellular and molecular mechanisms related to carcinogenesis, a multistep process involving the transformation, survival, proliferation, invasion, angiogenesis, and metastasis of the tumour cells. Flavonoids also act as protein kinase inhibitors for cancer chemoprevention. Recent studies show that flavonoids can bind directly to some protein kinases and then alter their phosphorylation state to regulate multiple cells signalling pathways in carcinogenesis processes (Umadevi et al., 2012) [28] . Flavonoids are one of the most popular secondary metabolites possessing a variety of biological activities at nontoxic concentration. Dietary phytochemicals, mainly flavonoids hinder cell signalling responsible for multidrug resistance and cancer progression, primarily targeting cancer cells sparing normal cells. Such properties establish flavonoids as a potential candidate for synergistic therapy. However, due to low absorption and high metabolic rates, the bioavailability of flavonoids becomes a challenge. Such challenges may be overcome using novel approaches like derivatization and single or co-delivery nano-complexes of flavonoids with conventional drugs. It also highlights the application of flavonoids as a potential anticancer phytochemical class in combination with known anti-cancer drugs/nanoparticles (Hussain et al., 2020) [8] . The flavonoids have been shown to possess a wide variety of anticancer effects: they modulate reactive oxygen species (ROS)scavenging enzyme activities, participate in arresting the cell cycle, induce apoptosis, autophagy, and suppress cancer cell proliferation and invasiveness (Kopustinskiene et al., 2020) [12] . The biological potential of flavonoids, added to their bioavailability, cost-effectiveness, and minimal side effects, direct them as promising cytotoxic anticancer compounds in the optimization of therapies and the search for new drugs in the treatment of cancer, since some extensively antineoplastic therapeutic approaches have become less effective due to tumour resistance to drugs commonly used in chemotherapy. (Luna et al., 2023) [16] . Among the phytochemicals, flavonoids are very effective in treating a wide range of diseases from cardiovascular diseases and immunological disorders to cancer. They scavenge reactive oxygen species (ROS), inhibit cancer metastasis, modulate the immune system and induce apoptotic or autophagic cell death in cancers. (Mir et al., 2024) [18] . Cytotoxic effect of saponins was due to their ability to stimulate apoptotic process in tumour cells, usually through its intrinsic pathway. Moreover, non-apoptotic processes were also involved in saponin cytotoxic activity, such as cell cycle arrestment, autophagic cell death stimulation, inhibiting of metastasis and cytoskeleton disintegration (Podolak et al., 2010) [22] .

In vitro study with MTT assay

The in vitro cytotoxic activity of the crude extracts was determined from the mitochondrial activity of cell lines which represent the number of viable cells after the treatment, by using the MTT cytotoxic assay (Kaewpiboon et al., 2012) [10] . In the present in-vitro MTT assay, the polyherbal extract composed of A. muricata leaves, A. indica leaves, A. marmelos leaves, and N. sativa seeds exhibited an IC₅₀ value of 358 µg/mL against MCF-7 human breast cancer cell lines. These findings were in accordance with earlier reports of Gavamukulya et al. (2014) [6] who reported the ethanolic extract of A. muricata leaves extract had IC₅₀ value of 335 µg/mL against MDA cell lines. They also reported that an integrated part of cancer cell development is the resistance to programmed cell death (apoptosis) and therefore re-establishment of apoptosis in cancer cells is a target mechanism for anticancer agents. Some plant derived products are known to selectively induce apoptosis in cancer cells, which represent the ideal property for successful anticancer agents. The current study showed the highly effective action of the ethanolic leaves extract of A. muricata and can be used in the management and treatment of cancer. This is in line with a study which showed that any extract had anticancer and cytotoxic activity if it had an IC50 value less than 1 000 µg/mL after 24 h contact time, and that the smaller the IC50 value of a test compound, the more toxic the compound was. Sharma et al., (2014) [25] reported the antiproliferative effects of different concentrations of ethanolic neem leaf extract on MCF-7 cells, HeLa cells, and lymphocytes by MTT assay. MCF-7and HeLa cells treated with increasing concentrations of ethanolic neem leaf extract ranging from 10 to 500 𝜇g/mL showed a dose-and time dependent increase in cell death. In MCF-7 cells, the EC 50 (Effective concentration) was observed at 350 𝜇g/mL after 72 h treatment with ethanolic neem leaf extract, whereas in HeLa cells, it was found to be 175 𝜇g/mL in 48h. Mohammed et al. (2019) [21] reported that the treatment with different concentration of A. muricata leaves extract inhibited the proliferation of MCF-7 cells in concentration dependent manner.

In the present study, the significant morphological changes were characterized by reduced cell density, rounded up cells, loses of contact with neighbouring cells and cell shrinkage indicating apoptosis in MCF-7 cell lines which is in accordance with Sharma et al. (2014) [25] who reported similar changes along with detachment from the substrate while studying the effect of ethanolic neem leaf extract in MCF-7 cell lines. The results were also in strong agreement with Kumar et al. (2023) [14] who observed similar morphological changes and stated that Aegle marmelos inhibited the proliferation of MCF-7 cells, changed the cytoplasmic morphology, reduced the invasion and migration ability of MCF-7 cells and exhibited proapoptotic effects. Fathima et al. (2023) [5] reported that the cells undergoing apoptosis displayed morphological changes such as rounded up cells that shrink and lose contact with neighbouring cells. Some sensitive cells detached from the surface of the plates in A. marmelos treated osteosarcoma cell lines. The results were also in strong agreement with Lampronti et al. (2003) [15] who observed A. marmelos treated human tumour cells showed cell shrinkage and subsequently lost contact with neighbouring cells.

Conclusion

Overall, the findings support the potential of the polyherbal extract (Leaves of Annona muricata, Azadirachta indica and Aegle marmelos and seeds of Nigella sativa) as a promising natural anticancer candidate for mammary cancer therapy. Further studies involving apoptosis assays, cell cycle analysis, gene expression profiling, and animal models are warranted to confirm its molecular mechanisms and therapeutic efficacy.

References

1. Ahmad A, Husain A, Mujeeb M, Ali Siddiqui N. Physicochemical and phytochemical standardization with HPTLC fingerprinting of Nigella sativa L. seeds. Pak J Pharm Sci. 2014;27(5):1175-1182.
2. Aljabali AAA, Obeid MA, Bashatwah RM, Qnais E, Gammoh O, Alqudah A, et al. Phytochemicals in cancer therapy: a structured review of mechanisms, challenges, and progress in personalized treatment. Chem Biodivers. 2025;22(8):2025000.
3. Arumugam A, Agullo P, Boopalan T, Nandy S, Lopez R, Gutierrez C, et al. Neem leaf extract inhibits mammary carcinogenesis by altering cell proliferation, apoptosis, and angiogenesis. Cancer Biol Ther. 2014;15(1):26-34.
4. Daud NMNN, Ya'akob H, Mohamad Rosdi MN. Acetogenins of Annona muricata leaves: characterization and potential anticancer study. Integr Cancer Sci Ther. 2016;3(4):1-6.
5. Fathima NJ, Thangavelu L, Jeevitha R, Perumal E. in vitro antiproliferative and proapoptotic effect of Aegle marmelos fruit extract on osteosarcoma cell line. J Surv Fish Sci. 2023;10(1S):337-347.
6. Gavamukulya Y, Abou-Elella F, Wamunyokoli F, AElShemy H. Phytochemical screening, antioxidant activity and in vitro anticancer potential of ethanolic and water leaf extracts of Annona muricata (graviola). Asian Pac J Trop Med. 2014;7(Suppl 1):S355-S363.
7. Hasan AAZ, Bermawie N, Julistiono H, Riyanti EI, Hasim H, Suhender U. Phytochemical screening and anti-breast cancer activities of Annona muricata (L.) leaf extracts. Pharmacologyonline. 2020;2:23-30.
8. Hussain Y, Luqman S, Meena A. Research progress in flavonoids as potential anticancer drugs including synergy with other approaches. Curr Top Med Chem. 2020;20(20):1791-1809.
9. Jagetia GC, Venkatesh P, Baliga MS. Aegle marmelos (L.) Correa inhibits the proliferation of transplanted Ehrlich ascites carcinoma in mice. Biol Pharm Bull. 2005;28:58-64.
10. Kaewpiboon C, Lirdprapamongkol K, Srisomsap C, Winayanuwattikun P, Yongvanich T, Puwaprisirisan P, et al. Studies of the in vitro cytotoxic, antioxidant, lipase inhibitory and antimicrobial activities of selected Thai medicinal plants. BMC Complement Altern Med. 2012;12:217.
11. Khanal S. Qualitative and quantitative phytochemical screening of Azadirachta indica Juss. plant parts. Int J Appl Sci Biotechnol. 2021;9(2):122-127.
12. Kopustinskiene DM, Jakstas V, Savickas A, Bernatoniene J. Flavonoids as anticancer agents. Nutrients. 2020;12(2):1-24.
13. Kumar A, Gautam B, Dubey C, Tripathi PK. A review: role of doxorubicin in treatment of cancer. Int J Pharm Sci Res. 2014;5:4117-4128.
14. Kumar NB, Thangavelu L, Jeevitha R, Perumal E. Evaluation of pro-apoptotic effect of Aegle marmelos fruit extract on breast cancer cell lines. J Surv Fish Sci. 2023;10(1):518-529.
15. Lampronti I. in vitro anti-proliferative effects on human tumour cell lines of extracts from the Bangladeshi medicinal plant Aegle marmelos Correa. Phytomedicine. 2003;10:300-308.
16. Luna FCFD, Ferreira WAS, Casseb SMM, de Oliveira EHC. Anticancer potential of flavonoids: an overview International Journal of Advanced Biochemistry Research https://www.biochemjournal.com with an emphasis on tangeretin. Pharmaceuticals (Basel). 2023;16(9):1-29.
17. Bordeaux (France): University of Bordeaux II; 2005. p. 1-128.
18. Mir SA, Dar A, Hamid L, Nisar N, Malik JA, Ali T, et al. Flavonoids as promising molecules in cancer therapy: an insight. Curr Res Pharmacol Drug Discov. 2024;6:1-14.
19. Moe L. Population-based incidence of mammary tumors in some dog breeds. J Reprod Fertil Suppl. 2001;57:439-443.
20. Moghadamtousi SZ, Fadaeinasab M, Nikzad S, Mohan G, Ali HM, Kadir HA. Annona muricata (Annonaceae): a review of its traditional uses, isolated acetogenins and biological activities. Int J Mol Sci. 2015;16(7):1562515658.
21. Mohammed RMH, Noshy MM, Mohamed HRH. Inhibition of human breast cancer cell proliferation by graviola crude extract. Recent Res Genet Genomics. 2019;1(1):1-7.
22. Podolak I, Galanty A, Sobolewska D. Saponins as cytotoxic agents: a review. Phytochem Rev. 2010;9(3):425-474.
23. Ravi K, Bharavi K, Ravi Kumar P, Vamsi Krishna B. Phytochemical screening and antibacterial activity of ethanol extract of leaves and twigs of Azadirachta indica A. Juss. Pharm Biosci J. 2015;:56-59.
24. Salsabila IA, Nugraheni N, Ahlina FN, Haryanti S, Meiyanto E. Synergistic cotreatment potential of soursop (Annona muricata L.) leaf extract with doxorubicin on 4T1 cells with antisenescence and antireactive-oxygen-species properties. Iran J Pharm Res. 2021;20(2):57-67.
25. Sharma C, Vas AJ, Goala P, Gheewala TM, Rizvi TA, Hussain A. Ethanolic neem (Azadirachta indica) leaf extract prevents growth of MCF-7 and HeLa cells and potentiates the therapeutic index of cisplatin. J Oncol. 2014;2014:1-12.
26. Tinoco G, Warsch S, Gluck S, Avancha K, Montero AJ. Treating breast cancer in the 21st century: emerging biological therapies. J Cancer. 2013;4(2):117-132.
27. Ullah MF. Breast cancer: current perspectives on the disease status. Adv Exp Med Biol. 2019;1152:51-64.
28. Umadevi KJ, Varadharajan V, Krishnamurthy V. Physicochemical evaluation, phytochemical screening and chromatographic fingerprint profile of Aegle marmelos (L.) leaf extracts. n.d. 2012.
29. Veer B, Singh R. Phytochemical screening and antioxidant activities of Aegle marmelos leaves. Anal Chem Lett. 2019;9(4):478-485.