Study place and duration

This study was conducted after institutional ethical approval for six months at the Liaquat University of Medical and Health Sciences, Jamshoro in Pakistan in collaboration with Sindh Agricultural University Tando Jam Animal House and the Medical Research Center Jamshoro, Pakistan.

Animals: For this study, 60 adult female Wistar albino rats were purchased from the National Institute of Health, Karachi.  Healthy female rats weighing between 165gms to 175gms were included in this study.

Experimental design: Animals were handled according to protocol of animal’s care provided by guidelines of National Institute of Health Pakistan (NIH). Stainless steel cages were used for housing animals with 20 mm thick rice husk bedding in each cage; well ventilated and had a 12-hour light-dark cycle alternativelywith temperature 22±2 degrees Celsius, and relative humidity of 55±10% [7]. The environment of the cages and the surroundings were kept properly hygienic and adequately ventilated. Each cage contained four animals. The food and water were accessible to the animals to ad libitum. The standard chow was used as feed and tap water to drink.  Animals were divided in five equal groups, each group comprised of 12 animals, housed in separate cages. All animals were weighed and a blood sample was taken before, during and after experiment [8].

Animal Groups and Treatments:

Group A: PC (Positive control) – Control experiment with normal diet and water.

Group B: NC (Negative control) – DMBA induced rats only not treated.

Group C: TR1– DMBA Induced rats were treated with tamoxifen.

Group D: TR2– DMBA induced rats were treated with 4 β-Hydroxy withanolide E

Group E: TR3– DMBA induced rats were treated with tamoxifen and 4 β-Hydroxy withanolide E.

Plant Material and Extract Preparation: Physalis peruviana was obtained from Botanical store Karachi, Pakistan, certified by the Department of Botany of Sindh University Jamshoro. One hundred grams of Physalis peruviana was diluted in 1 liter boiling water. Filtered extract under the filter paper and dried for 3 hours and stored at -21°C until use.

Tumor Induction: DMBA (Dimethylbenz(a)Anthracene) is a yellowish green, odorless chemical derivative of phenanthrene; an immunosuppressant and a potent laboratory carcinogen purchased from Sigma Aldrich.

Soy oil:  purchased from the Departmental store.

A single dose of DMBA (20 mg) in soy oil (5 ml) given by intra-gastric gavage is sufficient to induce tumor in the mammary gland. Each rat was weighed and examined for tumor in the mammary gland by gross inspection, palpation weekly. The tumor was confirmed by biopsy on the 6th week after an administration of DMBA [9].

Physalis Peruviana administration: Extracted dried Physalis peruviana 100 mg/kg/week administered with diet.

Tamoxifen administration:  Tamoxifen (Sigma‐Aldrich) dissolved in soy oil at a concentration of 20 mg/ml by shaking overnight at 37°C.75 mg tamoxifen/kg body weight via intraperitoneal injection was administered.

Blood sampling:  The blood samples were collected before tumor induction and at weeks 1, 5, 7, and 10 after anesthetizing rats with ketamine injection. The blood (1ml) was collected by retro-orbital technique via the orbital sinus in heparinized micro-crit capillary tubes and transferred to the vacutainer blood collection tube [10].

Statistical analysis:

The data were analyzed using SPSS version 26.0. Numerical parameters like weight, hemoglobin, platelets, red blood cells, and white blood cells were presented as mean ± SD and one-way analysis of Variance (ANOVA) and T-test were applied to compare the means between the groups. P-value < 0.05 was considered as a statistically significant level for all comparisons.

We assessed the difference in body weight measurement (table 1) and hematological parameters; Hemoglobin (Hb), Red Blood Cells (RBCs), White Blood Cells (WBCs) and platelets with Mind-ray hematology analyzer – BC-2300 (table 2).

Weight measurement

Mean of the weight was compared among the five groups by applying one-way ANOVA, and there was a significant increase in the weight of rats of Group E versus Group B (p-value = 0.0001). Negative Control showed massive weight loss and worsening of all hematological parameters like pancytopenia, anemia and leucopenia. Six rats died during the 10th week because of the severity of the illness.

Hematological parameters

By applying the ANOVA test, significant decrease in RBC and hemoglobin in both B and C Groups (P-value = 0.0001) was observed. There was a significant increase in RBCs and hemoglobin in Group E.WBC and platelets have increased to normal levels after treatment with Physalis peruviana. We found a significant decrease in WBC and Platelets in Group B and Group D (P-value = 0.0001). A significant increase in WBC and platelets in Group E (P-value = 0.0001) was also observed after 10 weeks. There were no major variations between Group A and Group E After treatment, Group E revealed gross improvement in weight and all hematological parameters.

Abbreviations: PC, positive control (Normal); NC, negative control (Induced with DMBA, no treatment) – Control experiment; TR1, DMBA induced rats treated with tamoxifen; TR2, DMBA induced with 4β-hydroxy withanolideE; TR3, DMBA induced with tamoxifen and 4β-hydroxy withanolide E

Figures & Tables

Discussion

The phytochemicals found in leaves, fruits, and other parts of several plants have therapeutic potential for treating various diseases including cancers [11]. The incidence of breast cancer is increasing in developing countries [12]. Several factors contribute to cancer development, including inflammation [13]. In breast tissue, DMBA causes DNA damage and promotes cell proliferation [14]. Vitamin C& E, polyphenols, carotenoid compounds, lutein, and lycopene are antioxidants present in many plant extracts, helps in tumor suppression [15].

The complete blood count reflects the response of cellular immunity in a patient with cancer. [16]. In this study, after induction with DMBA (carcinogen), blood sampling at weeks 1, 5, 7, and 10 revealed drastic variations among Group A (Positive Control) and Group B (Negative control (Induced with DMBA no treatment). We found significant improvement in all parameters of Group E (DMBA induced rats treated with tamoxifen and 4β-hydroxy withanolideE) compared to Group B (Negative control (Induced with DMBA no treatment).These observations are line with Akuru [17] who reported indices of DMBA administered albino rats, given aqueous concentrate of different plants. For RBC and Hb concentrations, there was a significant increase (p≤0.05) in all the treated groups when contrasted with the control and DMBA groups (p≤0.05) [17].

In our results, there was a significant decrease in RBC and hemoglobin in all four groups induced by DMBA compared to the Group A. This contrasts with Akuru et al, who showed contrary results and reported that there was an additional increase in the RBC level in all groups when compared with the control and DMBA untreated group, however not significant (p≤0.05)[17].

This study also suggests anemia in cancer with low Hb level and red blood cell count, which may predict the death among cancer patients [18,19]. There is a high incidence of anemia observed in cancer patients, which may be caused by bleeding, nutritional deficiencies, damage to the bone marrow, or tumor infiltration [20].

Different plants have been shown to possess anti-anemic properties. Zingue et al. observed ethanoic extracts of plants were useful in increasing multiple biochemical markers of DMBA-induced mammary tumors, including MCV, MCHC, MCH, RBC, PCV, and hemoglobin concentrations, compared to DMBA-untreated animals [21].

We observed enhanced WBC representation of lymphocytosis in all groups induced by DMBA. White Blood Cells have increased to normal levels after treatment with physalis peruviana and tamoxifen in group E (DMBA induced rats treated with tamoxifen and 4 β-Hydroxywithanolide E) versus Group C (DMBA induced rats treated with tamoxifen) and D (DMBA induced with 4β-hydroxy withanolideE).  This was similar to the study conducted by Akuru et al, who revealed the white blood cells of DMBA administered to albino rats given different plant extracts. There was a significant increase (p≤0.05) in the white blood cell level of animals treated with 1000mg/kg of a plant when compared with the control and the DMBA-untreated group. The DMBA group had the most reduced white blood cell level(not significant, p ≤ 0.05) [17]. When the WBC count is below the normal level, infection is a serious risk, whereas higher counts increase the chance of developing invasive breast cancer. This is also consistent with the findings of Chen et al. [22], who found that plant extracts could reduce the levels of WBCs, neutrophils, and lymphocytes in DMBA-induced mammary cancer. In a study, Akuru et al (2019) measured blood platelet concentrations of albino rats given DMBA treatment and an aqueous extract from different plants. The DMBA-untreated group showed a significant increase (p≤0.05) when compared to the control group and the DMBA STD group. The DMBA-untreated group had lower values than the various treated groups (although not significantly, p≤0.05). According to Rochet, Markovic [23] because platelets release a variety of growth factors and cytokines that promote angiogenesis, high platelet counts are associated with poorer prognosis for cancer, which is crucial to breast cancer metastasis [24]. This study reveals that this extract reduced blood platelets and hence angiogenesis, as evidenced by the lower values of platelet count. Similar is reported by Zingue et al [21], that ethanolic extracts of the plant reduced platelet concentration in tumors induced by DMBA compared to the group not exposed to DMBA.

This study concludes Physalis peruviana aqueous extract was found to enhance all hematological parameters of DMBA-induced breast cancer in albino rats. It increases the red blood cells and platelet count and stabilizes white blood cells in their function to reduce the anemic burden caused by cancer. It decreases the risk of thrombocytopenia. Compared with different adverse effects and contraindications of traditional drugs, cultural values, efficacy, low cost, and easy availability encourage the use of medicinal herbs. The use of Physalis peruviana in breast cancer treatment has significant potential, especially the adjunct to tamoxifen. To evaluate these effects, further research is required, particularly clinical studies.

Conflict of Interest

The authors declare that there is no conflict of interest.

Author Contributions

Fahmida Gul: Designed the experiment, data collection and analyzes the study and drafting of the manuscript. Corresponding author
Samreen Memon: Conceived the idea,was the in-charge of data collection, final approval of the manuscript.
Ikramuddin Ujjan; Brought new ideas, helped intechnical issues.
Pushpa Goswami:Helped indata collectionand drafting of manuscript.
Kanwal Abbas:Helped in statistics fordataanalysis and final drafting of manuscript