INTRODUCTION:

Cancer is a disease characterized by uncontrolled cell division and the potential to invade surrounding tissues. It can originate in any part of the body's trillions of cells. When apoptosis, the natural process of cell death, malfunctions, damaged or abnormal cells can proliferate uncontrollably, forming tumors. These tumors can be either benign (non-cancerous) or malignant (cancerous), with malignant tumors having the ability to metastasize, or spread to other parts of the body.

While cancers like leukemia do not typically form solid tumors, many other types do. Unlike benign tumors, malignant tumors can recur after removal. Though benign tumors usually do not recur, some, like benign brain tumors, can cause severe or life-threatening symptoms. Breast cancer ranks as the second-leading cause of cancer-related deaths globally, with a disproportionate number occurring in developing countries.

MATERIALS AND METHODS:

Chemicals:

DMBA was obtained from Alfa Aesar Thermo Fisher Scientific India Pvt Ltd. (Powai), Corn oil from Eywe seed and exports, Jamnagar. All other chemicals employed in the study were of analytical grade. Laboratory animals: Wistar female rats weighing 160 to 200 g were used in the investigation. At the Appasaheb Birnale College in Sangli, Female wistar rats were obtained for experimentation. 30 rats were divided into five groups of six rats each. Approval for animal study has been obtained from IAEC of ABCP Sangli and IAEC approval number is IAEC/ABCP/03/2022-23

PLANT MATERIAL PROCESSING:

Collection And Authentication of Seeds:

Seeds of V. aconitifolia were collected and authenticated by Dr. M.D. Wadmare, Asso. Prof. and Head, Department of Botany, Kasturbai walchand college, Sangli.

Processing and Extraction:

The dried seed of V.aconitifolia were ground to a coarse consistency. Using the Soxhlet apparatus, a weighed amount of powder was extracted with acetone. The extract was air dried at room temperature till yellow semisolid mass was formed and stored in a desiccator.

TOXICITY STUDIES:

Referred OECD guideline no. 423 to perform acute oral toxicity.

Dose selection:

From acute toxicity study. 2000mg/kg, 200mg/kg body weight of acetone extract of seeds was chosen.

In vitro Cytotoxicity Study:

1) Brine Shrimp Lethality Assay:

Brine Shrimp assay was performed.³: The LC 50 values were estimated using dose-response data that had been transformed into a straight line using trend line fit linear regression analysis.

Number of dead nauplii

% Death = ----------------------------------------------- × 100

Number of dead naupalii + Number of live naupalii

2) MTT Assay:

MTT assay was performed. Following formula was used to calculate the IC50 value.⁵

Cytotoxicity formula:

(Control OD) - (test OD)

% Cytotoxicity = ------------------------------- × 100

(Control OD)

In vivo Cytotoxicity Study:

Preparation of chemical inducer:

DMBA was dissolved in corn oil. The concentration was then adjusted to 8mg/100g body wt./rat.

Experimental Design:

In this experimental study, mammary carcinoma was induced in female Wistar Albino ratsby IG route of DMBA (80mg/kg). The animals were weighed weekly and palpated in order to record the numbers, location, and size of tumours also blood was collected weekly by retroorbital route for evaluation of haematological parameters. After the completion of study animals were euthanized, livers and tumours from each animal were isolated and evaluated for antioxidant activity and tumorological parameters respectively.^4,7

Study Design:

Group I (Vehicle control): Rats were administered corn oil thrice a week for 12 weeks via Oral route.

Group II (Drug control): 200mg/kg dose of extract alone was given thrice a week for 12 weeks via oral route.

Group III (DMBA control): Animals were administered with single dose of 0.5 ml DMBA (80 mg/kg) in corn oil during week 3, via intragastric route and corn oil for 4-12 weeks.

Group IV(Pre-treatment): Animals were subjected to pre-treatment with 200mg/kg dose of extract thrice week from 1 to 12 weeks and DMBA (80mg/kg) in corn oil was given in week 3.

Group V (Post-treatment): Animals were administered with single dose of 0.5ml DMBA (80mg/kg) in corn oil during week 3. From week 8 to 12 animals were treated with 200mg/kg dose of extract via oral route

Evaluating Parameters:

Various parameters recorded in the In-Vivo anticancer study were described below.

A. Change in the body weight:

Animals were weighed weekly

B. Evaluation of Tumour Parameter:

i. Tumour yield:

Tumour yield is defined as average number of tumours per Rats.

ii. Tumour volume:

The tumour volume was measured by the formula

Tumor Volume = l × w²

iii. Tumour burden:

Tumour burden was calculated by multiplying tumour volume and the number of tumours/Rat.

iv. Tumour size:

The diameter of each tumour was measured at the end of experiment by using vernier calliper.

v. Tumour mass:

The weight of the tumour of each animal at the end of the experiment was measured.

C. Haematological Parameters:

The effect of test drug on haematological parameters of animal was examined by evaluating following parameters.

Haemoglobin (Hb) Estimated by using Sahli’s Method,

red blood cells (RBC):

Estimated by using haemocytometer,

White blood cells (WBC):

Estimated by using haemocytometer.

Differential leukocyte Count (DLC):

Estimated by using haemocytometer.

D. Evaluation of Tissue Antioxidant Biomarkers:

Estimation of superoxidedismutase (SOD) activity in liver is done by method of Marklund et.al, estimation of reduced glutathione (GSH) activity in liver is done by method of Ellman et.al

E. Liver Biomarker: Serum glutamic oxaloacetic transaminase (SGOT) Estimated by modified IFCC methodSerum glutamic Pyruvic transaminase (SGPT) Estimeted by modified IFCC method

F. Histopathological Study:

Histopathological slides were prepared and evaluated.

Statistical analysis:

Values are expressed as ±SEM for six rats in each group,statistical test was performed using one way ANOVA Followed by Dunnett’s test(Graph Pad Instant 8.00,USA pc0.05)was taken as the criterion of statistical significance

In vitro Cytotoxicity Studies:

Figure No. 01: Calculation of LC50 of Vigna Aconitifolia Seed extract

Brine Shrimp Lethality Assay:⁶

Table No.01: Brine Shrimp Lethality Assay

Sr. No.	Concentration	No. of shrimp dead out of 10			Total No. of shrimp dead out of 30	% Lethality	LC50 (µg/ml)
Sr. No.	Concentration	T1	T2	T3	Total No. of shrimp dead out of 30	% Lethality	LC50 (µg/ml)
1.	3	1	1	2	4	13.34	35.96
2.	6	3	2	3	8	26.66
3.	12	4	4	2	10	33.33
4.	25	3	4	4	11	36.66
5.	50	7	7	8	22	73.33
6.	100	10	10	10	30	100
7.	200	10	10	10	30	100

MTT Assay:

Table No.02: MTT Assay^5,11,12

Sr. No	Sample	Concentration (µl)	OD	Mean	% Inhibition	LC50
1	Control		1.340 1.236 1.272	1.282
2	Std. 5 FU	10	0.421 0.456 0.412	0.429	66.53	39.22
		40	0.312 0.352 0.361	0.341	73.40
		100	0.287 0.256 0.246	0.263	79.48
5	Vigna Aconitifolia	10	0.745 0.653 0.723	0.707	44.85	48.05
		40	0.659 0.654 0.685	0.666	48.04
		100	0.465 0.554 0.565	0.528	58.81

In vivo Cytotoxicity Studies

Physical Parameter (Body Weight in Gram)

Table No.: 03. Effect Vigna aconitifolia Extract on Weights of animals in comparison with Vehicle Control Group

Vehicle Control

DMBA Control

Drug Control

Pretreatment

Post treatment

Body weight

(gm)

165.7361±

1.4090

142.4861±

3.5787****

174.4306±

2.6497

149.4722±

4.7289***

147.4583±

2.4070***

Values are expressed as (Mean±SEM ) n=6 *P<0.05 Statistically significant when compared with vehicle control group by One way ANOVA followed by Dunnett's multiple comparisons test.

Table No.04: Effect Vigna aconitifolia Extract on Body Weights of animals in comparison with DMBA Control Group-

Vehicle Control

DMBA Control

Drug Control

Pretreatment

Post treatment

Body weight

(gm)

165.7361±

1.4090****

142.4861±

3.5787

174.4306±

2.6497****

149.4722±

4.7289*

147.4583±

2.4070*

Values are expressed as (Mean±SEM ) n=6 *P<0.05 Statistically significant when compared with DMBA control group by One way ANOVA followed by Dunnett's multiple comparisons test.

TUMOR PARAMETER:

Table No.05: Effect V. aconitifolia Extract on Tumor parameters of the animals.

Parameter	DMBA Control	Pretreatment	Posttreatment
Tumor Incidence (%)	100	100	100
Tumor Volume(cm³)	4.4265± 0.331815	1.035167±70.21028****	2.6745±0.458659**
Tumor Burden (%)	6.19±0.013944	1.19±0.002733****	2.66±0.005247****
Tumor Mass (gm)	5.7±0.444222	2.25±0.257876****	4.4±0.206559*
Tumor Yield	1.666667±0.210819	1.25±0.170783*	1.466667±0.224098*
Tumor Size(cm)	5.3±0.505688	1.676667±0.117804****	2.966667±0.172079****

Values are expressed as (Mean±SEM ) n=6 *P<0.05 Statistically significant when compared with DMBA control group by One way ANOVA followed by Dunnett's multiple comparisons test.

Mammary tumors isolated from various groups of female rats

Group Name	Tumor isolated from the DMBA control group	Tumor isolated from the pretreatment group	Tumor isolated from the posttreatment group
Tumor isolated

Haematological Parameter:

Table No.06: Effect Vigna aconitifolia Extract on Haematological parameters of animals in comparison with Vehicle Control Group-

Haematological Parameters

Sr No

Parameter

Vehicle Control

DMBA Control

Drug Control

Pretreatment

Posttreatment

Hb(gm/dl)

13.27222±

0.13095

11.58056±

0.055325****

13.44167±

0.122877

12.34722±

0.121872****

11.20833±

0.143454****

RBC

(Millions/cumm)

7.802778±

0.095465

6.116667±

0.074911****

7.869444±

0.130743

6.716667±

0.097257****

6.438889±

0.117753****

WBC

(Thousand/ cumm)

7608.889±

101.5566

5577.278±

62.075****

7558.806±

57.70821

6299.778±

1106.728***

5986.5±

56.640***

Lymphocytes (%)

60±1.247219

41.57143±

0.996024****

62±

1.374369

54±

1.374369**

47±

0.527046***

Neutrophils (%)

19.57143±

1.868706

10±1*

19.71429±

2.064438

15.85714±

1.657115*

12±

0.912871*

Values are expressed as (Mean±SEM ) n=6 *P<0.05 Statistically significant when compared with vehicle control group by One way ANOVA followed by Dunnett's multiple comparisons test.

Table No.07: Effect Vigna aconitifolia Extract on Haematological parameters of animals in comparison with DMBA Control Group

Haematological Parameters

Sr No

Parameter

Vehicle Control

DMBA Control

Drug Control

Pretreatment

Post treatment

Hb(gm/dl)

13.27222±

0.13095****

11.58056±

0.055325

13.44167±

0.122877****

12.34722±

0.121872**

11.20833±

0.143454*

RBC

(millions/cumm)

7.802778±

0.095465****

6.116667±

0.074911

7.869444±

0.130743****

6.716667±

0.097257**

6.438889±

0.117753*

WBC

(thousand/cumm)

7608.889±

101.5566****

5577.278±

62.075

7558.806±

57.70821***

6299.778±

1106.728***

5986.5±

56.6407*

Lymphocytes (%)

60±

1.247219****

41.57143±

0.996024

62±

1.374369****

54±1.374369****

47±

0.527046**

Neutrophils (%)

19.57143±

1.868706****

10±

19.71429±

2.064438**

15.85714±

1.657115**

12±0.912871*

Values are expressed as (Mean±SEM) n=6 *P<0.05 Statistically significant when compared with DMBA control group by One way ANOVA followed by Dunnett's multiple comparisons test.

LIVER BIOMARKER:

Table No.08: Effect Vigna aconitifolia Extract on Liver biomarkers of the animals in comparison with the Vehicle Control group-

Sr no

Parameter

Vehicle Control

DMBA Control

Drug Control

Pretreatment

Posttreatment

SGOT

31.5±

1.176152

80.33333±

2.155097****

29±

0.966092

48.33333±

2.951459****

67.83333±

2.151227****

SGPT

31.66667±

0.557773

60.5±

1.688194****

30.16667±

0.703167

39.16667±

1.621042**

50±

2.294922****

Values are expressed as (Mean±SEM) n=6 *P<0.05 Statistically significant when compared with vehicle control group by One way ANOVA followed by Dunnett's multiple comparisons test

Table No.09: Effect Aconitifolia Extract on Liver biomarkers of the animals in comparison with the DMBA Control group-

Sr no

Parameter

Vehicle Control

DMBA Control

Drug Control

Pretreatment

Posttreatment

SGOT

31.5±

1.176152****

80.33333±

2.155097

29±

0.966092****

48.33333±

2.951459****

67.83333±

2.151227***

SGPT

31.66667±

0.557773****

60.5±

1.688194

30.16667±

0.703167****

39.16667±

1.621042****

50±

2.294922****

Values are expressed as (Mean±SEM) n=6 *P<0.05 Statistically significant when compared with DMBA control group by One way ANOVA followed by Dunnett's multiple comparisons test

TISSUE ANTIOXIDANT BIOMARKER:

Table No.10: Effect Aconitifolia Extract on Tissue antioxidant biomarkers of the animals in comparison with the Vehicle Control group-

Sr. no

Parametr

Vehicle control

DMBA Control

Drug Control

Pre treatment

Post treatment

SOD

6.366667±

0.120185

3.066667±

0.054263****

6.316667±

0.151474

5.133333±

0.105409****

4.171667±

0.165336****

GSH

5.983333±

0.11949

3.076667±

0.178245****

5.7783333±

0.116667

5.416667±

0.177795***

4.6±

0.152753**

Valus are expressed as (Mean±SEM) n=6 *P<0.05 Statistically significant when compared with vehicle control group by One way ANOVA followed by Dunnett's multiple comparisons test.

Table No. 11: Effect Aconitifolia Extract on Tissue antioxidant biomarkers of the animals in comparison with the Vehicle Control group

Sr. no

Parametr

Vehicle control

DMBA Control

Drug Control

Pre treatment

Post treatment

SOD

6.366667±

0.120185****

3.066667±

0.054263

6.316667±

0.151474****

5.133333±

0.105409****

4.171667±

0.165336****

GSH

5.983333±

0.11949****

3.076667±

0.178245

5.7783333±

0.116667****

5.416667±

0.177795****

4.6±

0.152753****

Values are expressed as (Mean±SEM) n=6 *P<0.05 Statistically significant when compared with DMBA control group by One way ANOVA followed by Dunnett's multiple comparisons test.

HISTOPATHOLOGICAL STUDY:

It illustrates the histological examination of rat breast tissue samples from the experimental and control groups. Animal breast tissue sections with cancer have proliferative lesions and lobular alveolar hyperplasia. On the other hand, the group that received DMBA showed pericentral necrosis, and pleomorphism. Animals pre-treated with V. aconitifolia showed reduced lobular alveolar damage with nearly normal architecture, whereas animals post-treated with V.aconitifolia showed somewhat reduced lobular alveolar damage. Both the vehicle control and V.aconitifolia showed no histopathological alterations.

Sr. No.	Group Name	Slide observed
1.	Figure No.02: Histological structure of mammary tissue of rat (Vehicle control)
2.	Figure No.03: Histological structure of mammary tissue of rat (Drug control)
3.	Figure No.04: Histological structure of mammary tissue of rat (DMBA control)
4.	Figure No.05: Histological structure of mammary tissue of rat (Pretreatment)
5.	Figure No.06: Histological structure of mammary tissue of rat (Posttreatment)

DISCUSSION:

Breast cancer is a leading global health concern among women, with current treatments often facing limitations such as effectiveness and severe side effects. Consequently, there is a growing interest in exploring natural plant-based alternatives for cancer treatment. One such plant, Vigna aconitifolia (Matki), from the Fabaceae family, has been noted for its rich phytochemical composition, including alkaloids, phenols, flavonoids, phytic acids, trypsin inhibitors, and vicilins. These compounds have shown potential anticancer properties in preliminary studies but require further scientific evaluation.In this study, the acetone extract of V. aconitifolia seeds was evaluated for its anticancer activity through both in vitro and in vivo methods. Initial acute toxicity studies in rats, conducted according to OECD guidelines, confirmed the safety of the extract at a dose significantly lower than the lethal dose (LD50 determined as 2000 mg/kg).In vitro cytotoxicity studies utilized the brine shrimp lethality test and MTT assay. The brine shrimp test indicated an LC50 of 35.96 µg/ml, suggesting significant cytotoxic effects against brine shrimp nauplii. The MTT assay on MCF-7 cell cultures revealed an IC50 value of 48.05 µg/ml, indicating the extract's ability to inhibit cancer cell growth by assessing mitochondrial activity. Further evaluation included an in vivo study using a DMBA-induced mammary cancer model in rats, a model known to closely mimic human breast cancer. Rats pretreated with V. aconitifolia seed extract demonstrated reduced tumor size, volume, burden, and mass compared to the control group. Additionally, the extract appeared to help maintain or improve hemato-biochemical parameters such as Hb, RBC, WBC, SGOT, and SGPT levels, indicating potential protective effects on liver function and blood parameters.These observed effects are attributed to the presence of flavonoids, phenols, and other antioxidant compounds in the V. aconitifolia seed extract. These findings highlight the plant's potential as a natural source for developing new anticancer therapies or complementary treatments for breast cancer. Further research is warranted to fully elucidate its mechanisms of action and therapeutic potential.

In-vivo study:

DMBA-induced mammary cancer in rats closely mimics the histogenesis, morphology, and biochemical features of human breast cancer. DMBA, metabolically activated by cytochrome P450 enzymes to DMBA-DE, initiates mammary cancer by inducing DNA damage, free radical production, mutations, and NF-κB activation, leading to tumorigenesis. Wistar rats were orally administered DMBA (80 mg/kg dissolved in corn oil), resulting in 100% incidence of breast cancer within 8–10 weeks. The study included DMBA control, pre-treatment, and post-treatment groups monitored for tumor development until palpable tumors emerged, marked by physical symptoms like weight loss, dermatitis, and respiratory issues in the DMBA control group. Pre-treatment with Vigna aconitifolia seed extract significantly reduced body weight loss observed from week 4 onward compared to the DMBA control group, with a minor increase noted in later weeks. The extract's fatty acids (Linoleic acid, Linolenic acid, Oleeic acid, Palmitic acid, Palmitoleic acid) likely contributed to enhanced body weight in the pre-treatment group.At the end of the 12-week study period, all groups exhibited 100% tumor incidence, with significantly reduced tumor size, volume, burden, and mass observed in the pre-treatment group compared to the DMBA control group. Hematological parameters (Hb, RBC, WBC) were improved in the pre-treatment group, indicating mitigation of myelosuppression and anemia associated with cancer progression. Liver biomarkers (SGPT, SGOT) were elevated in the DMBA control group but significantly reduced in the pre-treatment group, indicating potential hepatoprotective effects of the extract.Antioxidant enzymes SOD and GSH levels were low in the DMBA control group but markedly increased in the pre-treatment group, attributed to flavonoids and phenolic compounds present in Vigna aconitifolia seed extract. GC-MS-MS analysis confirmed the presence of antioxidant components, reinforcing the extract's ability to protect breast tissue from oxidative damage and inhibit tumor formation. In conclusion, Vigna aconitifolia seed extract shows promising anticancer and antioxidant activities in DMBA-induced mammary cancer, suggesting its potential as a natural therapeutic agent for breast cancer treatment. Further research is necessary to elucidate its mechanisms and clinical applications.

CONCLUSION:

The current study shows that DMBA administration was linked to breast carcinoma growth, elevated levels of tumorigenicity, and elevated levels of oxidative stress indicators. Administration of Vigna aconitifolia assisted in lowering levels of oxidative stress and tumorigenicity. The findings clearly shows that Vigna aconitifolia is able to protect breast tissue from oxidative damage and also prevents tumor formation. Overall, the results of investigation concludes that Vigna aconitifoliaseed acetone extract ability to prevent DMBA-induced breast cancer in rats.

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Received on 03.10.2023 Revised on 15.04.2024

Accepted on 02.09.2024 Published on 24.12.2024

Available online from December 27, 2024

Research J. Pharmacy and Technology. 2024;17(12):5809-5815.

DOI: 10.52711/0974-360X.2024.00883