Evaluation of protective effects of Bacopa monnieri in Doxorubicin induced Testicular toxicity in rats

Ankitha K¹, Shivani Singh¹, Reena Sherin Parveen¹, Shalini Adiga¹, Mohandas Rao KG²,

Ravindra Maradi³, Aswatha Ram HN⁴, Shivaprakash Gangachannaiah¹*

¹Department of Pharmacology, Kasturba Medical College, Manipal,

Manipal Academy of Higher Education, Manipal, Karnataka, India – 576104.

²Department of Basic Medical Sciences,

Manipal Academy of Higher Education, Manipal - 576104, Karnataka, India.

³Department of Biochemistry, Kasturba Medical College,

Manipal, Manipal Academy of Higher Education, Manipal, Karnataka, India – 576104.

⁴Department of Pharmacognosy, Manipal College of Pharmaceutical Sciences, Manipal,

Manipal Academy of Higher Education, Manipal, Karnataka, India – 576104.

*Corresponding Author E-mail: shiva.g@manipal.edu

ABSTRACT:

Background Doxorubicin is widely used as chemotherapeutic agent in many cancers along with other anticancer drugs. However, its use is hampered by its adverse effects. The present study aimed to evaluate the protective effect of Bacopa monnieri in ameliorating Doxorubicin(Dox) induced testicular toxicity. Materials and Methods: Male rats (n=36) were distributed into 6 groups as normal control, Doxorubicin, Bacopa, and three test groups B75,B150 and B250. Normal control group received distilled water, Dox group was administered Doxorubicin at 5mg/kg on days 7, 14 and 21 and distilled water on all the days. Bacopa group was administered ethanolic extract of bacopa at 250mg/kg and test groups received bacopa extract at 75mg/kg,150 mg/kg and 250mg/kg daily along with Doxorubicin 5mg/kg on days 7,14 and 21. The duration of treatment was 21 days. Animals were sacrificed on day 22 and testis was dissected and collected for bio chemical and histological analysis. Results: The treatment groups (Bacopa, B75, B150, B250 mg/kg) have shown a significant increase in testicular weight as compared to the Dox group. There was increase in testicular index (TI) in bacopa compared to Dox group. The sperm count and motility was decreased in Dox group compared to control and bacopa group. Compared to Dox group, bacopa treated groups have shown significant increase in the sperm count and sperm motility. Malondialdehyde (MDA) levels significantly increased and antioxidants GSH and SOD levels decreased in Dox group, whereas MDA levels decreased and antioxidant levels increased in B75, B150, B250 groups compared to DOX group. However, the levels of antioxidants werelesser when compared to control and bacopa only group in B75, B150 and B250 groups. Histopathology of testis in bacopa treated groups demonstrated partial restoration of testis from Dox induced toxic effects. Conclusion: The study demonstrated the ability of Bacopa monnieriin ameliorating Doxorubicin induced testicular toxicity. Bacopa monnieri extract was able to partiallyrestore the testicular structure and function by its antioxidant activity when used concommitantly with Doxorubicin.

KEYWORDS: Doxorubicin, testicular toxicity, Bacopamonnieri.

INTRODUCTION:

Chemotherapy with doxorubicin (Dox) has been highly effective in the treatment of haematological and solid tumours. Its effective use in cancer chemotherapy is limited due to its toxicity. The major processes involved in doxorubicin cardiotoxicity and testicular toxicity involve oxidative stress, apoptosis, lipid peroxidation, apoptosis along with dysregulation of autophagy.

Increase in oxidative stress and the lipid peroxidation process cause enhanced production of reactive oxygen species, while autophagy guards the cell from stress stimuli or contributes to cell death alternatively^1,2. The cell nucleus being the primary site of action, Dox interacts with DNA and inhibits enzyme topoisomerase II. This leads to inhibition of DNA replication. Dox is also known to increase the production of quinone type free radicals which are mainly involved in exertion of its cytotoxic effects. Histone separation from active chromatin caused by Dox has shown to cause substantial DNA damage. These cytotoxic effects lead to significant adverse effects of Doxleading to cardiac dysfunction, nephrotoxic effect and testicular toxicity^3-5. Oxidative stress markers are the biological indicators that measures the level of oxidative damage and refer to an imbalance between reactive oxygen species (ROS) and antioxidants in the body, leading to potential damage to cells, proteins, and DNA⁶. Oxidative stress marker Malondialdehyde (MDA) (Lipid Peroxidation Marker) level indicate the extent of membrane lipid damage⁷. Antioxidant enzyme Superoxide Dismutase (SOD), catalyzes the dismutation of superoxide radicals into oxygen and hydrogen peroxide, Catalase converts hydrogen peroxide into water and oxygen, and Glutathione (GSH) is the major antioxidant that protects cells from ROS. These are the antioxidant defense markers whose activities can be altered in response to oxidative stress⁸.

Bacopa monnieri (B.monnieri.) being a medicinal plant from the family Scrophulariaceae has a common name known as Brahmi. It is an aquatic plant typically found in India and other Asian countries. B.monnieri has been used in Ayurvedic medicine since time immemorial, particularly in the treatment of medical conditions such as anxiety, cognitive deficit and deficient memory. The active elements of this plant consist mainly of alkaloids and saponin glycosides among which bacoside A is the main constituent⁹.

Earlier animal toxicity studies in rats have demonstrated the safety of B.monnier¹⁰. The effect of Brahmi has been demonstrated in various neurodegenerative disorders. The neuroprotective properties of this plant include reduction in neuroinflammation, reactive oxygen species (ROS) reduction, inhibition of aggregation of Amyloid-β and enhancement of cognitive and learning behaviour. Previous reports also shows that treatment with Brahmi extract caused betterment in sperm quality, steroidogenic indices, the spermatogenic cell density and reduction in the testis peroxidative damage^10,11.

B. monnieri was found to have many beneficial activities such as antioxidant, anti-inflammatory, hepatoprotective, anti-apoptotic, vasodilatory, calcium antagonistic and cardioprotective by increasing the rat coronary flow properties^12-15.

Though the previous reports suggest favourable testicular actions there are hardly any studies to prove its benefits in doxorubicin toxicity. Also, because it contains many beneficial alkaloids and flavonoids, has favourable actions on blood vessels, and its proven ability to increase antioxidant activity we have conducted this study with the aim to determine its biochemical and histological effects indoxorubicin induced testicular toxicity¹³.

MATERIALS AND METHODS:

Animal and Ethics approval:

The study was approved by Institute Animal Ethics Committee (IAEC/KMC/68/2021), MAHE, Manipal. A total of 36, healthy male albino rats of Sprague Dawley strain weighing 180-250gm and 10 – 12 weeks, old were procured from the “Central Animal Research Facility, of the institute. In accordance to Committee for the Purpose of Control and Supervision of Experimentation on Animal (CPCSEA) guidelines, the animals were housed in polypropylene sterile cages of size 41cm x 28cm x 14cm, with sterile paddy husk (procured locally) as bedding, 12hr alternate light and dark cycle in 25±3˚C temperature and 50% humidity and food pellets and water ad libitum was provided.

Drugs and chemicals:

Drugs Doxorubicin hydrochloride, Ketamine hydrochloride and Xylazine were purchased from pharmacy store. All other reagents used in the experiment were procured from sigma company and of analytical grade.

Bacopa monnieri extract:

The ethanolic extract of B.monnieriwas received as a gift sample containing a dried powder formulation which was greenish brown in color (Batch number RD/22017) from Natural Remedies Private Limited, VeersandraIndustrial Area, Electronic City Phase 2, Bangalore 560100 Karnataka. B.monnieriwas certified as the pure extract under the product name Bacomind extract.Phytochemical analysis showed to contain mainly Triterpene glycosides, BacopasideI, BacopasideII, Bacoside A_3,Jujubogenin isomer of Bacopasaponin C and Bacopasaponin C. The dried powder of B. monnieri was suspended in distilled water and administeredorally.

Experimental design:

A total of 36 rats equally distributed into six groups: Control group was administered distilled water; Dox group received Doxorubicin at 5mg/kg by IP route; Bacopa group received bacopa extract 250mg/kg orally; test groups received bacopa extract at 75mg/kg(B75), 150mg/kg(B150), 250mg/kg(B250) orally daily along with Doxorubicin at 5mg/kg by IP route on day 7, 14 and day 21. (Figure 1) All the groups were treated for 21 days. Bacopa monnieri extract was administered orally 1 hour before Dox. After 24 hours of last dose of treatment, body weight was checked. Rats were sacrificed under 125mg/kg of ketamine anaesthesia and 10mg/kg of xylazine by intraperitoneal route on day 22 and testis was dissected out for assessment.

Sperm count:

To create a suspension, the collected epididymis was first manually chopped and then homogenised in 1 ml of pH 7.2 PBS (phosphate buffer solution). Semen analysis was done using the suspension that was collected. A nylon mesh filter (80 m) was used to filter the suspension. The filtrate was used to conduct the sperm count using the Neubauer chamber in accordance with industry standards. In the leukocyte hemocytometer, an aliquot of the suspension was quickly drawn up to the 0.5 mark and diluted with PBS until the mark 11 was reached. After thoroughly mixing the suspension, it was charged into the Neubauer counting chamber. The total sperm count multiplied by 5x10⁴ to determine the number of spermatozoa in each epididymis in eight squares of 1 mm2 each (excluding the central erythrocyte area)¹⁶.

Sperm motility:

It was done by the method as described by Sonmezet al.(2005)¹⁷.Epididymis was macerated and mixed with 1 ml PBS. An aliquot of this solution was taken on the slide and the percentage of motility was evaluated microscopically at a magnification of 400 X.

Testicular index (TI):

Left testis was dissected out and weight was measured. Testis index was calculated by the following formula¹⁸.

Test is weight

TI= -------------------------- x 100

Bodyweight

Biochemical parameters:

The phosphate buffer with heart and testis tissue will be homogenised using glass homogenizer. The homogenate was centrifuged at 10,000 rpm for 10minutes and the pellet was discarded and the supernatant obtained was used to assay the following biochemical parameters.

Malondialdehyde (MDA):¹⁹

MDA is involved in breakdown of unsaturated fatty acids. Thiobarbituric acid and MDA reacts to give pink colour, this is quantified at 532nm.

Superoxide Dismutase (SOD):²⁰

SOD, an antioxidant enzyme catalyze the dismutation superoxide anions to hydrogen peroxide, which is then reduced to H₂O and oxygen. The ability of superoxide dismutase to inhibit the auto-oxidation of epinephrine to adrenochrome at pH 10.2 has been used as the basis for the assay of this enzyme.The absorbance was read at 480nm using spectrophotometer.

Glutathione (GSH):²¹

Glutathione reductase containing sulfhydryl group reduces 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) to yellow compound. The absorbance was measured at 412 nm.

Histopathology:

The histological examination of testiswas done using paraffin-embedded specimens. Qualitative analysis is performed. Sections were stained with haematoxylin and eosin. After Incubation, tissue sections were cooled and kept in xylene for thirty min to remove wax from it. Hydration of sections was done through alcohol series (90%, 70% and 50%). Distilled water is used for washing for five minutes and staining is done with haematoxylin. Sections washed in tap water for blueing. Staining with Eosin is done. Dehydration through different alcohol series is performed. Xylol for five to ten minutes and then mounted using DPX under a cover slip. Once the slides are dried, Hematoxylin and Eosin (H & E)-stained tissues are observed under light microscope

Statistical analysis:

Statistical analysis was performed by R Version 4.3.2. One-way analysis of variance (ANOVA) followed by post hoc “Tukey test” for intergroup comparison and student t-test for within group comparison. Data were expressed as mean ± S.D and probability value (P) was fixed at ≤0.05.

RESULTS:

Body weight:

The weight of each animal was recorded on the Day 1 and Day 21

Fig 2. Changes in body weight within the groups at day 21.

Control received distilled water; Dox=Doxorubicin at 5mg/kg; B75=Bacopa at 75mg/kg dose; B150=Bacopa at 150mg/kg; B250=Bacopa at 250mg/kg. Analysis was done using student t-test and values are expressed as Mean±S.D ^* denotes p-value<0.05 vs baseline values, ^**denotes p-value< 0.01 vs baseline values.

Significant increase in body weight in control group at Day 21 was observed compared to Day 1. In doxorubicin group and other test groups there was decreased weight at Day 21 compared to baseline values. (Fig 2)

Control received distilled water; Dox=Doxorubicin at 5mg/kg; B75=Bacopa at 75mg/kg dose; B150=Bacopa at 150mg/kg; B250=Bacopa at 250mg/kg.Comparison is done by one way ANOVA, post hoc Tukey’s test, all values are expressed as Mean±S.D.* denotes p-value < 0.05 vs Dox group, $denotes p-value <0.05 vs Control group, # denotes p-value <0.05 vs Bacopa alone group

Testicular weight in Doxorubicin group was reduced (p< 0.05) when compared to control group. The treatment groups (Bacopa, B75, B150, B250mg/kg) have shown a significant increase in testicular weight as compared to the Dox group.(Fig 3a)There was no significant difference in the TI of the animals in the treatment group (B75, B150, B250) when compared to Dox group. But significant increase was observed between the Bacopa 250mg/kg group compared to the dox group. (Fig.3b) Sperm count of each rat in the different groups was calculated. Decreased sperm count was observed in Dox group compared to control and bacopa group. Compared to Dox group all the treatment groups have shown significant increase in the sperm count. However, there was significant decrease in sperm count in B75, 150, 250 groups compared to control and Bacopa only group. Sperm motility was decreased in Dox group compared to control and bacopa group. Compared to Dox group, bacopa treated groups have shown significant increase in the sperm motility. There was significant decrease in motility in B75, 150, 250 groups compared to control and Bacopa only group. (Fig 3c and 3d)

Biochemical analysis:

Table 1: Estimation of Oxidative stress markers in testis

Groups	MDA (nM/g protein)	GSH (μM/mg protein)	SOD (U/mg protein)
Control	35.72 ± 0.75*	0.05159 ± 0.001*	3.548 ± 0.31*
Dox	91.52 ± 4.23^$#	0.02280 ± 0.002^$#	1.098 ± 0.06^$#
Bacopa	35.72 ± 2.56 *	0.05025 ± 0.001*	3.128 ± 0.34 ^*$
B75+Dox	58.07 ± 3.89 *^$#	0.02953 ± 0.001*^$#	2.160 ± 0.20 ^*$#
B150+Dox	58.02 ± 4.78 *^$#	0.02869 ± 0.004*^$#	1.913 ± 0.24 ^*$#
B 250+Dox	68.55 ± 5.46^*$#	0.02754 ± 0.001*^$#	1.795 ± 0.39 ^*$#

Control received distilled water; Dox=Doxorubicin at 5mg/kg; B75=Bacopa at 75mg/kg dose; B150=Bacopa at 150mg/kg; B250=Bacopa at 250mg/kg; Comparison is done by one way ANOVA, post hoc Tukey’s test, all values are expressed as Mean ± S.D. * denotes p-value < 0.05 vs Dox group, $ denotes p-value < 0.05 vs Control, # denotes p-value < 0.05vs Bacopa group.

MDA levels significantly increased in Dox group, its levels significantly decreased in the control, Bacopa, B75, B150, B250 compared to Dox group. MDA levels significantly increased in B75, B150, B250 when compared to control and Bacopa only group.

GSH levels significantly increased in control, Bacopa, B75, B150, B250 compared to Dox group. GSH levels significantly decreased in B75, B150, B250 when compared to the control and Bacopa only group.

SOD levels significantly increased in control, Bacopa, B75, B150, B250 compared to Dox group. SOD levels significantly decreased in B75, B150, B250 when compared to the control and Bacopa only group. (Table 1)

Histopathological changes:

Fig. 4. Effect of Bacopa monnierion testicular tissue from different groups

Representative photomicrographs of Hematoxylin and Eosin (H&E) stained sections of testis showing cross sections of seminiferous tubules and interstitial cells with connective tissue between the tubules inControl (Group 1), Doxorubicin induced (Group 2), 250mg/kg Bacopa treated (Group 3), 5mg/kg Doxorubicin + 75mg/kg Bacopa treated (Group 4), 5mg/kg Doxorubicin + 150mg/kg Bacopa treated (Group 5) and 5mg/kg Doxorubicin + 250mg/kg Bacopa treated (Group 6) [Magnification: 10x10 X]

Hematoxylin and Eosin (H&E) stained sections of testisof rats belonging to control group showed normal looking cross sections of seminiferous tubules with very little inter-tubular space (green arrow) containing normal looking collection of Leydig cells. Epithelial height and density of sperms in the seminiferous tubules appeared normal. Sections of testis of doxorubicin treated rats (Group 2) showed cross sections of seminiferous tubules with diminished size (diameter) and increased inter-tubular space (yellow arrow). Seminiferous tubular epithelial height appeared to be decreased (red arrow) indicating germ cell loss. Interstitial cells of Leydig also looked sparse in their distribution.Sections of testis of rats belonging to groups 4, 5 and 6 exhibited mostly normal seminiferous tubule structure almost at par with control(Group 1) with some areas where seminiferous tubules were diminished in size (diameter) with increased inter-tubular space (yellow arrow). However, Bacopa only group (Group 3) showed mostly normal seminiferous tubule structure as that of group 1. (Fig 4)

DISCUSSION:

This study has been conducted in order to evaluate the effect of herbal product Bacopa monnierion Dox induced testicular toxicity in animal model using Sprague Dawley male rats. In the present study, Dox has induced testicular toxicity and the toxicity was ameliorated by different doses of B. monnieri. Testicular toxicity was found in our study as there was decreased testicular weight and testicular index on receiving Dox. The testicular index is used to assess the proportionate change in weight of testis with respect to change in the body weight. It is a very useful parameter to assess testicular toxicity and reduced in Dox group²².

Studies have reported that Dox induced testicular toxicity resulted in vacuolar degeneration, reduced testicular weight, shrinkage of seminiferous tubules, decreased sperm count, motility and viability²³. Dox causes direct injury to DNA and cause lipid peroxidation. There are two pathways for the free radical formation: NADPH dependent reductases producing semiquinone free radical which in the presence of O₂- redox cycling gives superoxide radicals and secondly nonenzymatic mechanism which involves iron²⁴. Similarly in our study administration of Dox affected sperm functions and increased oxidative stress.

Oxidative stress is the leading factor that is responsible for the infertility. The effect of Bacopa monnierion testicular functions is still not known. In a study, rats treated with Bacopa monnieriresulted in the improvement in quality of the sperm, spermatogenic cell density and steroidogenic parameters due to the decreased oxidative stress in the testis and reduced lipid peroxidation levels in the gonad¹⁰. On the contrary, in another study treatment of Bacopa monnieri adversely affected sperm functions like motility, viability and the sperm count. It was believed to be due to actions on sertoli cells. Sertoli cells are important in maintaining spermatogenesis process, and damage to them causes suppression of spermatogenesis activity²⁵.However, in our study there was no adverse effect observed in Bacopa monnieri treatment group and there was recovery in the sperm parameters like count and motility when compared to the Dox induced group.

Previous study has demonstrated the free radical scavenging property and prevention of DNA damage by B.monnieri. Treatment with B.monnieriwas shown torestore the levels of non-enzymatic antioxidants like Vit-C and Vit-E²⁶.Similarly in our study, treatment with B.monnieri has significantly increased the levels of antioxidant enzymes (SOD, GSH), and inhibited lipid peroxidation activity (MDA). The restoration of testicular structure and functions was demonstrated in histopathological summery.

Phytochemical screening has shown the presence of flavonoids, saponins, triterpenoids which were responsible for antioxidant activity. They contribute for the protective action on lipid peroxidation and enhance cellular antioxidant defence which protects against oxidative damage^27,28. Due to the presence of the above components in Bacopa monnieri extract its administration demonstrated a significant beneficial effect in the Dox- induced testicular toxicity.

CONCLUSION:

The study demonstrates the ability of Bacopa monnieri in ameliorating Doxorubicin induced testicular toxicity. Bacopa monnieri reduced the tissue damage caused by Doxorubicin and was able to partially restore thetesticular structure and function by its antioxidant activity.

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Received on 02.01.2024 Revised on 20.05.2024

Accepted on 22.07.2024 Published on 20.01.2025

Available online from January 27, 2025

Research J. Pharmacy and Technology. 2025;18(1):17-23.

DOI: 10.52711/0974-360X.2025.00003

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