Skip to main content
NCBI home page
Toxicology Research logoLink to Toxicology Research
. 2024 Aug 22;13(4):tfae133. doi: 10.1093/toxres/tfae133

Assessment of cytoprotective and genoprotective effects of Moringa oleifera and Tinospora cordifolia extracts in vitro

Preeti Bagri 1,✉, Vinod Kumar 2, Kanisht Batra 3
PMCID: PMC11339162  PMID: 39184218

Abstract

Background

Moringa oleifera and Tinospora cordifolia is extensively used as an ingredient of food and in traditional medicine for the management of a variety of diseases.

Material and methods

The extracts of leaf of Moringa oleifera and stem of Tinospora cordifolia were assessed to examine their ability to inhibit the oxidative DNA damage (by DNA protection assay), cytoprotective and genoprotective potential (by Comet assay) in V79 cells individually and in combinations.

Result

It was found that these extracts could significantly inhibit the OH-dependent damage of pUC18 plasmid DNA. M. oleifera extract (160 and 320 μg/mL) and Tinospora cordifolia extract (640, 1,280 and 2,560 μg/mL) individually showed higher DNA protection activity. M. oleifera (1,280 μg/mL) combined with Tinospora cordifolia (640 μg/mL) showed best cytoprotective and genoprotective activities among different concentration combinations and various concentrations of individual plants in V79 cell line against hydrogen peroxide induced cytotoxicity and genotoxicity.

Conclusion

This study demonstrates the cytoprotective and genoprotective activity of M. oleifera and Tinospora cordifolia individually or in combination.

Keywords: Moringa oleifera, Tinospora cordifolia, V79 cells, comet assay, plasmid DNA

Introduction

Plants are important repositories of vital elements that humans require for survival, which are abundantly provided by nature. They have been the main source of medicinal compounds for millennia. Studies on folk medicine traditions have shown that medicinal plants and their derivatives have been used by all human civilizations throughout history.1 Plants are being used as inhabitant management in tradition or long-established system of remedy for management of various kinds of disease including cancer.2 In recent times, a greater significance has been given towards exploration on complementary and alternative medicine that deals with cancer treatment.3 In traditional medicine, plants are being used for curative purposes and are important as they hold biologically active components that are not toxic.4 Ayurveda, a conservative division of Indian system of medicine mainly based on plant drugs has been doing well since very early times for preventing or suppressing the disease.5 Early documentation about the use of medicinal plants has been mentioned in Discorides and Ayurveda. Epidemiological studies recommend that use of diets containing fruits and vegetables having phytochemicals and micronutrients decrease the threat of developing cancer. Certain products from plants are known to induce apoptosis in neoplastic cells but not in healthy cells. Medicinal plants and their bioactive compounds constitute the fundamental basis of traditional pharmacological methodologies.6

M. oleifera Lam. (drumstick tree, horseradish tree) is a native tree from Northwestern India. It has spread to Africa and is extensively cultivated in Cameroon.7 Moringa, valued mainly for its leaves, tender pods, seeds, and flowers, is considered an important source of β-carotene, vitamin C, minerals, and phytochemicals that show a confirmed antioxidant activity.8–10 In addition, M. oleifera leaf extracts demonstrate important pharmacological activities against inflammation, cancer, genotoxicity, diabetes and hyperglycemia, and neurodegeneration.9,11–17 Therefore, they compose a potential material for nutraceutical formulation.

T. cordifolia, commonly known as Guduchi or Giloy, is used as a remedy for centuries in the Ayurvedic and Unani systems of the medicine.18T. cordifolia extract contains many constituents such as alkaloids, steroids, glycosides, and polysaccharides.19 It has been shown to possess antidiabetic, antihepatotoxic, antioxidant, genoprotective and immunomodulatory properties.1,12,18,20,21 Both the species Moringa and Tinospra are promising in the treatment of cancer as already studied by researcher.22–24

The estimation of genotoxicity indicators is important in determining the association between the harm caused and the substance to which the organism was exposed. Although there are facts that both plants show therapeutic properties, it is essential to carry out research studies to explore their safety for therapeutic use. This study was undertaken to assess the cytoprotective and genoprotective effects of aqueous extract of leaves of M. oleifera and methanolic extract of Tinospora cordifolia stem extracts in vitro in V79 cells.

Materials and methods

Chemicals

Different chemicals were obtained from the following suppliers: Luria Bertani Broth, Luria Bertani Agar, Miller (LB media) and slides were purchased from Himedia Laboratories Pvt. Ltd, Mumbai. pUC18 plasmid DNA (Invitrogen), Fetal bovine serum (FBS), Dulbecco’s Modified Eagle’s Medium—high glucose, Nutrient Mixture F-10 Ham, Trypsin–EDTA solution, ampicillin and ethidium bromide were purchased from Sigma Chemical Co., USA. Ferric chloride and Hydrogen peroxide (Sisco Research Laboratories, Mumbai), Antibiotic-Antimycotic (100X) (Gibco), Ascorbic acid (CDH) were used in the research. The cell culture flasks of V79 Cell line were purchased from cell culture repository of NCCS, Pune.

Collection of plant material and extract preparation

Leaves of M. oleifera and stem of Tinospora cordifolia were collected from Medicinal, Aromatic and Under-utilized plant section of Department of Genetics and Plant Breeding, College of Agriculture, CCS HAU, Hisar, Haryana, India. The extracts were prepared as per standard established protocols.25,26 Briefly, the leaves of M. oleifera and stem of Tinospora cordifolia were cleared with running water to take away any dust impurities and shade dried at room temperature. The leaves and stem of plants were grinded, powdered and stored at room temperature till further process. The powder of leaves of M. oleifera were soaked in distilled water and stem of Tinospora cordifolia were soaked in methanol (1:10) for 48 h at 40 °C with intermittent shaking. The solutions were then filtered through Whatman filter paper. The filtrates were dried in vacuum rotary evaporator at 40 °C followed by further drying on water bath for obtaining the extracts. Finally, the dried extracts were collected and stored at 4 °C till further analysis. The cytoprotective and genoprotective assays were carried out in three independent experiments.

DNA protection assay (in-vitro gel electrophoresis using plasmid DNA)

A DNA protection assay was performed using supercoiled pUC18 plasmid DNA. Plasmid DNA (5 μg) was incubated with Fenton’s reagents (30 mM H2O2, 50 mM ascorbic acid and 80 mM FeCl3) containing test sample (10 μg/mL to 2,560 μg/mL in pUC18 DNA protection studies respectively) and the finally volume of the mixture was raised up to 20 μL. The mixture was then incubated for 30 min at 37 °C followed by addition of loading dye and electrophoresis was carried out in TAE buffer (40 mM Tris base, 16 mM acetic acid 1 mM EDTA, pH 8.0) at 60 V for 2.5 h. DNA was analyzed followed by ethidium bromide staining using Gel Doc XR system (Bio-Rad, USA).

Cell culture and maintenance

V79 cells were maintained as monolayers in plastic culture flasks (25 cm2) containing HAM-F10 and DMEM (1:1) medium supplemented with 10% fetal bovine serum, antibiotics (0.01 mg/mL streptomycin and 0.005 mg/mL penicillin;), and 2.38 mg/mL HEPES, at 37 °C in a CO2 incubator. The normal cell cycle time was 12 h, in these conditions. The experiments were carried out using V79 cells between the 6th and 12th culture passage after thawing. In-vitro cytoprotective (cell line based) activity of both plants individually and in combination by dye exclusion method was assessed in V79 cell lines. Cells were seeded (3 × 106 cells) in 5 mL complete medium in a 25-cm2 flask, and grown for 2 days up to 60%–70% confluence before treatment with the test substance. Plant extracts was dissolved in PBS and added to culture medium to achieve the differently designed concentrations. Oxidative challenge with 1 mM H2O2 was carried out for 12 h in the dark in FBS-free medium.

For the experiments, 300,000 cells were seeded in tissue culture flasks, incubated for two cycles (24 h) in complete HAM-F10/DMEM medium, and then treated with each concentration of plant extracts alone or in combination with the H2O2 (1 mM) for 12 h in serum-free medium. At the end of treatment, the cells were washed with PBS at 37 °C and trypsinized with 1 mL trypsin. After 3 min, the cells were suspended in PBS and 1 mL of the cell suspension was immediately used for the test. Cytoprotection was evaluated by the determination of cell viability using the Trypan blue exclusion method. Briefly, a solution of 20 μL 0.4% Trypan blue, freshly prepared in distilled water, was mixed with 20 μL of each cell suspension for 5 min, taken in Neubauer counting chamber. Non-viable cells appear blue. At least 200 cells were counted per treatment.

In this experiment, cytoprotective effect on H2O2 induced cytotoxicity was assessed. For this, H2O2 at 1 mM concentration was used to induce cytotoxic effects in positive control and while others it was incubated with different plant extracts concentration individually and in combination for 12 h. After this cells were trypsinized as per standard protocol, collected and used for dye exclusion method. In this test different concentration of both plant extracts were tested and finally three concentrations of each plant were selected for further studies. In M. oleifera, 640, 1,280 and 2,560 μg/mL concentration were selected and used in cytoprotective and genoprotective studies. Among this 640 and 1,280 μg/mL were showed good results so further selected for combination studies. In Tinospora cordifolia 320, 640 and 1,280 μg/mL concentration were selected and used in cytoprotective and genoprotective studies. Among this 320 and 640 μg/mL were showed good results so further selected for combination studies.

Different concentrations used are

  1. Control cells without any treatment

  2. M. oleifera I- 640 μg/mL + H2O2 - 1 mM

  3. M. oleifera II- 1280 μg/mL + H2O2 - 1 mM

  4. M. oleifera III- 2560 μg/mL + H2O2 - 1 mM

  5. Tinospora cordifolia I- 320 μg/mL + H2O2 - 1 mM

  6. Tinospora cordifolia II- 640 μg/mL + H2O2 - 1 mM

  7. Tinospora cordifolia III- 1280 μg/mL + H2O2 - 1 mM

  8. M. oleifera I- 640 μg/mL + Tinospora cordifolia I- 320 μg/mL + H2O2 - 1 mM

  9. M. oleifera II- 1280 μg/mL + Tinospora cordifolia II- 640 μg/mL + H2O2 - 1 mM

  10. M. oleifera II- 1280 μg/mL + Tinospora cordifolia I- 320 μg/mL + H2O2 - 1 mM

  11. M. oleifera I- 640 μg/mL + Tinospora cordifolia II- 640 μg/mL + H2O2 - 1 mM

  12. H2O2 - 1 mM

In-vitro genoprotective assay (cell line based) by comet assay (single-cell gel electrophoresis) was assessed in V79 cell lines

For the experiments, 300,000 cells were seeded in tissue culture flasks, incubated for two cycles (24 h) in complete HAM-F10/DMEM medium, and then treated with each concentration of plant extracts alone or in combination with the H2O2 (1 mM) for 12 in serum-free medium. At the end of treatment, the cells were washed with PBS at 37 °C and trypsinized with 2 mL trypsin. After 3 min, the cells were suspended in complete medium and 2 mL of the cell suspension was immediately used for the test. The comet assay was carried out according to27,28 with some modifications.

Base slide was prepared in 1% normal melting agarose (NMA). Slides were dipped into molten NMA up to two-thirds of their frosted end. The cells were isolated in Dulbecco’s phosphate buffer saline (DPBS). Cell pellets were washed thrice with 1 mL of DPBS. Cells were then re-suspended in 1 mL DPBS. Viability assay was done by 0.4% Trypan blue dye. Cell count and number of cells was adjusted to 1.0x106 cells/mL with DPBS. On the first layer of base slides, 90 μL of diluted sample (100 μL cell suspension mixed with 100 μL of 1% LMA) was added to form the second layer. Cover slips were placed gently on the slides to evenly spread the cells in the agarose. The slides were kept at 4 °C for 15–20 min to allow the gel to solidify. After solidification, the cover slips were removed and a third layer of 0.5% LMA (80 μL) was added on the slides and kept at 4 °C for 15–20 min for gel solidification. Finally, the cover slips were removed and the slides were immersed in freshly prepared chilled lysing solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, 1% Triton X 100 and 10% dimethylsulfoxide, pH 10 adjusted with NaOH). The slides remained in the lysing solution overnight at 4 °C. Electrophoresis was done in chilled electrophoretic buffer (300 mM NaOH, 1 mM Na2EDTA, pH > 13). Initially slides were left in this solution for 25 min for unwinding. Electrophoresis was conducted at 24 V (0.7 V/cm) and a current of 300 mA using a power supply (Biorad Power Pac Basic) for 20 min on ice in dark. After this, neutralization buffer (0.4 M Tris base pH 7.5) was added drop-wise to the slide and left for 5 min to neutralize excess alkali. Neutralizing of slides was repeated thrice. Slides were stained with 80 μL EtBr for 5 min and dipped in chilled distilled water to wash off excess EtBr and cover slip placed over. Slides were dipped in 100% chilled ethanol for 20 min for storage purpose. Scoring of slides were done by Randomly selecting 100 cells (50 cells from each of the two replicate slides) per treatment at 200x magnification using a fluorescent microscope equipped with green filter. Slides from different groups were scored using an image-analysis system (TriTek Comet-Score™ Freeware v1.5 software). The comet parameters recorded were tail length (μm), tail DNA (%), tail moment, Olive tail moment (OTM, arbitrary units), and comet length (μm).

Results

DNA protection assay (In-vitro gel electrophoresis using plasmid)

Aqueous extract of leaves of M. oleifera and methanolic extract of stem of Tinospora cordifolia were tested individually at various concentration (10 μg/mL to 2,560 μg/mL) for genoprotective activity against DNA damage induced by Fenton’s reagent (30 mM H2O2 + 50 μM Ascorbic acid +80 μM Ferric Chloride) using this test. The purified pUC18 plasmid DNA was used for DNA protection study. Both plant extracts individually showed DNA protection activity in concentration dependent manner. M. oleifera at 160 and 320 μg/mL concentrations whereas Tinospora cordifolia at 640, 1,280 and 2,560 μg/mL showed its appreciable DNA protection effect (Fig. 1a and b).

Fig. 1.

Fig. 1

Open in a new tab

a) Effect of various concentrations (10 μg/mL to 2,560 μg/mL) of M. oleifera on DNA damage induced by Fenton’s reagent. Lanes M: DNA marker; 1: pUC18 plasmid without any treatment (control-1); 2: Plasmid treated with Fenton’s reagent (control-2); 3–11: Plasmid treated with Fenton’s reagent and 2-fold concentration of M. oleifera extract. Lanes 7 and 8 indicates the most protective concentrations of plant extract and the least damage of the plasmid DNA by Fenton’s reagent is observed as compared to the controls (lane 1 and 2). b) Effect of various concentrations (10 μg/mL to 2,560 μg/mL) of Tinospora cordifolia on DNA damage induced by Fenton’s reagent. Lanes M: DNA marker; 1: pUC18 plasmid (control-1); 2: Plasmid treated with Fenton’s reagent (control-2); 3–11: Plasmid treated with Fenton’s reagent and 2-fold concentration of Tinospora cordifolia extract. Lanes 9, 10 and 11 indicates the most protective concentrations of plant extract and the least damage of the plasmid DNA by Fenton’s reagent is observed as compared to the controls (lane 1 and 2).

In-vitro cytoprotective (cell line based)

The activity of aqueous extract of leaves of M. oleifera and methanolic extract of stem of Tinospora cordifolia plants individually and in combination was assessed in V79 cell line (Hamster lung fibroblast origin) by dye exclusion method.

In M. oleifera, 640, 1,280 and 2,560 μg/mL concentration were selected and used in cytoprotective and genoprotective studies (Fig. 2a). Among this 640 and 1,280 μg/mL were showed good results so further selected for combination studies. In Tinospora cordifolia 320, 640 and 1,280 μg/mL concentration were selected and used in cytoprotective and genoprotective studies (Fig. 2b). Among this, 320 and 640 μg/mL showed good results so further selected for combination studies.

Fig. 2.

Fig. 2

Open in a new tab

a) Effect of different concentrations of aqueous extract of leaves of M. oleifera on H2O2 induced cytotoxicity in V79 cell line. MO: M. oleifera. b) Effect of different concentrations of methanolic extract of stem of Tinospora cordifolia on H2O2 induced cytotoxicity in V79 cell line. TC: Tinospora cordifolia. c) Effect of different concentrations of plant extracts on H2O2 induced cytotoxicity in V79 cell line. Data are presented as mean ± SEM. MO-I, II, and III: M. oleifera Treatment group-I, II and III, respectively. TC_I, II and III: Tinospora cordifolia treatment group- I, II and III, respectively. The values are compared using one-way ANOVA followed by Tukey post-hoc test. Means bearing a, b, c, d, e, f, g, h, i, j and k superscripts differ significantly (P ≤ 0.05) as compared to the (a) control, (b) M. oleifera (640 μg/mL) + H2O2, (c) M. oleifera (1,280 μg/mL) + H2O2, (d) M. oleifera (2,560 μg/mL) + H2O2, (e) Tinospora cordifolia (320 μg/mL) + H2O2, (f) Tinospora cordifolia (640 μg/mL) + H2O2 (g) Tinospora cordifolia (1,280 μg/mL) + H2O2, (h) M. oleifera (640 μg/mL) + Tinospora cordifolia (320 μg/mL) + H2O2, (i) M. oleifera (1,280 μg/mL) + Tinospora cordifolia (640 μg/mL) + H2O2, (j) M. oleifera (1,280 μg/mL) + Tinospora cordifolia (320 μg/mL) + H2O2, and (k) M. oleifera (640 μg/mL) + Tinospora cordifolia (640 μg/mL) + H2O2, respectively. d) Effect of Moringa Oleifera and Tinospora cordifolia in combination on H2O2 induced cytotoxicity in V79 cell line. MO: Moringa Oleifera; TC: Tinospora cordifolia.

In dye exclusion assay % viability of cells was reported and presented in Fig. 2c. It has been observed that M. oleifera showed good results at 640 and 1,280 μg/mL than 2,560 μg/mL. So these concentrations were further selected for combination studies. In Tinospora cordifolia 320 and 640 μg/mL concentration showed good results so further selected for combination studies. In combination studies both concentrations were used in different combination as details are given and it has been observed that M. oleifera (1,280 μg/mL) + Tinospora cordifolia (640 μg/mL) showed 92.34% viability best results among different combination and individual plant (Fig. 2d).

In-vitro genoprotective assay (cell line based) by comet assay (single-cell gel electrophoresis) was assessed in V79 cell lines

After collection of cells comet assay was carried out with standard protocol. Duplicate slides per treatment were made and 100 cells were randomly selected and scored per treatment to get a reproducible data at 200× magnification using a fluorescence microscope equipped with green filter. Slides from different treatment were scored using an image-analysis system (TriTek Comet-ScoreTM Freeware v1.5 software). The comet parameters recorded were tail length (μm), tail DNA (%) and comet length (μm) and represented in Fig. 3a–c. Different concentrations used are mentioned in cytoprotective part. In comet assay analysis, it has been observed that M. oleifera showed good results at 640 and 1,280 μg/mL than 2,560 μg/mL and Tinospora cordifolia at 320 and 640 μg/mL concentration were showed good results. In combination studies both concentrations were used in different combinations as details are given and it has been observed that M. oleifera (1,280 μg/mL) + Tinospora cordifolia (640 μg/mL) showed best genoprotective results among different combination and individual plant.

Fig. 3.

Fig. 3

Open in a new tab

a–c) COMET assay showing the effect of different concentrations of plant extracts on H2O2 induced genotoxicity in V79 cell line. a) Comet length; b) tail DNA and c) tail length. Data are presented as mean ± SEM. MO-I, II, and III: Moringa oleifera treatment group-I, II and III, respectively. TC_I, II and III: Tinospora cordifolia treatment group- I, II and III, respectively. The values are compared using one-way ANOVA followed by Tukey post-hoc test. Means bearing a, b, c, d, e, f, g, h, i, j and k superscripts differ significantly (P ≤ 0.05) as compared to the (a) control, (b) M. oleifera (640 μg/mL) + H2O2, (c) M. oleifera (1,280 μg/mL) + H2O2, (d) M. oleifera (2,560 μg/mL) + H2O2, (e) Tinospora cordifolia (320 μg/mL) + H2O2, (f) Tinospora cordifolia (640 μg/mL) + H2O2 (g) Tinospora cordifolia (1,280 μg/mL) + H2O2, (h) M. oleifera (640 μg/mL) + Tinospora cordifolia (320 μg/mL) + H2O2, (i) M. oleifera (1,280 μg/mL) + Tinospora cordifolia (640 μg/mL) + H2O2, (j) M. oleifera (1,280 μg/mL) + Tinospora cordifolia (320 μg/mL) + H2O2, and (k) M. oleifera (640 μg/mL) + Tinospora cordifolia (640 μg/mL) + H2O2, respectively.

Discussion

In the current study, we examined whether M. oleifera and Tinospora cordifolia can prevent oxidative damage in V79-4 lung fibroblasts and found that these plants have the ability to escape H2O2- induced cytotoxicity while having ROS scavenging activity, genotoxicity via DNA damage.

In case of plasmid, cleavage of the phosphodiester chains of the supercoiled DNA is result of DNA damage that produces a relaxed open circular form. Further linear double-stranded DNA molecules formed from cleavage near the first breakage. Single-strand breaks suggested formation of circular form of DNA whereas double-strand breaks indicate the formation of linear form of DNA. Since the Fenton’s reagents are composed by Ascorbic Acid, H2O2 and Fe3+. They can produce OH. according to the following reaction.29

graphic file with name DmEquation1.gif (1)
graphic file with name DmEquation2.gif (2)
graphic file with name DmEquation3.gif (3)

The formation of Fe2+ after reduction of Fe3+ in the presence of potent reducing agent Ascorbic Acid, initiate the decomposition of H2O2 leading to generate highly reactive FR species OH. via Fenton-like reaction (Eqs. (1) and (2).30 Leaves of M. oleifera and stem of T. Cordifolia have been well known as outstanding antioxidants and excellent scavengers of reactive metabolites such as OH., O2.-, and ROO.31–33 FRs caused pBR322 plasmid DNA damaged by a mechanism of electron or hydrogen-atom transfer could protected by polyphenols rich red onion peel.34 In this work we evaluate the oxidative DNA damage protective activity of leaves of M. oleifera and stem of T. Cordifolia against pUC18 plasmid DNA damaged induced by OH. using in vitro method of.29 As shown in fig. 1a and b incubation of pUC18 plasmid DNA with Fenton’s reagent for 45 min resulted in the cleavage of supercoiled to give open circular and linear forms of plasmid DNA, indicating that OH. generated from iron-mediated decomposition of H2O2 produced both single-strand and double-strand DNA breaks. Gutteridge35 reported that H2O2 and O2.- are potentially cytotoxic; most of the oxidative damage in biological systems is caused by the OH., which is generated by the reaction between H2O2 and O2.- in the presence of redox-active metals. Standard catalase enzyme reduced this type of oxidative damage that converts H2O2 into water and oxygen. Addition of plant extracts to DNA and Fenton’s reagent mixture induced the significant reduction in the formation of open circular and linear forms and increased supercoiled or native form of plasmid DNA (Fig. 1a and b). Previous scientific studies have shown that redox- active metals in solution might bind to phenolic antioxidant compounds and form the complexes, consequently it prevents the reduction of redox-active metal ions with H2O2.29,32,36M. oleifera and T. Cordifolia extracts might prevent the reaction of Fe ions with H2O2. On the other hand, natural polyphenols can influence the FRs-mediated oxidation of DNA through simple mechanism including quenching of ROS by donating hydrogen atom or electron.37 This type of action might diminish the reduction potential of Fe ions, leading to the inhibition of Fenton-like reaction, and might also directly scavenge OH. and therefore protecting OH.- dependent strand breaks in the supercoiled plasmid DNA. Various plant extracts (Tabernaemontana divaricata, Azadirachta indica, Anthocephalus cadamb, M. oleifera, Tinospora cordifolia, Terminalia chebula) were used to assess the genoprotective activity against H2O2-induced oxidative damage on different plasmid DNA32,38–42 and it was concluded that these plants are good antioxidant so prevent H2O2-induced oxidative damage of plasmid DNA. Bioactive constituents and their derivative are directly/indirectly involved in slowing down the effect of carcinogenic agents and could be a remedy for DNA damage protection.43

H2O2 is an effective genotoxin, capable to induce oxidative DNA damage that includes DNA-strand breakage and base modification.44 The genotoxic effects of ROS, in particular H2O2 that induces lesions similar to those resulting from ionizing radiation, have been well documented in V79 cells.45 Indeed, H2O2 treatment was cytotoxic and genotoxic in V79 cells (Figs 2 and 3). Both plant extracts had a protective effect against H2O2-induced cytotoxicity and genotoxicity. Consistent with this protective effect, plants displayed a significant protective effect against H2O2-induced DNA breaks, verified in the comet assay. Sreelatha and Padma46 explored the modulatory effect of moringa leaf extracts against H2O2-induced cytotoxicity and oxidative damage in the HeLa-derived KB cell line and concluded that moringa leaf extract can prevent H2O2-induced DNA damage in KB cells. Sikder et al.47 studied protective action of M. oleifera leaf extract against oxidative stress induced DNA damage and concluded that it possesses significant DNA protective activity. Chandrashekar et al.48 studied the protective effect of Tinospora cordifolia against pro-oxidant induced DNA fragmentation and its in-vitro anti-oxidant activity. Ghate et al.32 studied the DNA protecting effect of Tinospora cordifolia. These studies support our result.

Conclusion

This study demonstrates that M. oleifera and Tinospora cordifolia individually or in combination, is cytoprotective and genoprotective to V79 cells.

Acknowledgments

The study reported in the present paper was funded by state scheme of Department of Veterinary Pharmacology and Toxicology, LUVAS, Hisar. We are also thankful to Department of Veterinary Pharmacology and Toxicology, College of Veterinary Sciences, LUVAS, Hisar for providing infrastructural facilities.

Contributor Information

Preeti Bagri, Department of Veterinary Pharmacology and Toxicology, College of Veterinary Sciences, Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar, Haryana 125004, India.

Vinod Kumar, Department of Veterinary Pharmacology and Toxicology, College of Veterinary Sciences, Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar, Haryana 125004, India.

Kanisht Batra, Department of Animal Biotechnology, College of Veterinary Sciences, Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar, Haryana 125004, India.

Author contributions

Dr Preeti Bagri conceived of the study. Dr Preeti Bagri performed data collection, data analysis, and produced the figured and scripts, with overall guidance from Dr Vinod Kumar. All authors wrote the manuscript. Kanisht Batra helped in conducting experiments.

 

Conflict of interest statement: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

References

  • 1. Kimmy G, Verma DK, Prabhakar P, Tripathy S, Dadrwal BK, Kumar P, Srivastav PP, Banerjee M. In vitro and In silico evaluation of the antioxidant, anti-microbial and antihyperglycemic properties of giloy (Tinospora cordifolia L.) stem extract. Biocatalysis and agricultural. Biotechnology. 2024:56:103059. [Google Scholar]
  • 2. Pandey M, Debnath M, Gupta S, Chikara SK. Phytomedicine: an ancient approach turning into future potential source of therapeutics. J Pharmacog Phytother. 2011:3:27–37. [Google Scholar]
  • 3. Sawadogo WR, Schumacher M, Teiten MH, Dicato M, Diederich M. Traditional west African, pharmacopoeia, plants and derived compounds for cancer therapy. Biochem Pharmacol. 2012:84(10):1225–1240. [DOI] [PubMed] [Google Scholar]
  • 4. Duffy R, Wade C, Chang R. Discovery of anticancer drugs from antimalarial natural products: a MEDLINE literature review. Drug Discov Today. 2012:17(17–18):942–953. [DOI] [PubMed] [Google Scholar]
  • 5. Liu WJH. Traditional herbal medicine research methods: identification, analysis, bioassay, and pharmaceutical and clinical studies.New Jersey: John Wiley & Sons; 2011. pp. 1–26. [Google Scholar]
  • 6. Silva MJD, Périco LL, Da Silva MA, Rinaldo D, Gambero A, Santos LCD, Hiruma-Lima CA, Vilegas W, Verma DK, Thakur M. Phytomedicinal study on Brazilian medicinal plants for their potential in chronic diseases treatment. In: Verma DK, editors. Phytochemicals in food and health: perspectives for research and technological development. USA: Apple Academic Press; 2021. [Google Scholar]
  • 7. Agamou JAA, Fombang EN, Mbofung CMF. Particular benefits can be attributed to Moringa oleifera Lam leaves based on origin and stage of maturity. J Exp Biol Agric Sci. 2015:3(6):541–555. [Google Scholar]
  • 8. Nobosse P, Fombang EN, Mbofung CMF. The effect of steam blanching and drying method on nutrients, phytochemicals and antioxidant activity of Moringa (Moringa oleifera L.) leaves. American. J Food Sci Technol. 2017:5(2):53–60. [Google Scholar]
  • 9. Rastogi S, Farswan TS, Pandey MM. Seasonal variation in the phytoconstituents and antioxidant activity in Moringa oleifera Lam. Leaves of North India. S Afr J Bot. 2024:166:492–502. [Google Scholar]
  • 10. Sreelatha S, Padma PR. Antioxidant activity and total phenolic content of Moringa oleifera leaves in two stages of maturity. Plant Foods Hum Nutr. 2009:64(4):303–311. [DOI] [PubMed] [Google Scholar]
  • 11. Azad SB, Ansari P, Azam S, Hossain SM, Shahid MIB, Hasan M, Hannan JMA. Anti-hyperglycaemic activity of Moringa oleifera is partly mediated by carbohydrase inhibition and glucose-fibre binding. Biosci Rep. 2017:37(3):BSR20170059. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12. Bagri P, Kumar V. Determination of genoprotection against cyclophosphamide induced toxicity in bone marrow of Swiss albino mice by Moringa oleifera leaves and Tinospora cordifolia stem. J Toxic Environ Health A. 2024:87(16):647–661. [DOI] [PubMed] [Google Scholar]
  • 13. Boonsirichai K, Jetawattana S. Radiation-induced DNA double strand breaks and their modulations by treatments with Moringa oleifera Lam. Leaf extracts: a cancer cell culture model. Atom Indonesia. 2014:40(1):7–12. [Google Scholar]
  • 14. Fombang E, Willy Saa R. Antihyperglycemic activity of Moringa oleifera Lam leaf functional tea in rat models and human subjects. Food Nutr Sci. 2016:7(11):1021–1032. [Google Scholar]
  • 15. Galuppo M, Giacoppo S, De Nicola GR, Iori R, Navarra M, Lombardo GE, Mazzon E. Antiinflammatory activity of glucomoringin isothiocyanate in a mouse model of experimental autoimmune encephalomyelitis. Fitoterapia. 2014:95:160–174. [DOI] [PubMed] [Google Scholar]
  • 16. Hannan MA, Kang JY, Mohibbullah M, Hong YK, Lee H, Choi JS, Choi IS, Moon IS. Moringa oleifera with promising neuronal survival and neurite outgrowth promoting potentials. J Ethnopharmacol. 2014:152(1):142–150. [DOI] [PubMed] [Google Scholar]
  • 17. Kashyap P, Kumar S, Riar CS, Jindal N, Baniwal P, Guine RPF, Correia PMR, Mehra R, Kumar H. Recent advances in drumstick (Moringa oleifera) leaves bioactive compounds: composition, health benefits, bioaccessibility, and dietary applications. Antioxidants. 2022:11(2):402. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18. Kumar P, Verma DK, Kimmy G, Srivastav PP, Sandhu KS. Phytochemicals in giloy (Tinospora cordifolia L.): Structure, chemistry, and health benefits. In: Verma DK, editors. Phytochemicals in food and health: perspectives for research and technological development. USA: Apple Academic Press; 2021. [Google Scholar]
  • 19. Singh SS, Pandey SC, Srivastava S, Gupta VS, Patro B, Ghosh AC. Chemistry and medicinal properties of Tinospora cordifolia (Guduchi). Ind J Pharmacol. 2003:35:83–91. [Google Scholar]
  • 20. Panchabhai TS, Kulkarni UP, Rege NN. Validation of therapeutic claims of Tinospora cordifolia: a review. Phytother Res. 2008:22(4):425–441. [DOI] [PubMed] [Google Scholar]
  • 21. Sharma U, Bala M, Kumar N, Singh B, Munshi RK, Bhalerao S. Immunomodulatory active compounds from Tinospora cordifolia. J Ethnopharmacol. 2012:141(3):918–926. [DOI] [PubMed] [Google Scholar]
  • 22. Berehu HM, Patnaik S. Biogenic zinc oxide nanoparticles synthesized from Tinospora Cordifolia induce oxidative stress, mitochondrial damage and apoptosis in colorectal cancer. Nano. 2024:8(3):312–329. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23. Rojas-Armas JP, Palomino-Pacheco M, Arroyo- Acevedo JL, Ortiz-Sánchez JM, Justil-Guerrero HJ, Martínez- Heredia JT, Castro-Luna A, Rodríguez Flores C, Guzmán Duxtan AJ. Phytochemical profiling by UHPLC–Q-TOF/MS and chemopreventive effect of aqueous extract of Moringa oleifera leaves and benzyl isothiocyanate on murine mammary carcinogenesis. Molecules. 2024:29(6):1380. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24. Wu YY, Xu YM, Lau ATY. Anti-cancer and medicinal potentials of Moringa Isothiocyanate. Molecules. 2021:26(24):7512. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25. Awodele O, Oreagba IA, Odoma S, Silva JA, Osunkalu VO. Toxicological evaluation of the aqueous leaf extract of Moringa oleifera Lam. (Moringaceae). J Ethnopharmacol. 2012:139(2):330–336. [DOI] [PubMed] [Google Scholar]
  • 26. Jaiswal D, Kumar Rai P, Kumar A, Mehta S, Watal G. Effect of Moringa oleifera Lam. Leaves aqueous extract therapy on hyperglycemic rats. J Ethnopharmacol. 2009:123(3):392–396. [DOI] [PubMed] [Google Scholar]
  • 27. Dhawan A, Bajpayee M, Parmar D. Comet assay: a reliable tool for the assessment of DNA damage in different models. Cell Biol Toxicol. 2009:25(1):5–32. [DOI] [PubMed] [Google Scholar]
  • 28. Patel S, Pandey A, Bajpayee M, Parmar D, Dhawan A. Cypermethrim-induced DNA damage in organs and tissues of the mouse: evidence from the comet assay. Mutat Res. 2006:607(2):176–183. [DOI] [PubMed] [Google Scholar]
  • 29. Lee JC, Kim HR, Kim J, Jang YS. Antioxidant property of an ethanol extract of the stem of Opuntia ficus-indica var. Saboten. J Agric Food Chem. 2002:50(22):6490–6496. [DOI] [PubMed] [Google Scholar]
  • 30. Halliwell B. Human health and disease: a radical look. Biol Sci Rev. 1995:7(4):21–24. [Google Scholar]
  • 31. Chumark P, Khunawat P, Sanvarinda Y, Phornchirasilp S, Morales NP, Phivthong-ngam L, Ratanachamnong P, Srisawat S, Pongrapeeporn KUS. The in vitro and ex vivo antioxidant properties, hypolipidaemic and antiatherosclerotic activities of water extract of Moringa oleifera Lam leaves. J Ethnopharmacol. 2008:116(3):439–446. [DOI] [PubMed] [Google Scholar]
  • 32. Ghate NB, Chaudhur D, Mandal N. In vitro assessment of Tinospora cordifolia stem for its antioxidant, free radical scavenging and DNA protective potentials. Int J Pharm Bio Sci. 2013:4(1):373–388. [Google Scholar]
  • 33. Iqbal S, Bhanger MI. Effect of season and production location on antioxidant activity of Moringa oleifera leaves grown in Pakistan. J Food Compos Anal. 2006:19(6–7):544–551. [Google Scholar]
  • 34. Prakash D, Singh BN, Upadhyay G. Antioxidant and free radical scavenging activities of phenols from onion (Allium cepa). Food Chem. 2007a:102(4):1389–1393. [Google Scholar]
  • 35. Gutteridge JM. Reactivity of hydroxyl and hydroxyl-like radicals discriminated by release of thiobarbituric acid-reactive material from deoxysugars, nucleosides and benzoate. J Biochem. 1984:224(3):761–767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36. Wei QY, Zhou B, Cai YJ, Yang L, Liu ZL. Synergistic effect of green tea polyphenols with trolox on free radical-induced oxidative DNA damage. Food Chem. 2006:96(1):90–95. [Google Scholar]
  • 37. Prakash D, Upadhyay G, Singh BN, Singh HB. Antioxidant and free radical-scavenging activities of seeds and Agri-wastes of some varieties of soybean (Glycine max). Food Chem. 2007b:104(2):783–790. [Google Scholar]
  • 38. Kumar M, Chandel M, Kumar S, Kaur S. Studies on the antioxidant/Genoprotective activity of extracts of Koelreuteria paniculata Laxm. Am J Biomed Sci. 2012. 10.5099/aj120200132. [DOI] [Google Scholar]
  • 39. Manikandan P, Anandan R, Nagini S. Evaluation of Azadirachta indica leaf fractions for in vitro antioxidant and protective effects against H2O2-induced oxidative damage to pBR322 DNA and red blood cells. J Agric Food Chem. 2009:57(15):6990–6996. [DOI] [PubMed] [Google Scholar]
  • 40. Singh BN, Singh BR, Singh RL, Prakash D, Dhakarey R, Upadhyay G, Singh HB. Oxidative DNA damage protective activity, antioxidant and anti-quorum sensing potentials of Moringa oleifera. Food Chem Toxicol. 2009:47(6):1109–1116. [DOI] [PubMed] [Google Scholar]
  • 41. Soumya K, Haridas KR, James J, Kumar VBS, Edatt L, Sudheesh S. Study of In vitro antioxidant and DNA damage protection activity of a novel luteolin derivative isolated from Terminalia chebula. J Taibah Univ Sci. 2019:13(1):755–763. [Google Scholar]
  • 42. Thind TS, Agrawal SK, Saxena AK, Arora S. Studies on cytotoxic, hydroxyl radical scavenging and topoisomerase inhibitory activities of extracts of Tabernaemontana divaricata (L.) R. Br. Ex Roem. And Schult. Food Chem Toxicol. 2008:46(8):2922–2927. [DOI] [PubMed] [Google Scholar]
  • 43. Kaur P, Purewal SS, Sandhu KS, Kaur M. DNA damage protection: an excellent application of bioactive compounds. Bioresour Bioprocess. 2019:6(1):2. [Google Scholar]
  • 44. Valko M, Rhodes CJ, Moncol J, Izakovic M, Mazur M. Free radicals, metals and antioxidants in oxidative stress-induced cancer. Chem Biol Interact. 2006:160(1):1–40. [DOI] [PubMed] [Google Scholar]
  • 45. Bronzetti G, Cini M, Caltavuturo L, Fiorio R, Croce CD. Antimutagenicity of sodium selenite in Chinese hamster V79 cells exposed to azoxymethane, methylmethansulphonate and hydrogen peroxide. Mutat Res. 2003:523–524:21–31. [DOI] [PubMed] [Google Scholar]
  • 46. Sreelatha S, Padma PR. Modulatory effects of Moringa oleifera extracts against hydrogen peroxide-induced cytotoxicity and oxidative damage. Hum Exp Toxicol. 2011:30(9):1359–1368. [DOI] [PubMed] [Google Scholar]
  • 47. Sikder K, Sinha M, Das N, Das DK, Datta S, Dey S. Moringa oleifera leaf extract prevents in vitro oxidative DNA damage. Asian J Pharm Clin Res. 2013:6(2):159–163. [Google Scholar]
  • 48. Chandrashekar S, Umesha S, Chethan Kumar M, Chandan S. Inhibition of pro-oxidant induced DNA damage in isolated human peripheral lymphocytes by methanolic extract of Guduchi (Tinospora cordifolia) leaves. Int J Pharma Sci Res. 2010:1(10):445–450. [Google Scholar]

Articles from Toxicology Research are provided here courtesy of Oxford University Press

RESOURCES