Table of Contents    
ORIGINAL ARTICLE
Year : 2018  |  Volume : 9  |  Issue : 2  |  Page : 242-246  

Radioprotective potential of Asparagus racemosus root extract and isoprinosine against electron beam radiation-induced immunosupression and oxidative stress in swiss albino mice


1 Central Research Laboratory, K.S Hegde Medical Academy, Nitte (Deemed to be University), Mangalore, Karnataka, India
2 Department of Pharmaceutical Chemistry, NGSM Institute of Pharma Sciences, Nitte (Deemed to be University), Mangalore, Karnataka, India
3 Department of Biochemistry, K. S. Hegde Medical Academy, Nitte (Deemed to be University), Mangalore, Karnataka, India
4 Department of ENT, K. S. Hegde Medical Academy, Nitte (Deemed to be University), Mangalore, Karnataka, India
5 Department of Oncology, Nitte Leela Narayan Shetty Memorial Cancer Institute, Mangalore, Karnataka, India
6 Department of Radiation Oncology, Apollo CBCC, Ahmedabad, Gujarat, India

Date of Web Publication20-Jun-2018

Correspondence Address:
B Satheesh Kumar Bhandary
6th Floor, University Enclave, Medical Sciences Complex, Nitte (Deemed to be University), Deralakatte, Mangalore - 575 018, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jnsbm.JNSBM_225_17

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   Abstract 

Background: Radiotherapy is an important and the most common treatment modality for human cancers. Cancer radiotherapy is associated with unadorned side effects that results from normal tissue damage which is a major subject of concern. Radiation induces damage to living cells due generation of aqueous-free radicals. Therefore, there is a crucial need for the protection of normal cells surrounding the tumor from radiation injury; and hence, the identification of radiation-protecting agents is a chief goal for basic radiation biologists and oncologists. Aim: The aim of this present study was to assess the radioprotective potential of Asparagus racemosus root ethanolic extract (ARE), and isoprinosine (IPR) against electron beam radiation (EBR)-induced immunosuppression and oxidative stress in Swiss Albino mice. Materials and Methods: Swiss albino mice were used for the assessment of the radioprotective potential of ARE and IPR against EBR-induced immunosuppression and oxidative stress. Cytokine estimations, namely, interleukin-2, interferon-gamma, and tumor necrosis factor-alpha were performed in the liver homogenate using ELISA kits, and bone marrow cellularity was determined in the experimental animals. Results: The results of the present study demonstrated the radioprotective and immunostimulatory efficacy of ARE and IPR against EBR-induced cytokine and bone marrow cellularity alterations. Conclusion: The findings of our study demonstrate the potential of ARE and IPR in mitigating radiation-induced mortality by offering protection to mice against lethal dose of whole body EBR. The present study also demonstrates that ARE and IPR exert its radioprotection against EBR induced immunosuppression by regulating cytokines.

Keywords: Asparagus racemosus, electron beam radiation, immunosupression, isoprinosine, oxidative stress


How to cite this article:
Sharmila K P, Kumar Bhandary B S, Fernandes R, Kumari N S, Bhat VS, Shetty K J, Jose JM, Peter AJ. Radioprotective potential of Asparagus racemosus root extract and isoprinosine against electron beam radiation-induced immunosupression and oxidative stress in swiss albino mice. J Nat Sc Biol Med 2018;9:242-6

How to cite this URL:
Sharmila K P, Kumar Bhandary B S, Fernandes R, Kumari N S, Bhat VS, Shetty K J, Jose JM, Peter AJ. Radioprotective potential of Asparagus racemosus root extract and isoprinosine against electron beam radiation-induced immunosupression and oxidative stress in swiss albino mice. J Nat Sc Biol Med [serial online] 2018 [cited 2018 Dec 14];9:242-6. Available from: http://www.jnsbm.org/text.asp?2018/9/2/242/234714


   Introduction Top


Radiotherapy is a vital treatment modality for cancer; it may be used as a single modality or as an adjuvant along with surgery and/or chemotherapy.[1] Since ionizing radiation induces damage to normal tissue, its effective use is compromised by the side effects.[2]

Besides, radiation severely inhibits the function of immune system by suppressing bone marrow and depleting peripheral blood lymphocytes, thereby making the exposed animals vulnerable to opportunistic pathogens, easily to be infected, and sometimes to be lethal. Long-term immunosuppression is also a major concern in patients treated with radio or chemotherapy years after treatment.[3],[4]

Several studies have shown that radiation-induced anomalies in the bone marrow-derived cell populations can persevere for long-term after irradiation.[5] Therefore, there is a crucial need for the development of effective radioprotectors and radio recovery drugs in view of their potential application during both planned radiation exposure (e.g., radiotherapy) and unplanned radiation exposure (e.g., in the nuclear industry and natural background radiation originating from the earth or other sources).[6],[7],[8]

Radioprotective compounds, in addition to protecting the normal tissue, it will also permit the use of higher doses of radiation to obtain better cancer control and possible cure and therefore, is of immense use in recent years. However, till date, no ideal radioprotectors are available as most synthetic compounds, namely, aminothiol S-2-(3-aminopropyl-amino) ethyl phosphorothioic acid, (WR-2721, amifostine, ethiophos [USA], or gammaphos [former USSR]), which have been approved by the Food and Drug Administration, USA, are associated with toxicity at their optimum concentrations. Apparently, there has been limited success of these agents in clinical therapy.[7],[9]

Asparagus racemosus belongs to Asparagaceae family, locally known by the name Shatavari, is one of the well-known Ayurvedic drugs, reported to prevent ageing, increase longevity, impart immunity, and improve mental function. Pharmacological activities of A. racemosus root extract include antiulcer, antioxidant, antidiarrheal, and immunomodulatory activities.[10],[11] This plant which improves health by increasing immunity, imparting longevity, as well as protection against stress, has been grouped under the rejuvenator herbs.[12]

Isoprinosine (IPR) is an immunostimulator which enhances the production of cytokines such as interleukin-1 (IL-1), IL-2, and interferon-gamma (IFN-γ). It increases proliferation of lymphocytes in response to mitogenic or antigenic stimuli, increases active T-cell rosettes and induces T-cell surface markers on prothymocytes.[13]

IPR acts on the immune system to restore impaired cell-mediated immune response to its baseline level, in addition to enhancing humoral immune response.

However, till date, no evidences are available on the immunostimulatory effect of A. racemosus root extract and IPR against ionizing radiation-induced immunosuppression. Therefore, the present study was designed to investigate the possible radioprotective and immunostimulatory activity of A. racemosus root extract and IPR against radiation-induced oxidative stress and immunosuppression.


   Materials and Methods Top


Collection of plant material

A. racemosus roots were collected from Coorg in March 2015 and were identified by a Taxonomist from Mangalore University, Karnataka.

Procurement of isoprinosine

Isoprinosine tablets (product code: 78/158) were procured from Brandmedicines, European Union.

Ethical clearance

The Institutional Animal Ethics Committee of K S Hegde Medical Academy, Nitte (Deemed to be University) approved this study (Ref. KSHEMA/IAEC/19/2015 dated 27.11.2015).

Determination of LD50 of radiation

Female Swiss albino mice (Mus musculus) 6–8 weeks old, weighing 25–30 g were used for this study.

The guidelines set by the WHO (World Health Organization, Geneva, Switzerland) was followed towards animal care and handling.

Radiation work was carried out at Oncology Department, Nitte Leela Narayan Shetty Memorial Cancer Institute using the linear accelerator. The experimental animals were divided into six groups with 10 mice in each group. The animals were restrained in well-ventilated perspex box and exposed to 4, 6, 8, 10, 12, and 14 Gy of whole body electron beam radiation (EBR) at a distance of 100 cm from the beam exit point of the linear accelerator and at a dose rate of 3 Gy/min. The experimental animals were observed twice or thrice daily for 30 days to determine survival rates. The LD50 of radiation was calculated using probit analysis following the method of Finney.[14]

Survival assay for optimum drug dosage fixation

Experimental animals

The experimental animals (8 groups; n = 10 mice/group) were orally administered with 100 mg, 200 mg, and 400 mg/kg body weight of A. racemosus root ethanol extract (ARE) and IPR once daily for 15 consecutive days before exposure to lethal total body radiation of 10 Gy. Control and radiation control (RC) groups were also maintained.

On the 15th day, 1 h after drug administration, the animals were exposed to lethal total body EBR (10 Gy). From the day of onset of the experiment until the 30th day, the survival of mice from all the groups was monitored. The probabilities of the survival of all different groups until the 30th day were plotted as Kaplan–Meier survival curves.

Pre - radiation study

The animals were housed in the animal house and prior exposure to sublethal radiation dose; they were administered ARE–200 mg and IPR-400 mg/kg body weight orally once daily for 15 consecutive days. Meanwhile, control group and RC group were also maintained. Throughout the study period, food and water intake were recorded daily, whereas, body weight was recorded once in a week.

On the 15th day, 1 h after drug administration, the animals were restrained in well ventilated perspex box and exposed to sublethal dose of whole body EBR (6 Gy) at a distance of 100 cm from the beam exit point of the linear accelerator and at a dose rate of 3 Gy/min.

Estimation of cytokines

After the study period, the experimental animals were sacrificed by cervical dislocation. Liver tissue was excised and rinsed in phosphate buffer saline (PBS) and then 10% tissue homogenate was prepared in PBS which was further used for estimation of cytokines, namely, IL-2, IFN-γ, and tumor necrosis factor-α (TNF-α) using mouse ELISA kits (Koma biotech Inc.,) in an ELISA reader.

Determination of bone marrow cellularity

Bone marrow was collected using PBS containing 2% bovine serum albumin from the femurs of all the sacrificed animals. Trypan blue staining was used for the determination of cell viability. In this preparation, the number of live and dead cells was counted using a hemocytometer and expressed as total live cells per femur.[15]

Statistical analysis

The data are expressed as mean ± standard deviation. The analysis of variance was used to make statistical comparison between the groups followed by Tukey's multiple comparison test using Prism3.0 software(GraphPad Software, Inc. San Diego, CA 92121 USA). The significance between survival curves was analyzed by Kaplan–Meier survival analysis using SPSS 16.0 software(SPSS Inc., 1989-2007, IBM SPSS software). P< 0.05 was considered as a criterion for statistical significance.


   Results Top


Determination of radiation dose response curve (LD50)

The LD50 of radiation dose was determined by exposing the mice with different doses (4, 6, 8, 10, 12, and 14 Gy) of EBR. No radiation-induced mortality and toxicity was observed up to a dose of 6 Gy. 30% mortality was observed with an increase in radiation dose to 8 Gy. 70% reduction in the survival of mice was observed when exposed to 10 Gy whole body EBR, when the electron beam dose was increased to 12 and 14 Gy, 100% of the mice died. From the probit analysis, the LD50 of electron beam for acute radiation-induced mortality in mice was found to be 8.9 Gy [Figure 1].
Figure 1: Radiation Dose-response Curve (LD50) m = 0.95; LD50 = log 10 of 0.95 = 8.9

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Effect of Asparagus racemosus root ethanolic extract and isoprinosine on 30-day survival

Preadministration of 200 mg/kg b.wt of ARE increased the 30-day survival of the irradiated mice by 30% [Figure 2] by reducing the radiation sickness characteristics whereas IPR-400 mg/kg b.wt was found to offer protection to mice against radiation-induced toxic effects by increasing the survival period of 21 days [Figure 3].
Figure 2: Survival function of Asparagus racemosus root ethanolic extract

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Figure 3: Survival function of isoprinosine

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Effect of Asparagus racemosus root ethanolic extract and isoprinosine on cytokines

In our present investigation, whole body EB irradiation elevated the level of pro-inflammatory cytokines IL-2 and TNF-α in liver homogenate and decreased the level of IFN-γ in irradiated control animals. The administration of ARE and IPR before irradiation caused a reduction in the elevated levels of IL-2 [Figure 4]; RC vs. ARE and IPR, P < 0.05] and TNF-α [Figure 5]; RC vs. ARE, P < 0.05, RC vs. IPR, P > 0.05] in the pretreated animals compared to RC. Similarly, reduced level of IFN-γ was also enhanced in the pretreated animals compared to RC [Figure 6]; RC vs. ARE, P < 0.05, RC vs. IPR, P > 0.05].
Figure 4: Interleukin-2 levels of irradiated and drug-treated animals

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Figure 5: Tumor necrosis factor-α levels of irradiated and drug-treated animals

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Figure 6: Interferon-gamma levels of irradiated and drug-treated animals

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Effect of Asparagus racemosus root ethanolic extract and isoprinosine pretreatment on bone marrow cellularity

There was a drastic reduction in bone marrow cellularity in irradiated animals compared to normal control. Treatment with ARE and IPR significantly increased (P< 0.05) the bone marrow cellularity similar to untreated controls [Table 1].
Table 1: Effect of Asparagus racemosus root ethanolic extract and isoprinosine pretreatment on bone marrow cellularity

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   Discussion Top


Recent research in radiation biology mainly focuses on the identification and development of nontoxic and effective radioprotective compounds that can reduce the deleterious effect of radiation. Such compounds could possibly protect the biological system against the harmful effects of ionizing radiation which includes genetic impairment, mutation, and variation in the immune system which acts through the generation of free radicals. A single total body exposure of mammals to ionizing radiation results in a complex set of disorders whose onset, nature, and severity are determined by both total radiation dose and radiation quality. Ionizing radiation can induce damage in biologically important macromolecules such as DNA, proteins, lipids, and carbohydrates in various organs at the cellular level.

The present study revealed that preadministration of ARE and IPR improved the 30-day survival of the irradiated mice after exposure to lethal total body irradiation (TBI) by reducing the radiation sickness characteristics [Figure 2] and [Figure 3] which is in agreement with the study carried out by Li et al., 2015.[16] It also indicated that the administration of ARE and IPR were able to minimize the radiation-induced sickness features, thus confirming the protective effect of ARE and IPR on total body radiation-induced injury.

The maximum survival was observed in the experimental mice pretreated with 200 mg/kg body weight of ARE and 400 mg/kg body weight of IPR, which also reduced the radiation sickness characteristics. This dose was considered as an optimal dose for radioprotection. Treatment of mice with ARE and IPR before irradiation delayed the onset of mortality as compared with the untreated irradiated controls. The determination of LD50 of radiation in mice provided further direction for utilization of sublethal dose to carry out in vivo radiation studies.

The RC group showed the signs of radiation sickness including reduced intake of food and water, weight loss, lethargy, epilation, and complete mortality within 13 days. On the other hand, mice that were administered with ARE and IPR for 15 consecutive days before exposure of acute lethal TBI (10 Gy) had reduced signs of radiation sickness and improved survival rates.

TNF-α and IL-2 are pro-inflammatory cytokines, their synthesis is induced by a variety of stimuli including ionizing radiation. IFN-γ stimulates macrophage for its phagocytic activity and causes differentiation of T cells and cytotoxic effects.[17]

TNF-α is thought to be an important intermediary for the pathogenesis of radiation pneumonitis because it shows several spectra of biological activities in particular pro-inflammatory effects by inducing the expression of adhesion molecules that recruit leukocytes into the sites of tissue damage and inhibits anticoagulatory mechanisms and thus promotes thrombotic processes.[18] IL-2, chiefly produced by monocytes and macrophages is a pro-inflammatory cytokine. A number of stimuli including endotoxin, other cytokines and microbial and viral antigens, as well as ionizing radiation promotes the synthesis of IL-2.[19] Phagocytic activity of macrophage and differentiation of T cells and cytotoxic effects stimulated by IFN-γ.[20]

A number of synthetic and natural compounds are reported to provide radioprotection to normal cells which is mediated through free radical scavenging activity.[21]

Even low doses of radiation can induce damage to the hematopoietic system as well as the hematocytes since they are known to be sensitive to radiation.[21] Treatment of the experimental animals with ARE and IPR effectively sustained the bone marrow cellularity which is affected by the sublethal dose of radiation. This demonstrates the protective effect of the extract on stem cell proliferation.

Numerous pathways have been proposed for the mechanism of radioprotective action in mammalian cells against the deleterious effects of ionizing radiation.[22] Radioprotectors scavenge the reactive oxygen species (ROS), generated by ionizing radiation before they can interact with biochemical molecules, thus reducing the harmful effects of radiation.

The results of the present study demonstrated the radioprotective and immunostimulatory efficacy of A. racemosus ethanol root extract and IPR against EBR induced biochemical alterations and also cytokine variations.In vitro antioxidant potential of ARE and IPR has been previously reported in our study which might be responsible for its radioprotective and immunostimulatory activity.

Several mechanisms may have been contributed for the normal cell protection by ARE and IPR against harmful effects of radiation including potent antioxidant activity, immune response, and enhanced recovery of bone marrow.[23]


   Conclusion Top


Together, the findings of our study demonstrate the potential of AREand IPR in mitigating radiation-induced mortality by offering protection to mice against lethal dose of whole body EBR. The present study also demonstrates that ARE and IPR exert its radioprotection against EBR induced immunosuppression by suppressing the formation of ROS generated as a result of irradiation, stimulating the antioxidant defense and hematopoietic system of the body and regulating cytokines.

Further scientific evaluation and validation is needed to prove the radioprotective and immunostimulatory potential of AREand IPR against ionizing radiation-induced immunosuppression.

Acknowledgement

The authors would like to extend earnest gratitude to the Board of Research in Nuclear Sciences, Department of Atomic Energy, Government of India for providing the financial support (Sanction No. 34(1)/14/32/2014-BRNS). The authors remain grateful to Nitte (Deemed to be University) for providing laboratory facilities and also would like to acknowledge Dr. K.R Chandrashekar, Chairman, Department of Applied Botany, Mangalore University, Karnataka, India for the identification of the study plant.

Financial support and sponsorship

Board of Research in Nuclear Sciences, Department of Atomic Energy, Government of India.(Sanction No. 34(1)/14/32/2014-BRNS).

Conflicts of interest

There are no conflicts of interest.

 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
 
 
    Tables

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