|Year : 2012 | Volume
| Issue : 4 | Page : 327-332
Radioprotective property of an aqueous extract from valeriana wallichii
Omika Katoch1, Shikha Kaushik2, Mysore Sadashiv Yogendra Kumar3, Paban K Agrawala1, Kshipra Misra3
1 Institute of Nuclear Medicine and Allied Sciences, Defence Research and Development Organisation, Lucknow Road, Timarpur, Delhi, India
2 Institute of Nuclear Medicine and Allied Sciences, Defence Research and Development Organisation, Lucknow Road, Timarpur, Delhi; University Institute of Engineering and Technology, Maharshi Dayanand University, Rohtak, Haryana, India
3 Defence Institute of Physiology and Allied Sciences, Defence Research and Development Organisation, Lucknow Road, Timarpur, Delhi, India
|Date of Submission||08-Nov-2011|
|Date of Decision||30-Dec-2011|
|Date of Acceptance||11-May-2012|
|Date of Web Publication||07-Nov-2012|
Paban K Agrawala
Institute of Nuclear Medicine and Allied Sciences, Defence Research and Development Organisation, Lucknow Road, Timarpur, Delhi
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Objectives: Preparations of herbal drugs have drawn considerable interest in scientific community in recent years for the treatment of several stress related health problems including radiation-injury. Materials and Methods: An aqueous extract from Valeriana wallichii containing hesperidin as one of its major constituent was evaluated for its ability to protect against radiation-injury in model systems like plasmid deoxyribonucleic acid (DNA) and cultured human fibroblast cells. Results: The extract was found to significantly counter radiation-induced free radicals at 4 h after 5 Gy irradiation, reduced prolonged oxidative stress led increase in mitochondrial mass, enhanced reproductive viability of cultured cells and protected against radiation-induced DNA damage in solution. Discussion: Further studies are required to validate the radioprotective ability of the extract and to develop a safer radioprotective agent.
Keywords: DNA damage, herbal preparation, mitochondrial mass, radioprotection, ROS
|How to cite this article:|
Katoch O, Kaushik S, Yogendra Kumar MS, Agrawala PK, Misra K. Radioprotective property of an aqueous extract from valeriana wallichii. J Pharm Bioall Sci 2012;4:327-32
|How to cite this URL:|
Katoch O, Kaushik S, Yogendra Kumar MS, Agrawala PK, Misra K. Radioprotective property of an aqueous extract from valeriana wallichii. J Pharm Bioall Sci [serial online] 2012 [cited 2022 Jul 2];4:327-32. Available from: https://www.jpbsonline.org/text.asp?2012/4/4/327/103272
Of late, the traditionally used herbal medicines are gaining tremendous attention world-wide because of their inherent advantages such as no or minimal toxicity, easy availability, significant bioactivity and cost-effectiveness. The inherent problems like lack of scientifically documented evidences in support of their bioactivity, variability in the constituent molecules depending upon several other factors like geographical distribution of the plant, proper collection of plant material, storage and processing techniques of the material etc. are the difficulties faced for the acceptance of herbal medicines by the scientific community. With an increased technological advancement, it is now possible to identify and quantify selected bioactive components, present in herbal extracts, prepared using standard protocols. Isolation of major bioactive components and possibility of synthesizing them under laboratory conditions also has added herbal medicine research in deciphering the possible mechanism of action (s) of herbal formulations. This paper reports the results of the study on the possible radioprotective action of an aqueous extract from Valeriana wallichii, prepared in our laboratory, and attempts were made to identify some of the active components thereof.
Valeriana wallichii DC (Family-Valerianaceae) is a small 14- 45 cm height perennial herb, growing in India and Pakistan. The major known active principles of this herb are valpotriates, dihydrovaltrate,  isovalerianate,  6-methylapigenin, hesperidins  and sesquiterpenoids.  Its rhizome and root contains volatile oil (valerianic oil), which is composed of alkaloids, bornyl isovalerianate, chatinine, formate, glucoside, isovalerenic acid, 1-camphene, 1-pinene, resins, terpineol and valerianine.  From the rhizomes, some important compounds, such as citric acid, malic acid, maliol, succinic acid and tartaric acid have also been isolated.  The herb has been used successfully in traditional systems of medicine like Ayurveda and Unani against Leishmania,  diseases of eye and liver, hysteria, hypochondriasis, nervous unrest and emotional arrest; it has also been found useful in clearing voice and acts as a stimulant in advance stage of fever and nervous disorder,  inflammatory conditions like one observed after scorpion stings and jaundice  and in pain conditions  epilepsy, insomnia, neurosis, sciatica. [3,5] The plant is widely used in the treatment of anxiety and depression either alone or in combination with other herbs, specifically St. John's Wort. ,, The plant is also used in habitual constipation,  antispasmodic  and as cytotoxic.  An herbal preparation (Dhanya Panchaka Kashaya), containing Valeriana wallichii has been found to be effective in dyspeptic symptoms.  It's essential oil exhibited antimicrobial activity against large number of pathogenic bacteria and potent antifungal activity against different human and plant fungal pathogens.  The herb has been reported to contain several bioactive flavonoids like Linarin- isovalerianate  6-methylapigenin and hesperidins. 
Ionizing radiation-induced damage is mainly attributed to its ability to generate free radicals, and therefore, compounds with the ability to effectively quench or scavenge free radicals are considered to impart beneficial effects against ionizing radiation exposure.  In recent years, several herbal extracts and formulations have been reported to render radioprotective effects in vitro and in vivo. , Radioprotectors are agents, capable of protecting from the radiation injury / lethality when administered at certain time before radiation exposure, and hence their application is limited to planned exposure scenarios, like radiotherapy and rescue operations in the event of radiation accidents. Radiation mitigators on the other hand are capable of reducing radiation injury or mortality when administered during or certain time after an exposure to radiation and have a bigger role in radiation accidents, terrorist events etc. However, both the agents have their own importance and are indispensable owing to our dependence on use of radiation, increasing day by day. Hesperidin, one of the active components present in Valeriana wallichii, has been shown to render protection against radiation-induced cellular damage. 
Since the extract we prepared contains a significantly high amount of hesperidin, we considered evaluating its radioprotective potential. Besides, other components present in the extract may exert additional synergistic or antagonistic actions, and it might be possible to achieve better protection with reduced toxicity.
| Materials and Methods|| |
Valeriana wallichii root material was procured from Numero-uno Natural Herbs, Delhi, India.
100 g of powdered Valeriana wallichii (VW) root sample was soaked in 500 ml of water at room temperature. After 24 h, supernatant was decanted, and the residue was soaked again in the fresh solvent. The process was repeated for 4 times in order to sufficiently complete the extraction, and the supernatants were pooled, filtered through muslin cloth and stored in amber colored bottle. The solution was centrifuged at 8000 rpm for 10 min., the supernatant solution was lyophilized, and the dried extract was stored at 5°C for the further studies. 
Waters HPLC system (Waters Corporation, USA), equipped with Waters 515 HPLC pump, Waters 717 plus auto-sampler and Waters 2487 UV detector was used. The separation was performed on a Symmetry C18 250 X 4.6 mm ID; 5 μm column (Waters, USA) by maintaining the gradient flow rate 0.75 ml/min of the mobile phase (solution A; Water: O-phosphoric acid 99.7: 0.3 and Solution B; acetonitrile:methanol 75: 25) and peaks were detected at 285 nm wave length. Identification of hesperidin in the prepared extract was performed on the basis of the co-injections and retention time matching with the standard. The calibration curve was prepared using standard stock solution of hesperidin (1 mg/2 mL) in DMSO. The stock was filtered through 0.22 μ m filters (Millipore), and appropriately diluted (0.01 - 100 μ g/mL) to obtain the desired concentrations in the quantification range. The calibration graph was plotted after linear regression of the peak areas versus concentrations. The HPLC profile of the extract has been shown in [Figure 1]a-b represents the structure of hesperidin. The concentration of hesperidin in the extract was estimated from the calibration curve as depicted in [Figure 1]c and [Table 1]. A 2.5 mg/ml solution of the extract was used for the estimation of the hesperidin content in the extract.
|Figure 1a: HPLC fingerprint of Valeriana wallichii aqueous extract obtained at described in "Material and Methods" section. Hesperidin was used as an internal control |
Figure 1b: Chemical structure of hesperidin
Figure 1c: Calibration curve for estimation of hesperidin concentration in the extract
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Cell culture and deoxyribonucleic acid (DNA)
SV-40 transformed human embryonic lung fibroblast cells, MRC-5 V1, were cultured in MEM containing 10% FBS, 2 mM L-glutamine and antibiotics in a 5% CO2 environment at 37 o C and 70% - 80% humidity. For all the experimental processes, cells were seeded at 0.1 x 10 6 cells/ ml, and all the treatments were carried out when the cells are in the exponentially growing phase. Plasmid DNA (pUC 18) was purchased from Life Technologies, India Ltd. and diluted in Tris buffer (pH 7.4) as per requirement.
Irradiation of cells was performed using Bhabhatron II (Panacea) cobalt teletherapy unit at a dose rate of 1.17 Gy/min (SSD 120 cm, field 20 × 20 cm). Irradiation of DNA solution was performed using Gamma Cell-5000 (Atomic Energy, Canada) at a dose rate of 40 Gy/min.
Plant extract and hesperidin treatment
The lyophilized plant extract was dissolved in double distilled water to obtain the desired concentrations and 0.22 μ sterile filtered before adding to the cultures. Commercially available analytical grade hesperidin standard compound, obtained from M/s. Merck India Ltd., was dissolved in DMSO and further diluted in double distilled water to obtain desired concentrations, sterile filtered and used.
Plasmid relaxation assay
The induction of strand breaks in plasmid DNA was determined as described earlier  with minor modifications. Briefly, pUC 18 plasmid DNA (250 ng) was incubated at 37°C for 30 min in Tris buffer, pH 7.4, with different concentrations of the extract in a final volume of 10 μl and exposed to 150 Gy γ-radiation thereafter. Normal un-irradiated control was kept for comparison. 2.5 μl of neutral gel loading buffer was added to the DNA solution, and the different forms of plasmid DNA [supercoiled (SC), open circular (OC) and linear (L)] were separated on 0.8% agarose gels in Tris-acetate- EDTA (TAE) buffer. The gels were stained with ethidium bromide and photographed. Band intensity was quantified using image analysis and Image Quant program. A correction factor of 1.4 was applied to correct for low fluorescence yield of ethidium bromide in SC DNA.
The clonogenic assay measures the reproductive viability of cells as a function of various stresses and was performed as described earlier.  Briefly, 150 cells were plated in triplicate 60-mm culture dishes (Nunc) for each treatment groups as mentioned in the figures, and the number of colonies containing more than 50 cells each were counted 12 - 14 days after various treatments.
Estimation of reactive oxygen species
The level of ROS after various treatments were estimated flowcytometrically  using the fluorescence dye DCF-DA (Molecular Probes, USA). Briefly, cells were treated with different concentrations of the plant extract with or without 5 Gy γ-irradiation, 10 μM DCF-DA was added to each plate 30 min before harvesting, and at least 10000 cells were acquired for each sample on a Beckton-Dickinson FACS Calibur flowcytometer equipped with suitable optics.
Determination of mitochondrial mass
Prolonged oxidative stress leads to an increase in mitochondrial mass that can be measured flowcytometrically using fluorescent dyes like mitotracker green. MRC-5 V1 cells were harvested at 96 h after various treatments following incubation with 10 μM mitotracker green for 30 min, and cells were fixed in 1% para-formaldehyde in PBS. At least 10000 cells were acquired for each group on a Beckton-Dickinson FACS Calibur flowcytometer, equipped with suitable optics. 
Cell cycle analysis
Distribution of the cells in different phases of the cell cycle was analyzed flowcytometrically, based on the cellular DNA content using propidium iodide.  Briefly, cells were harvested at different time intervals following various treatments, fixed in chilled 70% alcohol, washed and stained with 50 μg/ml PI in the presence of 200 μg/ml RNase A for 30 min at 37° C before acquired on a Beckton-Dickinson FACS Calibur flowcytometer, equipped with suitable optics.
All the experiments were repeated at least 3 times, and the data presented is mean ± SD of 3 independent experiments. Statistical significance was determined by performing students' t-test, and P < 0. 05 was considered significant.
| Results|| |
HPLC analysis of the extract
The concentration of hesperidin, one of the known and bioactive component present in the extract, was estimated based on a standard calibration curve taking high purity hesperidin was found to be 6.18 ± 0.26 mg/g extract [Figure 1]c.
2 Gy γ-irradiation led to significant (more than 50%) decrease in the number of reproductively viable colonies as compared to untreated control cells [Figure 2]a. All the concentrations of hesperidin rendered significant increase (80 - 90%) in the number of colonies, 20 μg/ml being most effective. This observation correlates well with the published data of Kalpana et al. (2009) where they have found a similar concentration (16.38 μM) to be most effective against radiation exposure using different end-points. The plant extract also exhibited a similar pattern, and a concentration of 50 μg/ml [Figure 2]b of the extract was found to be most effective in protecting against radiation-induced reproductive cell death (~ 70% colonies) compared to un-irradiated control.
|Figure 2a: Clonogenic assay was performed with 150 cells seeded onto 60 mm culture dishes and number of visible colonies containing more than 50 cells counted 14 days after various treatment. The number of colonies obtained in untreated control group was considered as one, (a) The cells were exposed to 2 Gy γ-radiation with or without 10, 20 or 30 μg/ml hesperidin administered 30 min before the time of irradiation. |
Figure 2b: Clonogenic assay was performed with 150 cells seeded onto 60 mm culture dishes and number of visible colonies containing more than 50 cells counted 14 days after various treatment. The number of colonies obtained in untreated control group was considered as one, (b) The cells were exposed to 2 Gy γ-radiation with or without 25, 50 or 100 μg/ml of the plant extract administered 30 min before the time of irradiation
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Estimation of reactive oxygen species
The quantum of free radical yield 4 h after 5 Gy irradiation was about 1.4 times higher [Figure 3] compared to the un-irradiated controls. The plant extract alone (50 μg/ml) did not exhibit any increase in ROS compared to control group while it could significantly counter radiation-induced free radicals bringing back the values comparable to control values.
|Figure 3: The cells growing exponentially in 35 mm culture dishes were exposed to 5 Gy γ-radiation with or without 50 μg/ml of the plant extract administered 30 min before irradiation. Cells were harvested 4 h after irradiation following a treatment with 10 μg/ml DCF-DA to each culture and live cells were analyzed flowcytometrically. The mean fluorescence intensity of DCF-DA for each group was plotted|
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Determination of mitochondrial mass
The relative fluorescence intensity of mitotracker green was significantly higher 96 h after 5 Gy radiation exposure (nearly 9 folds) as compared to the control group [Figure 4], however, none of the extract alone treated groups showed any increase in fluorescence intensity, indicating the extract to be non-toxic. Plant extract, treated 30 min before irradiation at all concentration, could bring back the fluorescence value close to the control value, 50 μg/ml of the extract being most effective.
|Figure 4: The cells growing exponentially in 60 mm culture dishes were exposed to 5 Gy γ-radiation with or without 25, 50 or 100 μg/ml of the plant extract administered 30 min before irradiation. Cells were harvested 96 h after irradiation following a treatment with 10 μM mitotracker green to each culture and live cells were analyzed flowcytometrically. The mean fluorescence intensity of mitotracker green for each group was plotted|
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Cell cycle analysis
Cell cycle analysis was performed only for the short time intervals (up to 6 h post-irradiation), during which no significant change in cell cycle distribution was observed for any of the treatment groups [Figure 5].
|Figure 5: Cells were grown in 60 mm culture dishes and exposed to 2 Gy γ-radiation with or without 25, 50 or 100 μg/ml of the plant extract administered 30 min before irradiation. Cells were harvested at 2, 4 or 6 h after irradiation time, fixed in chilled 70% alcohol for overnight and acquired on a flowcytometer following staining with propidium iodide. The percentages of cells in different cell cycle stages were estimated using MOD-FIT software|
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Plasmid relaxation assay
The control sample showed about 90% DNA, existing in super coiled (SC) form with no linear form (L) of DNA, representing double strand break and only about 10% of open circular (OC) form, representing single strand break [Figure 6]b. Treatment with any of the 3 concentrations of the extract exerted significant change in the fractions of different forms of DNA, indicating the extract alone to have no effect on the integrity of DNA. An exposure to 150 Gy of γ-irradiation induced severe DNA damage as was evident from the appearance of a large fraction of linear form and significant increase in the fraction of open circular form at the expanse of the SC form (6.5 ± 2.5% against 87 ± 3.5% in control sample). The maximum amount of DNA retained in SC form (75.32 ± 3.87%) was observed for the DNA treated with 50 μg/ml of the extract 30 min prior to irradiation though all the 3 concentrations of the extract rendered significant protection of plasmid DNA against radiation-induced single and double strand break formation [Figure 6]a.
|Figure 6b: The band (super coiled-intact DNA, open circular- DNA with single strand break and linear-DNA with double strand break) intensities were obtained using a densitometer following ethidium bromide staining and quantity of different form of plasmid DNA were estimated as described in text. The fraction of intact (super coiled) DNA calculated and plotted against corresponding treatments|
Figure 6a: pUC-18 plasmid was irradiated with 150 Gy γ-radiation with or without 25, 50 or 100 μg/ml of the plant extract and separated on a 0.8 % agarose gel in TAE buffer. Lane 1-control pUC-18 plasmid DNA, lane 2-pUC-18 DNA exposed to 150 Gy, lane 2-5-pUC-18 DNA treated with 25, 50 or 100 μg/ml of the plant extract for 30 min and lane 6-9- pUC DNA treated with 25, 50 or 100 μg/ml of the plant extract for 30 min before 150 Gy γ-irradiation. OC = Open circular, L = Linear and SC = Super coiled forms of the plasmid DNA,
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| Discussion|| |
The ability of ionizing radiation to generate high levels of free radicals in the cells has been attributed to majority of the radiation-induced injury to living systems.  Those free radicals can damage to virtually all bio-molecules including DNA, the genetic material and most critical target of radiation injury. Any compounds, having the ability to neutralize the free radicals and/ or to repair the free radical-induced damage to critical target molecules have, therefore, been considered beneficial in the events of radiation exposure.  Several antioxidants and flavonoids have also shown promising results in vitro and pre-clinical studies  in rendering protection against ionizing radiation-induced injuries. Herbal extracts contain several secondary plant metabolites including compounds with high antioxidant potential, such as flavonoids, and many flavonoids from plant origin have been shown to render radioprotection. 
Hesperidin, one of the active component and major constituents identified in the extract, recently has been reported to protect cultured human lymphocytes against radiation-induced cellular damage, including genotoxicity.  In this study, a concentration of 16.38 μM hesperidin was observed to be most effective in rendering radioprotection. In our study, we observed similar doses (10 and 20 μg/ml) of hesperidin to be most effective in protecting MRC5 V1 cells against radiation-induced reproductive death [Figure 2]a. Similar results were obtained with the plant extract pretreatment, and a concentration of 50 μg/ml of the extract was found most effective. The estimated hesperidin concentration in 50 μg of the extract is about only 0.31 μg, which is significantly (about 32 times) less than the effective concentration for hesperidin alone. The already reported effective concentration for hesperidin in terms of protecting against cytogenetic damage equals to a concentration of 10 μg/ml (16.38 μM). Therefore, the radioprotective ability of the extract cannot be attributed solely to the hesperidin content alone, and other components present in the extract must be contributing significantly to its radioprotective efficacy. However, it should be noted that the biological end points under investigation and the model system of choice in the current study are not the same cytogenetic damage analysis, and hence the likely effective concentrations for the radioprotective efficacy need not be the same as has been reported earlier. Nevertheless, hesperidin, at a similar concentration showing significant protection in our study as well, warrants further investigation for its future radioprotective applications. Same (i.e. 50 μg/ml) concentration of the extract was found to counter radiation-induced free radicals and increase in mitochondrial mass significantly. It could also protect naked plasmid DNA in solution against radiation-induced strand breaks significantly. Since strand break in irradiated plasmid DNA solution can arise predominantly due to free radicals generated as a result of hydrolysis of water by radiation exposure, it can be assumed that the extract possess high free radical scavenging potential. Treatment with the extract alone was observed to have no effect on the level of DNA damage in plasmid DNA, cellular free radicals and mitochondrial mass. The reduction in radiation-induced ROS and mitochondrial mass in cultured cells also support this assumption. The protection of DNA is crucial not only for the immediate survival of the cells and organism as a whole, but also important in the manifestation of delayed effects of ionizing-radiation exposure like mutagenesis and carcinogenesis. Any compound that can protect DNA from damage induction or repair existing damages faithfully while not inducing any damage by itself, therefore, can be a good candidate for radioprotection.
Further studies are warranted to validate the radioprotective potential of the extract. Most important is the identification and quantification of components other than hesperidin besides their role in isolation and in combination needs to be elucidated. It is also possible that the plant can be exploited as a potential source for hesperidin extraction.
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