Neuroprotective effect of essential oils from Nardostachys jatamansi and Valeriana jatamansi

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Neuroprotective effect of essential oils from Nardostachys jatamansi and Valeriana jatamansi against Methyl mercury induced toxicity using rat brain mitochondrial fraction

Abstract

Methyl mercury (MeHg) is a form of organic mercury in the environment and has become a threat to mankind over the past decades. It is supposed to have associations with several neurological disorders like Hunter Russell syndrome and Autism. As there is no proper treatment, combining the holistic approach and biochemical interventions may be useful in identifying drug for several neurological disorders. The traditional Indian herbs like Nardostachys jatamansi and Valeriana jatamansi, were taken in the present study for assessing their neuroprotective effect against MeHg induced toxicity using rat mitochondrial fractions. The results showed that MeHg decreased mitochondrial viability in MTT assay and the IC50 value was found to be 10µM. Our results also showed that MeHg significantly inhibited the activities of glutathione, catalase and increased the levels of TBARS in rat brain mitochondrial enriched fractions. These alterations were prevented by the preincubation with essential oil extracts of N. jatamansi and V. jatamansi. It also reduced GSH oxidation caused by MeHg confirming its chelating effect, one of the molecular mechanisms of protection against oxidative damage. GC-MS analysis of these extracts detected seven compounds in each. The presence of these essential oils is one of the factors which might reveal neuroprotection against MeHg.

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Keywords: Nardostachys jatamansi , Valeriana jatamansi , Methyl mercury, Oxidative stress, Neuroprotection.

1. INTRODUCTION

Methyl mercury is apervasive organic form of mercury. It is an environmental pollutant which provokes neurological, developmental and molecular malfunction in humans and animals [1]. It is most prevalent in freshwaters and marine ecosystems. For most of the people, this type of exposure,

materializes when consumed with MethylMercury contaminated fish or shellfish, as a result of bioaccumulation [2]. One of the most serious consequences of such exposure was pictured back in 1953 where consumption of MeHg by the people residing near Mina Mata Bay, Japan resulted in an epidemic of its poisoning.Upon ingestion, MeHg gets easily absorbed in the digestive tract, complexing with cysteine. This results in easy absorption by the cells and finally gains access to bloodstream. From where it gets distributed to various parts organs especially brain and cause damage to the nervous system.Once in the brain, it hampers the overall nerve cell growth and differentiation hence becoming one of the most potent neurotoxicant [3]. Its neurotoxicity has been allied with mechanism of Glutathione imbalance and oxidative stress. MeHg induced reduced levels of enzymatic and non enzymatic antioxidants, caused lipid oxidation and enhanced the formation of Reactive Oxygen Species (ROS) [4].MeHg oral intoxication in experimental animals led to impaired developmental growth, disruption of the brain organization, ataxia, altered calcium homeostasis and generalized weaknesses[5].Hence it is essential to find a possible treatment of such toxicity and removal of the accumulated metals in the tissues. One of the promising approaches towards MeHg toxicity is the use of chelating agents in a view to eliminate the metal. However, the use of such drugs is not completely characterized and it has been implicated with adverse side effects [6].

Present day world has witnessed the importance of the Traditional system of medicines like Ayurveda, Siddha and Unani. These folk-lore based systems since remote ages have shown to contribute in healing many diseases and disorders. Nardostachys jatamanasi and Valeriana jatamanasi referred in Ayurveda are indigenous herbs found in the Himalayan regions. The decoction of N. jatamansi exhibits therapeutic approach towards epilepsy, hysteria, mental weakness, convulsions and cardiac diseases [7] while that of V. jatamansi aids in the treatment of epilepsy, skin diseases, snake bite and is considered to have remarkable sedative effects in nervous unrest, stress and neuralgia [8]. Neural injury is supposed to be allied with oxidative stress induced by free radical formation and deflated anti-oxidant activity. This kind of prognosis is the prime mover in neurological disorders like Autism and Hunter Russell syndrome. Methyl mercury is the major effecter of such conditions and is responsible for its pathogenesis [9]. Hence it is requisite to capitalize the potency of medicinal drugs like N. jatamansi and V. jatamansi in assessing such neuroprotective effects. The basis of in vitro studies carried out on rat using the essential oils from these herbs as a remedy against the methyl mercury generated stress state has been elicited this work.

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2. MATERIALS AND METHODS:

2.1 Experimental Animals

Adult Wistar rats 9 months old were bred in Animal House of VIT University .All the experiments were approved by the Institutional Animal Ethics Committee (VIT/IAEC/VIIth/14). The animals were maintained at 23°C on a 12h light/dark cycle with free access to water and food (VIT, Vellore, India).

2.2 Chemicals

MeHg (II) chloride, DTNB, GSH reduced form and MTT were purchased from Sigma-Aldrich (St-Louis, MO). All other chemicals used were analytical grade.

2.1. Plant Material

The plant material which is the rhizome of Nardostachys jatamanasi was collected from Tungnath, Rudraprayag, Uttarakhand at an altitude of 3500 masl whereas Valeriana jatamanasi was collected from Khirsu, Pauri Garhwal, Uttarakhand at an altitude of 1800 masl during the month of October.

2.2. Extraction of Volatile oil

The rhizome of both the plants were cleaned from redundant matter, washed and air dried separately. The dried rhizomes were metamorphosed to coarse particles through grinding. The resulting weights of N.jatamansi and V.jatamansi were 120 grams and 68 grams respectively. Both the jatamansi species were hydro distilled in a Clevenger apparatus with time ranging from 4-6 hours to yield their respective oils [10].

2.3. Isolation of mitochondria from rat brain

The animals were sacrificed and their brains were dissected out. Mitochondrial enriched fractions were prepared essentially as described by Anderson et al. 2003 [11]. The brain homogenate was subjected to centrifugation at 17,500 rpm for 10 min at 4°C, resulting in a myelin-rich supernatant and a pellet consisting of synaptosomes and free mitochondria. The supernatant was discarded and the pellet was resuspended in the isolation medium but without albumin. Pellets were further centrifuged at 10,700 rpm for 15 min. The mitochondrial enriched fractions were kept on ice until the experiments were performed.

2.4. Protein estimation

The total protein content of the brain homogenate was assayed by using Folin- Ciocalteau colorimetric method described by Lowry et al. (1951) [12].The standard graph was obtained by taking bovine serum albumin as standard.

2.5. Incubation

The pellets (2mg of protein) were incubated in 10µM of MeHg and the essential oils (0.5, 1.0, 1.5 and 2.0µl) from N.jatamansi and V.jatamansi exclusively in isolation medium, in a total incubation volume of 150 µL. The incubation was carried out at 25°C for 30 min and biochemical assays were performed.

2.6. MTT cell viability assay

Mitochondrial function was assessed by the conversion of the dye 3- (4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetra zolium bromide (MTT) to dark purple formazan [13]. After incubation of the pellets with MeHg and oils, (30 min at 25 °C), the reaction medium (150 µL) was incubated with 150 µL of 1.2 mM MTT for 20 hours at 25°C. The purple formazan crystals were pelleted by centrifugation, and the supernatant was discarded. The pellets were dissolved in DMSO and absorbance was measured at 550nm. Data were expressed as percentage of control

2.7. Assay for Thiobarbituric acid reactive substance, a marker of lipid peroxidation

Lipid peroxidation levels were measured as TBARS according to the method described by Ohkawa and collaborators (1979) [14]. Briefly, tissue homogenate along with the oil was reacted with 2-thiobarbituric acid (0.375%), trichloroacetic acid (5%) and HCl (0.25N) in the ratio of 1:1:1. The mixture was incubated at 95 °C for 60 min. The absorbance of pink color formation in the supernatant was measured at 535 nm. Data were expressed as percentage of control.

2.8. Assay for reduced glutathione content

Reduced glutathione content was determined by the method followed by Allen et al. 2001 [15]. Briefly, the assay mixture contained 0.5 ml of the homogenate, 4 ml of 0.1 M phosphate buffer (pH 7.0) and 0.15 ml DTNB (34 mg in 100 ml of 0.1 % sodium citrate). The absorbance was measured at 412nm using GSH as a standard. Data was expressed as percentage of control.

2.9. Determination of Catalase activity

The catalase activity of the extracted oils was determined according to Sinha 1972 [16]. The reaction mixture consisted of 0.01M phosphate buffer (pH 7.0), 0.2 M H2O2 and 2 ml acetic acid: potassium dichromate (3:1) along with the 0.1 ml homogenate, in a final volume of 3.5 ml. This mixture was kept in boiling water bath for 10 minutes and the O.D was recorded at 620 nm. Data was expressed as percentage of control.

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2.10. GC-MS analysis

The essential oils analysis was carried out on PerkinElmer Claurus 600 mass spectrometer coupled with Claurus 680 gas chromatograph. The mass spectrometer was operated in the electron impact (EI) mode with the ionization energy of 70eV.The Helium was used as carrier gas at a flow rate of 1mL/min, while Elite-5MS (30.0, 0.25mmID, 250μm df) capillary column was used. The column oven temperature was programed between 60°C to 300°C.The initial temperature was programmed at 60°C for 2 min at the rate of 10°C/min and then 300°C for 6 min, with a total run time of 32 min. The injector and detector temperature were maintained at 250°C and 240°C respectively.1μL of the sample was injected and resolution of the components were attained. The identity qestablished using retention time and peak enhancement with the standard samples in gas chromatographic mode and comparing with the National Institute of Standard and Technology(NIST), USA mass spectral database. The relative amounts of detected compounds were calculated based on GC peak areas.

2.11. Statistical Analysis

Statistical differences were analyzed by one-way analysis of variance. Statistical significant difference were considered when P < 0.05.

3. RESULTS

3.1. MTT assay

The effect of essential oils of N.jatamansi and V.jatamansi on cell viability in mitochondria of rat brain has been shown in Fig 1. Treatment of cells with MeHg significantly showed cytotoxicity. It is inferred that N.jatamansi and V.jatamansi protects the cells even in low concentration (0.5µL N.jatamansi and V.jatamansi exhibited 51% and 72 % cell viability respectively). With the increase in concentration, it showed higher protection to the cells against MeHg. On the other hand at lower concentration i,e 0.5µL -1µL, V.jatamansi showed more protection than N.jatamansi but interestingly N.jatamansi from 1.5 -2µL exhibited higher protection i.e 109%.

Fig 1.MTT cell viability assay

3.2. TBARS

The protective role of N.jatamansi and V.jatamansi against oxidation of lipids has been interpreted in the Fig 2. Oxidation of lipid was increased upon inducing the cell with MeHg.The results showed that both N.jatamansi and V.jatamansi showed protection against lipid peroxidation. The level of protection against lipid peroxidation was not concentration dependent. At all the concentration, N.jatamansi was slightly more protective than V.jatamansi.

Fig 2.TBARS assay

3.3 .Glutathione content

The effect of N.jatamansi and V.jatamansi on glutathione level of the cells has been shown in Fig 3. The results showed that the level of glutathione is concentration dependent. At all concentrations, the level of Glutathione was higher in N.jatamansi in comparison to V.Jatamansi at a concentration of 2ug/mL, N.jatamansi exhibited 23% higher glutathione than V.jatamansi.

Fig 3.Glutathione content

3. 4. Catalase activity

Figure.4 shows the results of catalase activity of the N.jatamansi and V.jatamansi. The level of catalase activity was reduced in cells treated with MeHg, but elevated level of catalase was observed in cells treated the both the oil and MeHg. From the figure, it can be inferred that catalase activity increased with concentration for both the oil. In all the concentration, 0.5µL, 1µL, 1.5µL and 2µL, V.jatamansi showed 8% more catalase activity in comparison to N.jatamansi. Therefore, V.jatamansi is more protection than N.jatamansi.

Fig 4. Catalase activity

3. 5. GC-MS analysis

The GC-MS analysis of essential oils of N.jatamansi and V.jatamansi allowed the detection of 7 compounds. The compounds were identified on the basis of retention time.

Out of the 7 compounds identified in N.jatamansi and V.jatamansi, 3 were sesquiterpene. Patachouli alcohol was the major sesquiterpene identified in both the oil.

The chromatogram of N.jatamansi and V,jatamansi is given in Figure 5 and Figure 6 representing time on the X- axis and relative abundance on the Y-axis.

Fig.5. Chromatogram representing GC-MS analysis of N.jatamansi

Fig.6. Chromatogram representing GC-MS analysis of V.jatamansi

4. DISCUSSION

The damage of the mitochondria is caused by intracellular generated ROS. The level of ROS was found to be increased in damaged mitochondrial cells [17]. In the present study, when the mitochondrial cell of rat brain was exposed to MeHg, low cell viability was observed. The cell viability of the cells was seen to be concentration dependent. When mitochondrial cells, preincubated with low concentrations (0.5µL and 1µL) of oil obtained from N.jatamansi, the cell viability was still low. However, in comparison, at low concentrations (1.5µL and µL), V.jatamansi showed a higher protection to the cells. On the other hand, N.jatamansi showed protection to the cells against MeHg at higher concentrations (1.5-2µL).

Glutathione plays an important role in the functioning of mitochondria. Studies have shown that damaged mitochondrial cells have low GSH levels [18]. The glutathione level in cells on treatment with the oils gives us the idea about its antioxidant capacity. The glutathione level of the cells on treatment with both the oils showed significant increase with the increase in the concentration.

Catalase is an anti-oxidative enzymes. Reduction of this enzyme is seen in damaged mitochondrial cells. To determine the activity of the essential oil of N.jatamansi and V.jatamansi, catalase assay was carried out. On comparing the catalase activity of the cells pre-treated with oil obtained from N.jatamansi and V.jatamansi, it was observed that V.jatamansi was more protective at all the concentrations.

In comparison of our results with the literature it was indicated that in both the oils, monoterpenes and coumarin derivates were not identified. The major sesquiterpine was patachouli alcohol.

5. CONCLUSION

From our study, it is inferred that the essential oil from N.jatamansi and V.jatamansi have a potential neuroprotective effect against MeHg in rat brain mitochondrial enriched fractions. The study also reveals the relation between the different compounds present in N.jatamansi and V.jatamansi and their degree of neuroprotective action. In conclusion, based on the chemical profile and biochemical assays of the two essential oil obtained from Katmandu valley were found to be of comparable quality. Further studies are required to determine the mechanism of action of the essential oil against neurodevelopmental disorders.

6. ACKNOWLEDGMENTS

The authors are thankful to VIT University management for infrastructure, constant support and encouragements.

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