Parkinson s disease is a progressive disorder of the nervous system primarily affecting the motor system of the body and is also known as Shaking palsy (Bendick, 2002). Parkinson's disease is the second most common neurodegenerative disorder and the most common movement disorder. worldwide studies indicate a wide range of occurrence of PD and its prevalence rates ranges from 31 to 347 per 100,000 (Coffey and Cummings 1994). Parkinson s disease was described as early as 5000 B.C. by Indians practicing the medical system of Ayurveda (Vedas). They referred to the disorder as Kampavata, kampa meaning tremor and vata meaning lack of muscular movement. The suggested remedy for PD in Ayurveda is of use of Mucuna pruriens seeds. Continued research in this direction revealed L-DOPA as the active principle present in seed responsible for relief. Since then, seeds of M. pruriens are widely used for medication as chemical synthesis of this drug is costly and hindered with disadvantage of racemic mixture which inhibits the dopa decaboxylase activity in human body (Blaschko 1942). As the demand of L-DOPA is constantly increasing, it is a need of a time to discover new sources for L-DOPA which is cost effective. In addition it is observed that the prolonged medication has resulted in L-DOPA toxicity in patients due to oxidative stress. Hence, use of antioxidant in combination with L-DOPA is required. Therefore, a L-DOPA source with a good antioxidant potential is a need of an hour. First chapter introduces a underutilized source of L-DOPA i.e. Mucuna monosperma. The chapter presents results on Mucuna monosperma, which is a promising candidate for L- DOPA production, anti- Parkinson s drug. Seed powder of M. monosperma contains 5.48% of (dry weight) L - DOPA whereas intact seeds soaked in distilled water contains 6.579%. Different elicitors enhanced the level in seed up to 11.8%. The possible rationale behind this increase was confirmed by increase in tyrosinase activity in the seeds. Presence of L- DOPA was confirmed using various analytical techniques, HPLC, HPTLC and NMR. This chapter reports for the first time maximum yield of L- DOPA from intact seeds by using a novel method. Thus, study projects M. monosperma as an efficient natural source of L- DOPA which can be a good Page 159
alternative to M. pruriens. This study also underlines the use of soaked seeds rather than seed powder, as it allowed manipulation of biochemical reactions to achieve higher production from a limited source. Attempts were also made to evaluate antioxidant potential of seed extracts. Oxidative stress is supposed to be responsible for the onset and progression of Parkinson Disease (PD). Treatment for PD will be conducive when L -DOPA is administered in presence of antioxidants. Hence, present study was conducted to understand the efficacy of antioxidant potential of Mucuna monosperma seed. Analysis of antioxidant potentiality of seeds revealed that the extract of seed powder has the DPPH scavenging activity (89.20%), FRAP activity (30.205 µg AAE/ mg), NO scavenging activity (60.06%), H 2 O 2 scavenging activity (43.41%). Principal component analysis showed that PC1 explained 86.17 % of the total variation in data set whereas PC2 accounted for 12.47%. Ability of the extract to inhibit the DNA damage due to UV radicals has also been confirmed. The significance of present findings is that M. monosperma seeds, while possessing its anti-parkinson properties, have antioxidant properties, which is established by scavenging commercially available free radicals such as DPPH, reactive oxygen species, reactive nitrogen species, and by reducing Fe +++. These effects may be due to the presence of polyphenols and flavonoids, which can undergo redox reactions to scavenge hydroxyl radicals. Various MMP extracts prevented the H 2 O 2 and UV induced DNA damage that can eventually induce apoptotic cell death and neurodegeneration as seen in PD. Thus, this study promotes the use of M. monosperma seeds which can provide added advantage of antioxidant activity and DNA damage protective effect to anti Parkinson drug. In second chapter, green leafy vegetables are evaluated as a source of L-DOPA. An easily accessible, economic source present in daily diet is vegetables. Hence, incorporation of such vegetables in diet will reduce the cost of medication significantly. Among the different vegetables screened, Anethum graveolens was found to contain near about 1.6% L-DOPA which was after optimizing the extraction buffer composition, found to be 2.2 %. Different parameters like ph, ascorbic acid and NaCl concentration were optimized for efficient extraction of L-DOPA. Consumption of the A. graveolens as Page 160
a food ingredient will also provide necessary primary and secondary metabolites. Detailed biochemical analysis af this vegetables showed that, it is a good source of carbohydrates ( 54.37%), proteins (23.76%) and fibers (13.1%), whereas, fat level is within the acceptable range. Mineral content of this plant was studied and was found to contain good levels of Ca, Mg, Fe, Na, K, Mg and Zn. This vegetable also showed presence of secondary metabolites such as phenolics and flavonoids and exhibited antioxidant potential required for treatment of PD. Presence of L-DOPA in this plant was confirmed by using HPTLC and LC-MS analysis. In third chapter, optimization of a cost effective biotransformation medium for production of L-DOPA and melanin using M. monosperma callus with the help of response surface methodology was studied. We have optimized the L-DOPA and melanin production in simple buffer containing L-tyrosine as precursor. This study is related to optimization of biotransformation performance of callus for L-DOPA and melanin production. Ascorbic acid, ph, copper sulphate and L-tyrosine concentration were identified as process affecting parameters whereas SDS was used instead of ascorbic acid for melanin production. Three-level Box-Behnken factorial design with four factors was employed to optimize significant correlation between the effects of these variables on the production of the L-DOPA and melanin. Mathematical models were developed for L-DOPA and melanin showing the effect of each factor and their interactions on colour removal. To construct the quadratic model 29 experiments were conducted. The "Pred R-Squared" of 0.9539 was in reasonable agreement with the "Adj R-Squared" of 0.9824 for L-DOPA.For melanin, The "Pred R-Squared" of 0.9972 was in good agreement with the "Adj R-Squared" of 0.9990. This indicated suitability of the employed model and the success of RSM. The optimized levels predicted by the model were tyrosine 0.894 g l -1, ph 4.99, ascorbic acid 31.62 mg l -1 and copper sulphate 23.92 mg l -1. The predicted yield of L-DOPA with these concentrations was 0.319 g l -1, while the actual yield obtained was 0.309 g l -1. In case of melanin, the optimized levels predicted by the model were tyrosine 0.778 g l -1, ph 5.85, SDS 34.55 mg l -1 and copper sulphate 21.14 mg l -1. The predicted yield of L-DOPA with these concentrations was Page 161
0.894 g l -1, while the actual yield obtained was 0.887 g l -1. According to analysis of variance (ANOVA) results, the proposed model can be used to navigate the design space. Forth chapter reveals the different aspects of the enzyme tyrosinase, the key enzyme responsible for biotransformation of L-tyrosine to L-DOPA. The tyrosinase was purified by ion exchange chromatography using DEAE cellulose column. The enzyme was eluted at the eluent (0.1 M NaCl) showing 6844 U mg -1 specific activity with an 8.3 purification fold and 9.9 % yield. The molecular weight of the purified tyrosinase was estimated by polyacrylamide gel electrophoresis was near about 13 kda. The activity staining by using L-DOPA confirmed the protein was tyrosinase. The characterization of enzyme resulted in optimum ph 7 and temperature 40 C with tyrosinase activity of 8333 U mg -1. Substrate specificity was determined using three substrates, among these substrates the enzyme showed higher specific activity towards catechol with activity of 6066 U mg -1 than L-DOPA (3466 U mg -1 ) and L-tyrosine (2311 U mg -1 ). The effect of different metal ions was tested in the present study which indicates that the activity of tyrosinase enhanced to 9157 U mg -1 in presence of CuSO 4 and it was inhibited by FeCl 3. The kinetic characterization of tyrosinase was performed using Lineweaver-Burk plot. The Km and Vmax values for tyrosinase were found to be 2.28 mm and 1724 U mg -1 respectively. Spectroscopic analysis revealed the presence of type 3 copper centre, characteristic of tyrosinase and absence of type 1 copper centre. Enzyme showed presence of RNA moety as its integral part as suggested by PAGE and agarose gel electrophoresis analysis after treatment with RNase. Activity was reduced after removal of RNA and is supported by reduction in molecu;ar weight and change in intrinsic fluroscene. In conclusion, use of natural sources like M. monosperma and A. graveolens is a need of time for treatment of PD and treatment can be made cost effective using these different methods. Page 162