INTRODUCTION:

The L- isomer of dopa [3-(3,4-dihydroxyphenyl)alanine] is being used for symptomatic relief of Parkinson's disease. L-dopa is a neurotransmitter; content of dopa in brain tissues is reduced due to blockade conversion of tyrosine to L-dopa. While reaction, tyrosinase and dopa oxidases eliminates an e- pair from the –OH l groups of a diphenolic substrate and generate quinones, laccases remove single e- from the reducing group of the substrate and the products generated are usually free radicals.

DOPA treatment improves motor function at the onset of disease and stimulates human growth hormones released by the pituitary glands that are used as anti-hypertensive.

(Hussain G. et. al. 1997). Perumal Siddhuraju (2000) studied on the nutritional composition of dopa and its antinutritional factors of three different germplasm seed materials of an under-utilized tropical legume, mucuna pruriens Var. Perumal Siddhuraju et al (1996) showed dopa Chemical composition and protein quality of the little-known legume, velvet bean (Mucuna pruriens L.(DC.) In bio-adhesion studies, the catecholic amino acid 3,4-dihydroxyphenylalanine (DOPA) has been identified as an important molecule in bio-adhesive proteins such as those used by mussels to attach to rocks. (Guvendiren, M.; 2009 Waite 1999; Lin, Q. 2007; Hwang 2010) This discovery has spurred numerous investigations of potential applications of dopa to the development of new adhesives and antifouling materials.(Statz, 2005; Lee 2007) surface complexion study of dopa from rutile (r-TiO2) in NaCl solution also observed for adhesive property (Salima Bahri et al 2011). Catechol redox induced formation of metal core-polymer shell nanoparticles showing diverse function; the ability to adhere to almost any material of either organic or inorganic origin. ( Kvar C.et al 2011)

Plants belonging to Solanaceae family have great potential for the neuroprotective activity due to presence of phenolic substances. In India, these plants are used are as male tonic and for male vitality in traditional medicine. Anti-Parkinson property probably due to presence of L-dopa and other active components may increase effectiveness of it from herbal source in comparison to synthetic L-dopa and its conversion from tyrosine (Misra L, Wagner H. (2007). Due to its relatively high demand and spiraling price in the market and for huge scale production; need to intervene environment sources.

Leaves of Solanum melangena is thrown as waste after its fruits productions therefore it is selected for the

1. Extraction of L-dopa and subsequently application of it, as a substrate of enzyme to validate Alium Cepha L. o-diphenol oxidase activity.

2. Were evaluated for their physicochemical properties as well as their nutritional and antinutritional characteristics.

Since the UV absorption spectra of L-tyrosine and L-Dopa are very similar, spectrophotometric methods are not suitable (Gulendem et.al.,1999). The colorimetric method based on chemical procedures (Arnow 1937) is time consuming, requires specific reagents and is influenced by substances such as ascorbate which are likely to be present in the medium. HPLC, currently the most common method, requires sophisticated instruments and expensive reagents (Huizing et.al.1984). In this paper a rapid and sensitive quantitation method based on TLC along with UV, LCMS, ESR and ICPAES is presented for structural elucidation of dopa.

Dopa oxidase

Spontaneous conversion

MATERIALS AND METHODS:

All solutions and suspensions were made from Milli-Q-water. Solanum melangena L. were collected from Dhule, and Nanded District, Maharashtra. Alium Cepa L was collected from local market Nasik for the source o-diphenol oxidase.

Methods:

Extraction of L-dopa from Solanum xanthocarpum L and Solanum melangena L.⁵:

Extraction of L-dopa with water- ethanol (1:1) under ascorbic acid protection was carried out using dried leaves of Solanum melangena L. The dried leaves (100 g) were defatted with acetone (300 ml) by shaking for 48 hours at room temperature and defatted material was extracted with water-ethanol (1:1) with 0.1% ascorbic acid (3 X 500 ml) by shaking overnight. The residue was removed by filtration and filtrates were pooled and concentrated to yield crude L-Dopa.

Crystallization of L-dopa:

The above crude L-Dopa, was crystallized and further re-crystallization in hot water to procured pure crystals.

Characterization of isolated dopa:

Pure crystals of dopa from Solanum melangena leaves were subjected to thin layer chromatography (TLC), UV spectra IR spectroscopy and ESR.

Thin layer chromatography:

A rapid and sensitive quantitation of amino acid, TLC is quick monitoring tool.TLC plates were run in n-butanol-n-propanol-water, acetic acid (3:3:2:1) and spots were visualized by ninhydrin reagent and it was compared with standard amino acids which were run as co-TLC to ascertain the presence of amino acids (Rf in Parenthesis)

UV:

UV maxima for isolated L-dopa were determined by dissolving in proper solvent system and compared with aqueous media. The concentrations of amino acids in the supernatant were measured with UV-vis spectroscopy. Triplicate absorbance readings were performed for each sample.

DOPA was analyzed directly in the spectrophotometer without derivatisation using phosphate buffer pH 7. UV-vis spectroscopy has been shown previously to be a suitable technique for quantifying aqueous concentrations of DOPA at a wavelength of 280 nm. Hence, a calibration curve was determined for DOPA at this wavelength using a phosphate buffer medium to pH values less than 7 above which DOPA is known to oxidize. (Hamada at al 2007; Barreto et al 1999)

ESR Methods:

Samples in the form of nominally dry powders were weighed and placed in 3 mm ID quartz tubes, Stoppard and examined at SAIF- IIT (Powai) Mumbai.

For purposes of comparison, a normalized intensity (NI) was calculated using the expression (Leroy Chauffe, et al1975)

(NI) = h (LW) 2

(Mod)(Gain)(Wt.)

Where: h is peak to peak height of derivative spectrum; LW is line width measured between points of maximum slope; wt is sample weight; and mod, gain are the spectrometer modulation amplitude and gain settings. It was assumed that the line shape for all of the dopa spectra were the same. Microwave power levels of 5 mW or lower were used to minimize saturation effects. The electron g-values were determined. The magnetic field at the sample was determined by calculating a correction term to the Fieldial reading using a series of solid substances which g-values were accurately known.

Extraction of O-Diphenol Oxidase by solvent and salt method

Fresh onion leaves (300 g) were homogenized with 150 ml of ice-cold acetone, water with Citron X-100 (80:19:1, V/V) for about 24 hrs (-18°C).After filtration through a muslin cloth the residue was washed 3x with 150 ml of acetone (-18°C). The resulting acetone powder was dried for about 30 min. at room temperature and was suspended in 200 ml of 0.05 M phosphate buffer (pH 7.0), containing 0.01mM EDTA, and 0.1mM ascorbic acid. Sonicated for 1 hr. and then centrifuged at 5600 g for 15 mins. Solid ammonium sulfate was added to 60 % molar saturation, the solution was stirred for about 3 hrs and kept it for about 24 hr. at 4°C, centrifuged at 5600 g for 30 mins. The resulting pellet was homogenized in 50 mM sodium phosphate buffer (pH 7.0) and dialyzed for about 20 hrs against the same buffer at 4°C. The dialysate was used as active enzyme. O-diphenol oxidase activity and protein were measured at each stage.

Use of isolated dopa as substrate:

L-dopa isolated from Solanum melangena leaves was used as substrate and compared with standard L-Dopa (Merck). O-diphenol oxidase assay was carried out by Shimodo et al. (1997). Protein content was measured as per Lowry method. o-diphenol oxidase was isolated from onion leaves on natural column.

Quantitative analysis of dopa by colorimetric method:

Quantitative analysis of dopa was estimated by colorimetric method of Arnow⁶with slight modification. This method is based on simple chemical procedures, where no enzyme is required. Possible mechanism of melanin formation is nothing but the mixture of 3, 4-dihydroxyphenylalanine-tyrosine (Xu Zhang et al 2010). Interconversion of these amino acids are so rapid (Peter K. Macnicol 1977 ) ) therefore it is necessary to estimate tyrosine colorimetrically.

i) Determination of 3,4 – di-hydroxy phenylalanine :

One ml of standard L-dopa from Merck was used to determine the synthetic L-dopa from Solanum melangena for the comparison. 3,4-dihydroxyphenylalanine.

ii) Determination of tyrosine:

One ml of standard L-tyrosine from Merck was used to determine the synthetic L-tyrosine from Solanum melangena for the comparison. 3,4-dihydroxyphenylalanine.

iii) Determination of tyrosine from Allium cepa:

Since o-diphenol oxidase was isolated from Allium cepa and this enzyme is mediator of formulation of melanin hence need to isolate L-dopa from Allium cepa. To confine the concentration of L-dopa (substrate) from Solanum melangena, it was isolated by Arnow method (1937).

Result and Discussion:

Solanum melangena is a member of the Solanaceae family and has been cultivated for centuries. Leaves are among the most abundant sources of vital nutrients on the planet including an abundance of certain flavonoids-quercetin that are partly responsible for the color of many fruits and vegetables and offer a variety of potential therapeutic benefits. It inhibits PPO activity/ other allergic and inflammatory substances. (Goswami-Giri et.al. 2011) Dopamine is a hormone and neurotransmitter in the human body and L-Dopa. Both compounds are precursors to the well-known neurotransmitters, norepinephrine and epinephrine responsible to psychological stressors (Etsuko Kasuya 1t al 2013). Solanum melangena leaves are very dark green that may have good sources of vitamins/antioxidants. (Shalom Nwodo Chinedu et.al 2011).Oxidative damage from free radicals and the aging process are the factors responsible for parkinsonia which damage to dopamine-producing cells. Result of an excess of free radicals or the loss of ability of the body to inactivate the free radicals. To diminish excess of free radicals, L-dopa is crucial. Therefore it was extracted from Solanum melangena and compared with commercially available L-dopa. Enzymatic hydroxylation of L-tyrosine seems to be the best method for the synthesis of L-Dopa. Crystals of L-dopa obtained from Solanum melangena are as shown in figure 1. Needle shaped brown colored crystals were measured and observed under microscope. Qualitative characterization of these crystals were studied spectroscopically which are as enlisted in Table 1.

Figure 1 Crystal structure of L-Dopa extracted from and Solanum melangena leaves.

To prove isolated L-dopa acts as a substrate, o-diphenol oxidase was isolated from Allium cepa leaves. Colorimetric and spectroscopic method intended that Solanum melangena are good source of L-dopa. It was extracted with EtOH-H₂O mixture under the protection of ascorbic acid which gave higher yield of Solanum melangena L-Dopa (1.0952% Yield) as compared to water extract against oxidation. Their vitamins/antioxidant along with amino acid component was checked by TLC. Rf value indicates presence of amino acids like L-Dopa, tyrosine (0.36 and 0.49). Rf value of different samples are as listed in Table 1.While extraction of L-dopa, salt stress, as caused by the addition of NaCl, gives no induction of dopamine formation, whereas L-dopa is released into the medium which is evident from the visual spots on TLC plates. It suggest that if ample water were used along with sufficiently small particle size, extraction rates in water could improve and perhaps even more so at higher water temperature and lower pH.(Figure 2). Table 2 showed that the comparative data of the o-diphenol oxidase activity using L-dopa as substrate obtained from different sources also indicates the concentration of L-dopa and tyrosine isolated from Solanum melangena obtained from Dhule and Nanded but dried at different conditions. Onion leaves each value in the table corresponds to the mean of the 03 separate measurements.

Figure 2: Thin layer Chromatography of Solanum melangena leaves L-dopa . Leaves were collected from A: Dhule (wet leaves) B: Dhule air dried C: Dhule sun dried D: Nanded air dried E: Nanded Sun dried F: Standard Dopa.

All samples were subjected to inductively coupled plasma atomic emission spectroscopy (ICPAES) for qualitative analysis of different metals (table 3). Mass spectroscopy of isolated L-dopa from various samples is as shown in Figure 3. The electron spin resonances (ESR) of several native and modified dopa have been determined. Dopa isolated from Solanum melangena compared with synthesized from 3, 4-dihydroxy-L-phenylalanine (L-DOPA) and tyrosine all show similar ESR signals. The black and brown coloration found so widespread in nature are attributable to a class of pigments known as melanins. Better understanding of the chemical properties of melanins should lead to better methods of removing the color. However, characterization of melanins has been exceptionally difficult because of the intractability of the pigments towards usual physical and chemical probes. No melanin, natural or formed in vitro, has yet been isolated and fully characterized. One promising clue to the nature of melanins is the presence of an endogenous free radical, which can be detected by electron spin resonance. ESR spectra of several natural and synthetic melanins have been shown to be strikingly similar with respect to line width, line shape, and g-value ( i.e 2). The source of these signals is inferred to be free radicals trapped within the matrix of the melanin. However, the signals characteristically lack hyperfine structure, so that details of composition, structure, and environment of the radicals cannot be determined precisely. This lack of detail, coupled with the dark color of the pigment suggests that the signal might result from delocalization of the unpaired electron throughout a conjugated polymer system.

ESR of isolated L-dopa from various samples is shown in Figure 4. L-dopa and tyrosine isolated from i) Solanum melangena ii) onion leaves is shown in figure 5. UV-visible spectra showed evidence only of l-DOPA. A significant increase in intensity of the 280 nm band in these samples indicates that phosphate is outcompeting l-DOPA and causing desorption from the Solanum surface without DOPA being oxidized DOPA adsorption increases with pH, particularly at low surface loadings and higher ionic strength. However, at pH values of 5.6 the adsorption tends to plateau, particularly at high surface loadings and low ionic strength This behavior is similar to published studies of catechol; dopa adsorption (Salima Bahri,et al 2011). Figure 6 contains the electrophotogram of Dopa isolated from Solanum melangena leaves.

DOPA caused a color change for the solid from pure white to a tan color. This change is consistent with the formation of a charge transfer complex at the surface as documented in numerous studies of related molecules containing catechol entities. It was reversible by addition of phosphate in equal or larger amounts than the DOPA because the phosphate adsorption almost completely prevents DOPA adsorption DOPA adsorbed on rutile was re-suspended in phosphate solutions and UV-visible spectra showed evidence only of DOPA. A significant increase in intensity of the 280 nm band in these samples indicates that phosphate is outcompeting DOPA and causing desorption from the rutile surface without DOPA being oxidized. UV spectra of Solanum melangena L-dopa isolated from leaves is shown in figure 6. Figure 7 indicated the various geographical region samples having- A. Tyrosine from Allium Cepha B. Dhule air dried C. Dhule sun dried D. Nanded air dried E. Nanded Sun dried F. Dopa. a)L-dopa oxidase substrate b) o-diphenol oxidase reaction mixture.

UV explanation all fractions were screened for UV absorption, however, to detect possible sources of interferences with determination of L-dopa in the initial assays. Only minor amounts of UV absorption were encountered in fractions preceding L-dopa except for one peak that often appeared just before it on the refractometer recordings. The material responsible for this peak was not detected by ninhydrin on TLC but did show UV absorption. Amax 260 nm (H₂0) following the elution of L-dopa, all preparations gave one additional peak on the refractometer chart. This fraction gave a yellow-brown spot with ninhydrin on TLC and migrated at a rate very similar to that of L-dopa recovery from Mucuna Seed (Melvin E. Daxenbichler et.al.1972)



	Figure 3 Mass spectrum of l-dopa isolated from Dhule Solanum melangena leaves A. Air dried, B. Sun dried, and Nanded C. air dried D. sundried E. Standard l-Dopa.



Figure 4. ESR of isolated L-dopa from various samples. A. Dhule rainy season B. Dhule air dried C. Dhule sun dried D. Nanded air dried E. Nanded Sun dried F. Std.Dopa.

Table 1 Spectroscopic values of isolated L-dopa from Solanum melangena leaves. A. Dhule air dried B. Dhule sun dried C. Nanded air dried D. Nanded Sun dried and E. Standard dopa.

*Solanum melangena*	Rf	Amino acids detected	UV (nm)	IR (cm^-1)
Sample A	0.49	L-tyrosine 5-methoxytryptamine	Max:263.50 Min: 261.00	3779.21, 3464.15(1^oand 2^oamines and amides), 2919.54 (OH carboxylic acids), 2851.03 (Carboxylic acids), 2063.27(allenes), 1631.66(Amide), 1376.78(Sulfoxide), 1103.29(amines), 678.78 (Aromatics out of plane, 613.48 (Chloride)
Sample B	0.06	L-Lysine	Max: 193.50	3448.76(1^oand 2^oamines and amides), 1640.04(Alkenes), 669.82(Alkene out of plane)
	0.10	L-cysteine/glutathione
	0.17	L-glutamine
	0.32	L-DOPA
	0.37	L-DOPA
	0.50	5-methoxytryptamine
	0.59	Hydroxytryptamine
	0.64	Tryptamine
Sample C	0.43	Mixture L-dopa and L-tyrosine / Alanine	Max : 208.50	3453.90 (1^oand 2^oamines and amides stretch), 2105.32 (C≡C) alkyne, 1642.13 (Amide), 668.3(Aromatics out of plane)
	0.54	Hydroxytryptamine
	0.48	L-tyrosine
	0.43	Mixture L-dopa and L-tyrosine
Sample D	0.53	5-methoxytryptamine	Max : 201.50	3449.18 (1^oand 2^oamines and amides stretch), 2110.98 (C≡C alkyne), 1639.28 (Amide), 663.91(C-X)
Sample D	0.55	Hydroxytryptamine	Max : 201.50
Sample E	0.52	5-methoxytryptamine	Max : 264.00 Mim : 253.0	3445.27 (1^oand 2^oamines and amides stretch), 2093.25 (Allenes),1635.48 (1^oand 2^oamines and amides bend), 667.75(Aromatics)
	0.56	Hydroxytryptamine
	0.62	Tryptamine
	0.67	Leucine
	0.73	Leucine

Table 2 Comparative data of the o-diphenol oxidase activity using L-dopa as substrate and concentration of l-dopa obtained from different sources. A. Dhule air dried, B. Dhule sun dried, C. Nanded air dried, D. Nanded Sun dried, E Std.Dopa.

Sample	Activity (IU/ml)	Protein (mg/ml)	Specific activity (IU/mg)	Dopa (g %)	Tyrosine (g %)
Sample A	34	418750	811 X 10^-3	1.76	12.3
Sample B	-11	62500	-687.5 X 10^-3	1.4	21
Sample C	-85	481250	-17.6 X 10^-3	2.1	15.3
Sample D	6.6	368750	178 X 10^-3	1.96	12.6
Sample E	30	218750	13.7 X 10^-3	0.6	1.8

*Onion leaves : Onion leaves each value in the table corresponds to the mean of the 03 separate measurements.

0.63

15.6

Conclusion:

In conclusion, the present study demonstrated that L-dopa could be extracted in good yield from Solanum melangena leaves using ethanol-water (1:1) using ascorbic acid as protector. The extract can be thus authenticated for the industrial preparation of L-dopa. Further need to explain neuroprotective property and anti-psychotic property of L-dopa need to be explored.

Acknowledgment:

Greatly acknowledged University Grants Commission, New Delhi (India) for financial assistance through major research project no. 34-284(2008).

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Received on 19.09.2014 Modified on 10.10.2014

Asian J. Research Chem. 7(11): November, 2014; Page 954-963

Sample	Metal ions
A	Al,B,Ba,Ca,Cl,Fe,K,Li,Mg,Mn,Mo,Na,P,S,Si,Sr,Zn
B	Al,B,Ba,Ca,Cl,Cu,Fe,Hg,K,Li,Mg,Mn,Mo,Na,Ni,P,S,Si,Sr,Zn
C	Al,B,Ba,Ca,Cl,Fe,K,Li,Mg,Mn,Mo,Na,P,S,Si,Sr,Zn
D	Al,B,Ba,Ca,Cl,Fe,K,Li,Mg,Mn,Mo,Na,P,S,Si,Sr,Zn
E	Al,B,Ba,Ca,Cl,Fe,Li,Mg,Mn,Mo,Na,P,S,Si,Sr,Zn