INTRODUCTION:

More than 300 plants are having medicinal and commercial values and also commonly used by local people for ethanobotanical survey works¹. Solanum xanthocarpum ^l. is an Indian night shade, nontoxic, waste land weed found throughout India. It is belongs to family Solanaceae and is known to have numerous medicinal properties. The plant is bitter, pungent, thermogenic, anthelmintic, anti-inflammatory, anodyne, digestive, carminative, appetizer, stomach depurative, sudorific, febrifuge, expectorant, laxative, stimulant, dime, rejuvenating, emmenagogue and aphrodisiac. The Plant extracts exhibits excellent agricultural pests as repellent, contact poison and as molluscicide in public health.^2,3. Some organic compounds isolated by researchers from fruits are glucoalkaloid termed as solanocarpine. A sterol known as carpesterol and solanocarpidine, Potassium nitrate, a fatty acid, a resinous and phenolic substance, diosgenin and sitosterol are also present. Dry fruits contain traces of isochlorogenic, neochronogenic, chronogenic and caffeic acids from Solanum Xanthocarpum. Solasodine, solasonine and solamargine are present in fruits of Nepalese plant. Quercetin has also been isolated together with apigenin and sitosterol.

These alkaloids, phenolics flavonoids, sterols, saponin and their glycosides, have wide range of medicinal values^4-8. The leaves are applied externally as a pain relieving agent, good application for piles⁷. It also contains solasodine compounds which are used as a precursor in production of contraceptive⁹. The white flowers extract assisted to promote conception in females¹⁰. The root is pungent, bitter, heating; appetizer, laxative, stomachic, useful in bronchitis, asthma, fever, “vata”, and “kapha”, ozrena, strangury, lumbago, pains, piles, thirst, urinary concretions, and diseases of the heart ^11-13 and also useful in sore throat⁸. The seeds are anthelmintic good for boils, scabies, asthma, and cough.^7,14 Due to presence of phenolic substances it has great potential for the neuroprotective activity. 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.

In addition to the neuroprotective properties, L-DOPA has been extensively studied by researchers such as Dopamine has also been known to promote a positive mood and enhance mental alertness simultaneously improving sensitivity of the tissues. Perumal Siddhuraju²studied on the nutritional composition of DOPA and its antinutritional factors of three different germplasm seed materials of an under-utilized tropical legume, Mucuna pruriens. In bioadhesion studies, the catecholic amino acid 3,4-dihydroxyphenylalanine (DOPA) has been identified as an important molecule in bioadhesive proteins such as those used by mussels to attach to rocks^15-18. This discovery has spurred numerous investigations of potential applications of DOPA to the development of new adhesives and antifouling materials^19,20 Surface complexion study of DOPA from rutile (r-TiO2) in NaCl solution also showed adhesive property²¹. Catechol redox induced formation of metal core-polymer shell nanoparticles showed diverse function with the ability to adhere to almost any material of either organic or inorganic origin²². The L- isomer of DOPA [3-(3,4-dihydroxyphenyl)alanine] (L-DOPA ) is being used for symptomatic relief of Parkinson's disease. It is a neurotransmitter and its content in brain tissues is reduced due to blockade conversion of tyrosine to L-DOPA. 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 reduce it L-DOPA is necessary as it’s treatment improves motor function at the onset of disease. It also stimulates human growth hormones released by the pituitary glands that are used as anti-hypertensive.²³.

Due to these properties, DOPA has relatively high demand at spiraling price in the market. Hence Solanum xanthocarpum L. was selected as the source of L-DOPA for large scale production. With this attention, it was extracted and crystallized by green technology and its structural evaluation was performed by spectroscopic methods.

MATERIALS AND METHODS:

All solutions and suspensions were prepared in Milli-Q-water. Solanum xanthocarpum plant was collected from Ambernath, Thane District. Plant material was dried and powder was stored in amber color bottle till used.

Methods:

Extraction of L-DOPA from Solanum Xanthocarpum⁵:

Extraction of L-DOPA with water- ethanol (1:1) under ascorbic acid protection was carried out using dried leaves Solanum Xanthocarpum 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 by green technology:

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

CHARACTERISATION OF ISOLATED DOPA:

Pure crystals of DOPA from Solanum xanthocarpum L leaves were subjected to thin layer chromatography (TLC), UV spectroscopy, IR spectroscopy, Electron Spin Resonance (ESR), inductively coupled plasma atomic emission spectroscopy (ICPAES) and SDS-PAGE.

Thin layer chromatography:

A rapid and sensitive quantification of amino acid was achieved using TLC as it 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 and concentration of the isolated L-DOPA was determined by dissolving in ethanol and was compared with aqueous media using Shimadzu UV-Vis 1800 spectrophotometer. Triplicate absorbance readings were performed for each sample. DOPA was also analyzed directly in the spectrophotometer without derivatization using phosphate buffer medium to pH 7.

IR, ESR and ICP-AES methods:

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

Determination of 3, 4 – dihydroxyphenylalanine:

Standard L-DOPA (100µl) from Merck was used for relative quantification of the isolated 3,4-dihydroxyphenylalanine (L-DOPA) from Solanum xanthocarpum L .

SDS-PAGE:

The pure DOPA crystals dissolved in phosphate buffer (pH 7, 50µL) was subjected to SDS-PAGE. The soluble were analyzed by duplicate 15% SDS⁄PAGE gels. One gel was stained with Coomassie Brilliant Blue for total protein detection and other for its activity. Molecular weight of isolated dopa was also calculated by gel filtration chromatography.

RESULT AND DISCUSSION:

Solanum xanthocarpum is a waste land weed belongs to solanaceae family and is considered by some to be an herbal remedy^24,25. 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 when dopamine is degraded enzymatically or the loss of ability of the body to inactivate the free radicals. Indispensable role of L-DOPA condensed the action. Even though enzymatic and nonenzymatic metabolism of L-DOPA can produce hydrogen peroxide and oxygen free radicals, there has been controversy as to whether L-DOPA generates an oxidant stress in vivo. Therefore it was extracted from Solanum xanthocarpum and compared with commercially available L-DOPA. Needle shaped, brown colored crystals of L-DOPA obtained from Solanum xanthocarpum were measured and observed under microscope. Qualitative characterizations of these crystals were studied spectroscopically (Figure 1).

L-DOPA was extracted with EtOH-H₂O mixture under the protection of ascorbic acid which gave higher yield of Solanum xanthocarpum L. (1.0952% Yield) compared to aqueous extract against oxidation. 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. 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.

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 as above as Dopa is known to be oxidized ^26-27it 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. DOPA adsorption increases with pH, particularly at low surface loadings and higher ionic strength. However, at pH 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 adsorption²¹ (Figure 2)

Inductively coupled plasma atomic emission spectroscopy (ICPAES) of the sample showed the presence of following metals - Al,B,Ba,Ca,Cl,Fe,Li,Mg,Mn,Mo,Na,P,S,Si,Sr,Zn.

The electron spin resonances (ESR) of several native and modified DOPA have been determined (Figure 3) and compared with standard.

The vitamins/antioxidant along with amino acid component was checked by TLC. Rf value indicates presence of amino acids like L-DOPA, tyrosine, 5-methoxytryptamine, Hydroxytryptamine and Tryptamine. Rf value of these are enlisted in Table 1.

All repeatedly prepared samples 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 samples preceding l-DOPA except for sample that was not protected with ascorbic acid. The material responsible for this peak was not detected by ninhydrin on tlc but did show uv absorption, λmax 260 nm (H₂0). While other gave a yellow-brown spot with ninhydrin on TLC and migrated at a rate very similar to that of l-DOPA. It was also mentioned for L-DOPA recovery from Mucuna Seed ^28,29 .

On SDS-PAGE four bands were observed consistently, that are confirmed as cable metabolic amino acids of dopa. These amino acids are all aromatic dopa, tyrosine, 5-methoxytryptamine/ Hydroxytryptamine, and Tryptamine on TLC. Though dopa is acts as neurotransmitter; is not core amino acid but can be synthesized synthetically for medicinal purpose. Many investigators procured the result that tyrosine and dopa can be inter convertible. Contented of DOPA in brain tissues is reduced due to blockade conversion of tyrosine to L-DOPA responsible for psychological disorder. Molecular weight of dopa (197.19g mol⁻¹) was also calculated by gel filtration chromatography.

The dopa crystal (30µL) was loaded on HPLC it showed single peak which exactly match with standard dopa.

Table 1 Spectroscopic values of isolated L-DOPA from Solanum xanthocarpum leaves. A: B: C:

*Solanum xanthocarpum* l	Rf	Amino acids detected	UV (nm)	IR (cm^-1)
Sample A ( Air Dried )	0.49 0.52 0.37	L-tyrosine 5-methoxytryptamine L-DOPA	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 Std	0.39	L-DOPA	Max: 193.50	3448.76(1^oand 2^oamines and amides),1640.04(Alkenes),669.82(Alkene out of plane)
	0.37	L-DOPA
	0.56	Hydroxytryptamine
	0.62	Tryptamine
Sample C (Oven dried powder)	0.46	L-tyrosine	Max : 251.00	3473.41(1^oand 2^oamines and amides stretch), 2107.54 (C≡Calkyne) , 1642.75 ( C=N) ,1633.00 (Amide) ,717.70(Alkenes out of plane)
Sample C (Oven dried powder)	0.52 0.37	5-methoxytryptamine L-DOPA	Max : 251.00

CONCLUSION:

This study is the first report on dopa rich Solanum xanthocarpum l which is nontoxic versatile medicinal drug, grown in arid area. Thus after summer season leaves tumble, decompose and disturb the environmental balance. These leaves have good yield of DOPA, whose L-isomer is being used for symptomatic relief of Parkinson's disease but there are many other benefits as well. L-Dopa has also been shown to significantly increase the body's natural production of HGH (human growth hormone). Enhanced libido and sexual performance, increased energy levels, improved skin texture and appearance, decreased body fat and cellulite regeneration and healing of organs, strengthened immune system, increased lean muscle mass, improve mood and sense of well-being. Due to entire scenario and its availability has been very limited. Statistical data proves that dopa is reproducible. Thus, Solanum xanthocarpum l comparatively cheap source of L-DOPA may be consequently authenticated for the industrial preparation of L-DOPA and can be used as an alternative source of DOPA.

ACKNOWLEDGMENT:

Greatly acknowledged University of Mumbai, Mumbai MS (India) for financial assistance through minor research project no. 45 (2012-13).

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Received on 27.08.2013 Modified on 20.09.2013

Asian J. Research Chem. 6(11): November 2013; Page 1044-1048

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