Indian Journal of Traditional Knowledge Vol. 17(1), January 2018, pp. 148-154

Chemical evaluation of for L-dopa content - an anti-Parkinson’s drug yielding medicinal from India

Archana P Raina1* & RC Misra2 1Division of Germplasm Evaluation, ICAR - National Bureau of Plant Genetic Resources, New Delhi-110 012, India; 2ICAR - National Bureau of Plant Genetic Resources, Regional Station, Cuttack-753 006, Odisha, India E-mail: [email protected]

Received 23 November 2016, revised 2 June 2017

Mucuna species is an important medicinal plant of India which is the best known natural source of bioactive compound L-dopa (L-3,4-dihydroxy phenylalanine) used as potential drug for the treatment of Parkinson’s disease. The present study was undertaken for the chemical evaluation of four species of Mucuna, viz.; M. pruriens var. pruriens, M. pruriens var. utilis (Wall. ex Wight) L. H. Bailey, M. monosperma Wight, M. nigricans (Lour.) Steud., M. gigantea (Willd.) DC., collected from wild habitats of Odisha state of eastern India for pharmaceutically valued compound L-dopa. Results indicated a wide range of germplasm variability in size, color and L-dopa content. Among the four Mucuna species, highest L-dopa content was found in M. pruriens var. pruriens germplasm varying between 4.91 % - 7.09 %. Other Mucuna species having high L-dopa were M. gigantea (6.76 %), M. nigricans (6.16 %) and M. monosperma (4.61 %); while M. pruriens var. utilis showed least L-dopa content of 1.22 %. Promising accessions with high L-dopa content (> 6 %) were IC599290, IC599342, IC599336, IC599361 and IC599350. Superior accessions along with other rarely occurring Mucuna species can be exploited for large-scale cultivation of this drug plant for pharmaceutical use.

Keywords: Mucuna species, Germplasm, HPTLC, L-Dopa, , Parkinson’s disease IPC Int. Cl.8: A61K 36/00, A61K 38/00, A01D 8/46, A01D 8/00

Mucuna is a well-known medicinal and underutilised popularly known as Velvet , cowhage, kewanch legume plant which is widely exploited for its seed and atmagupta. All parts of Mucuna plant are pods. The Mucuna belongs to the reported to possess useful phytochemicals of high and has approximately 150 species reported medicinal value for human and veterinary importance from worldwide1. The plant is widely distributed and also constitute an important raw material in throughout the India and in other parts of the tropics Ayurvedic and folk medicines. It is a constituent of including Central and South America. In India, 14 more than 200 indigenous drug formulations. This species of Mucuna are found in the foothills of the plant is pharmacologically important for various Himalayas, the plains of West Bengal, Madhya activities reported like antidiabetic, aphrodisiac, Pradesh, Karnataka, Kerala, Andra Pradesh, Uttar antineoplastic, antiepileptic, antimicrobial activities, Pradesh and the Andaman and Nicobar islands2. etc4. Mucuna is an unconventional plant species Out of these, M. pruriens, M. monosperma and having promising nutritional, pharmaceutical and M. gigantea are widely distributed all over; while bioactive constituents. Mucuna are the best M. nigricans is distributed mainly in the North- nutritional source due to high content of protein, Eastern parts of India. Mucuna pruriens (L.) DC. is carbohydrate, lipids, fiber and minerals5. Researchers widely used in traditional Ayurvedic system of Indian from various countries have also identified Mucuna as medicine for the management of male fertility, a good nutritional supplement in livestock feed, as a nervous disorders and as an aphrodisiac3. It is fodder crop. The wild species of M. pruriens var. —————— pruriens and M. pruriens var. hirsuta are being *Corresponding author cultivated in India and Brazil as a cover and green RAINA & MISRA: CHEMICAL EVALUATION OF MUCUNA SPECIES FOR L-DOPA CONTENT FROM INDIA 149

manure crop. The demand for Mucuna is increasing in raw seed where it ranges from 4.47 % - 5.39 %12. day by day due to its pharmaceutical potency6. A wide range of variation in L-dopa content of Mucuna species have been known to have M. pruriens seeds have been reported by earlier bioactive compound L-dopa (L-3,4-dihydroxy phenyl workers6,7,11,13. However, little is known about alanine), a non-protein amino acid which is a the production of L-dopa in other species of precursor of the neurotransmitter dopamine7. It is Mucuna except for few reports on M. monosperma14,15, being used in the treatment of Parkinson’s disease, M. gigantea16, M. sanjappae17 and M. utilis18,20. a degenerative disease of the nervous system Keeping in view the increasing demand for herbal characterized by muscular rigidity, difficulty with drugs, importance of this genus and vast diversity of balance and walking, depressions and dementia. Mucuna germplasm available in Indian continent, L-Dopa, a precursor, either alone or efforts were made to collect diverse germplasm of in combination with aromatic amino acid Mucuna species from wild habitats of the state of decarboxylase inhibitor is the most effective drug for Odisha, India. These accessions have been conserved the treatment of Parkinson’s disease, since dopamine in the National Seed Genebank at ICAR-NBPGR, fails to pass through the blood brain barrier8. Use of New Delhi, India. The role of genetic resource biological sources for production of L-dopa, is always characterization in plant breeding endeavours is well desirable and advantageous because the chemical recognized. However, despite presence of rich synthesis results in racemic DL-mixture, which is genepool, such options are explored to minimal in inactive and furthermore separation of enantio- Mucuna species, problably due to lack of data on merically pure L-dopa from this mixture, is very germplasm attributes. Therefore, there is a need to difficult and cumbersome. In addition, D-dopa undertake detailed characterization of Mucuna genetic interferes with the activity of dopa decarboxylase, the resources in India to reinforce the local crop enzyme involved in the production of dopamine in the impovement program. In view of this, the present brain. L-dopa isolated from Mucuna was found to be study was undertaken with the main objective to more effective than the synthetic product9. The plant screen Mucuna species germplasm collected from sources have several advantages over the microbial different regions of Odisha state of India for L-dopa and chemically synthesized L-dopa, viz. it is natural, content and to identify high L-dopa yielding lines inexpensive source and provides additional benefits which can be used in future breeding programs and like antioxidant properties to reduce oxidative stress for pharmaceutical utilization. Four species of produced during Parkinson’s disease9,10. In a Mucuna namely, M. monosperma, M. nigricans, M. screening survey of more than 1000 species in 135 gigantea, M. pruriens var. pruriens and M. pruriens plant families, Mucuna was found as the only species var. utilis collected from wild habitats of Odisha from with sufficient L-dopa to suggest a possible use for its eastern India were analyzed for the presence of L- commercial production11. Various L-dopa yielding dopa. These plant species will be a potential candidate plant species such as M. holtonii, M. monosperma, M. for extraction of L-dopa and will be a good addition utilis, M. gigantea and M. pruriens were found to be a to the existing natural sources of anti-Parkinson’s promising source of L-dopa7. Demand for Mucuna drug since demand for L-dopa is continuously seeds for L-dopa is constantly increasing in the escalating. international drug market. M. pruriens has been Materials and methods profitably exploited for the extraction of this drug since long time12. Large-scale extraction of L-dopa Plant material from the wild populations of this plant has led to its Thirty accessions belonging to four species of limited availability in natural conditions. There is an Mucuna were collected by selective sampling urgent need for collection of diverse germplasm of method from wild regions of 10 districts of Odisha in Mucuna species from wild sources to identify superior India through explorations. Authentication and types and other species of Mucuna for exploitation for identification of collected Mucuna species was done anti-Parkinson’s drug. This will also ease the burden by Dr. R.C. Misra, Taxonomist at ICAR-NBPGR, on M. pruriens for L-dopa extraction. Distribution of Cuttack and voucher samples were preserved in the L-dopa in the species showed wide variation in herberaium. These germplasm collections comprised different parts of the plant with highest concentration of 26 accessions of M. pruriens var. pruriens (Kewanch) 150 INDIAN J TRADIT KNOWLE, VOL. 17, NO. 1, JANUARY 2018

and one accession each of M. pruriens var. utilis (20 cm × 20 cm) were purchased from E. Merck (Wall. Ex Wight) L. H. Bailey, M. monosperma (Darmstadt, Germany). A stock solution of L-dopa Wight (Negro bean), M. nigricans (Lour.) Steud. (1mg/mL) was prepared by dissolving an accurately (Black Jade vine) and M. gigantea (Willd.) DC. (Sea weighed 10 mg of L-dopa standard in 10 mL of 0.1N bean or Elephant Cowitch). The details of NBPGR HCl in a volumetric flask. Standard working solution accessions along with place of collection are given in was prepared by diluting it with 0.1 N HCl to make Table 1. These germplasm accessions were grown final concentration of 0.1 µg/µL. under uniform environmental conditions at NBPGR Regional Station, Cuttack, Odisha for evaluation Sample preparation during the years 2013-14 and 2014-15. Superior Matured seeds were harvested and dried at 60 °C. accessions were further evaluated and validated for Seeds were powdered to a mesh size of 60 after high L-dopa content in subsequent years. removing seed coats. Seed extract was prepared by refluxing 0.5 g of seed powder with 50 mL 0.1N HCl Chemicals on a boiling water bath for 30 min. The extract so Pure L-dopa was obtained from Sigma- obtained was filtered using Whatman filter paper and Aldrich. Other chemicals and solvents used were of the residue was again refluxed with fresh 50 mL of analytical grade and HPTLC plates silica gel 60F254 0.1N HCl for another 30 min. The extract was filtered

Table 1 — Germplasm of Mucuna species collected from twelve districts of Odisha, India S. No. Accession Collection site Village District 1 IC 599290 M. pruriens var. pruriens Gopalpur Jajpur 2 IC 599296 M. pruriens var. pruriens Indupur Kendrapara 3 IC 599300 M. pruriens var. pruriens Badasahi Khurda 4 IC 599301 M. pruriens var. pruriens Balabhadrapur Khurda 5 IC599306 M. pruriens var. pruriens Mahipur Nayagarh 6 IC 599310 M. pruriens var. pruriens Badasilinga Nayagarh 7 IC 599320 M. pruriens var. pruriens Janisahi Nayagarh 8 IC 599321 M. pruriens var. pruriens Talabasti Nayagarh 9 IC 599330 M. pruriens var. pruriens Purnakote Angul 10 IC 599331 M. pruriens var. pruriens Purnakote Angul 11 IC 599336 M. pruriens var. pruriens Gopalpur-Kolhasahi Angul 12 IC 599342 M. pruriens var. pruriens Megha-Savarsahi Cuttack 13 IC 599348 M. pruriens var. pruriens Dhainka pokhari Dhenkanal 14 IC 599349 M. pruriens var. pruriens Deogaon Dhenkanal 15 IC 599350 M. pruriens var. pruriens Biswanathpur Dhenkanal 16 IC 599356 M. pruriens var. pruriens Gaudakateni Dhenkanal 17 IC 599358 M. pruriens var. pruriens Gaudakateni Dhenkanal 18 IC 599361 M. pruriens var. pruriens Khamara Angul 19 IC 599366 M. pruriens var. pruriens Malyagiri Angul 20 IC 599369 M. pruriens var. pruriens Binjabal Keonjhar 21 IC 599282 M. pruriens var. pruriens Bissam Katak Rayagada 22 IC 599279 M. pruriens var. pruriens Bhadabhada Rayagada 23 IC 589204 M. pruriens var. pruriens Khemundikhol Ganjam 24 IC 589208 M. pruriens var. pruriens Baliganda Gajapati 25 IC 589220 M. pruriens var. pruriens Jeranga Gajapati 26 IC 589221 M. pruriens var. pruriens S. Karadasingh Gajapati 27 IC 599373 M. pruriens var. utilis Dhanwantari vatika Boudh 28 IC 599292 M. monosperma Indupur Kendrapara 29 IC 599297 M. gigantea Dangamal Kendrapara 30 IC 599365 M. nigricans Malyagiri Angul RAINA & MISRA: CHEMICAL EVALUATION OF MUCUNA SPECIES FOR L-DOPA CONTENT FROM INDIA 151

and both the filtrates were pooled to make total volume of the extract equal to 100 mL with 0.1N HCl for further estimation of L-dopa by HPTLC method standardized earlier in our laboratory19.

HPTLC analysis of L-dopa content A Camag high performance thin layer chromate- graphic (HPTLC) system equipped with linomat V automatic sample applicator, a CAMAG glass twin trough chamber (20 × 10 cm), TLC Scanner 3 and integrated WinCATS Software 4.03 was used for the Fig. 1 — A. Variability in seed characters of Mucuna species analysis. The seed extract (2 μL) was spotted on collected from wild habitats of Odisha, India and B. 1: HPTLC plates (20 × 10 cm) in 6 mm band width M. gigantea (Sea bean or Elephant Cowitch); 2: M. monosperma using Linomat V under a flow of Nitrogen gas, 10 (Negro bean); M. nigricans (Black Jade Vine); 4: M. pruriens var. mm from the bottom. The HPTLC plates were utilis (Velvet bean) developed in a presaturated twin trough chamber and medium in size with length of 10.64 mm, width using mobile solvent phase of n-butanol: glacial acetic of 11.21 mm, thickness of 7.95 mm and 100 seed acid: water (4:1:1, v/v) for 30 min up to 8 cm and then weight of 99.54 g. Seeds of M. pruriens var. pruriens air dried. After chromatographic development, the were smallest in size with length varying from 8.64 to peak areas of the individual bands were measured at 11.33 mm, thickness from 4.46 to 6.45 mm, width 280 nm UV using a Camag TLC Scanner 3 integrated from 6.74 to 8.54 mm and 100 seed weight from with WinCATS software at a slit width of 5 × 0.45 mm. 20.76 to 50.36 g was recorded. Seed color of The analysis was done in triplicate and the average M. pruriens var. pruriens accessions showed wide was taken for L-dopa estimation. Stock solution of variability ranging from light brown to dark brown, L-dopa standard (0.1 μg/μL) was used in different black and light green . concentrations (100 ng/spot - 1000 ng/spot) for HPTLC separation of L-dopa in seed extracts of preparing calibration graph of peak area versus Mucuna species showed a TLC spot and peak concentration. Concentration of unknown in the corresponding to pure L-dopa standard at Rf value of Mucuna seed extract was calculated using this standard 0.35 ± 0.02 (Figs 2A&B). Maximum absorption of linear calibration equation. Quantitative estimation of compound was observed at 280 nm wavelength under L-dopa in Mucuna germplasm was done by above UV and L-dopa peak was confirmed in seed extract described HPTLC procedure19. by overlaying spectra of sample with the pure compound spectra (Fig. 2C). A linear calibration Results and discussion equation developed for standard was used to quantify Mucuna species germplasm showed a wide range L-dopa in seed extracts of Mucuna species (Fig. 2D). of variability among seed characters like seed color, Among the four Mucuna species, L-dopa content was size and shape (Fig. 1). Among the four Mucuna highest in germplasm of M. pruriens var. pruriens species studied, maximum seed size was found in ranging between 4.91-7.09 % with standard deviation M. gigantea with length of 24.76 mm, width of 23.06 of 0.61 (Table 2). A perusal of results suggested mm and thickness of 14.30 mm and 100 seed weight comparatively high L-dopa content in the Mucuna was 464.46 g. Seeds were dark brown to black in pruriens germplasm under study than the earlier color, disk-like in shape with black hilum and reports by various workers from India13,14,18,21. Other almost hamburger like in appearance. Seeds of Mucuna species of M. monosperma, M. nigricans and M. monosperma were also very big in size with length M. gigantea showed moderate to high L-dopa content of 24.21 mm, width of 20.34 mm, thickness of 10.68 in seeds (Fig. 3). L- Dopa content in seeds of mm and 100 seed weight of 413.40 g. Seeds were M. monosperma (IC599292) was found to be 4.61 % dark brown in color and kidney shaped. Seeds of M. which was in agreement with the earlier report of nigricans were mottled brown in color and medium in Inamdar et al.14 from India. L-Dopa content in seeds size with length of 20.62 mm, width of 15.38 mm, of M. nigricans (IC599365) was 6.16 %, M. gigantea thickness of 11.12 mm and 100 seed weight of 258.34 (IC599297) was 6.76 %; while cream color seeds of g. Seeds of M. pruriens var. utilis were cream in color M. pruriens var. utilis (IC599373) showed very low 152 INDIAN J TRADIT KNOWLE, VOL. 17, NO. 1, JANUARY 2018

Fig. 2 — (A) HPTLC densitometry of L-DOPA standard; (B) Seed extract of Mucuna pruriens scanned at 280 nm; (C) Spectra of L-DOPA standard and seed extracts at 280 nm; (D) Calibration curve developed for quantification of L-DOPA in Mucuna pruriens seeds extract

Fig. 3 — Variation in L-DOPA content (%) in seeds of Mucuna species germplasm from Odisha, India

RAINA & MISRA: CHEMICAL EVALUATION OF MUCUNA SPECIES FOR L-DOPA CONTENT FROM INDIA 153

Table 2 — L-DOPA content (%) in seeds of Mucuna species collected from wild habitats of Odisha, India Mucuna species Seed color 100 Seed Seeds size (mm) L-DOPA (Accessions) Weight (g) Length Width Thickness (%) M. pruriens var. pruriens (26) Black, brown, green 20.76-50.36 8.64-11.33 6.74-8.54 4.46-6.45 4.91-7.09 M. pruriens var. utilis (1) Cream 99.54 10.64 11.21 7.95 1.22 (1) Dark brown to black 464.46 24.76 23.06 14.30 6.76 Mucuna nigricans (1) Mottled brown 258.34 20.62 15.38 11.12 6.16 (1) Dark brown 413.40 24.21 20.34 10.68 4.61 L-dopa content of 1.22 % (Fig. 3). Our results concluded that possibility of commercial cultivation reported comparatively higher L-dopa content in of M. monosperma, M. nigricans and M. gigantea M. gigantea, M. nigricans and M. monosperma for its L-dopa contents must be addressed which than earlier reports7,14-16. will ease the burden on M. pruriens for L-dopa A significant positive correlation was observed extraction. The promising accessions of Mucuna between 100 seed weight with seed length, seed identified in the present study can be exploited for thickness and seed width, however L-dopa content their pharmacological properties especially L-dopa was negatively correlated with all the other seed content. It may be an affordable alternative for characters indicating that seed size may not be a patients suffering from Parkinson’s disease. selection criterion for identifying a genotype with high L-dopa content21. M. pruriens var. pruriens Acknowledgement accession IC599290 having black coloured seeds The authors are grateful to the Director, showed highest L-dopa content of 7.09 ± 0.43 % ICAR-NBPGR for providing necessary help and after chemical evaluation and validation of superior encouragement to conduct the study. for four consequent years. Other promising accessions of M. pruriens identified for high L-dopa content (> 6.0 References %) were IC599342, IC599336, IC599361, IC599350. 1 Anonymous, The Wealth of India: Raw Materials, Vol 6, The range of L-dopa content recorded during (CSIR, New Delhi), 1985, 442. present study was found comparatively higher 2 Buckles D, Velvet been (Mucuna pruriens): A “new” plant with history, Econ Bot, 49 (1995) 13-25. than earlier reports18,20-22. Therefore, these high 3 Natarajan K, Naratanan N & Ravichandran N, Review on L-dopa yielding accessions of M. pruriens and other “Mucuna”- The wonder plant, Int J Pharm Sci Rev Res, 17 rarely occurring Mucuna species can be exploited (2012) 86-93. for commercial cultivaton of the plant for extraction 4 Sathiyanarayanan L & Arulmozhi S, Mucuna pruriens L.- of L-dopa drug. A comprehensive review, Pharmacogn Rev, 1 (2007) 157-162. Conclusion 5 Siddhuraju P, Becker K & Makkar HP, Studies on the The phenomenal increase in the demand for the nutritional composition and antinutritional factors of three different germplasm seed materials of an under-utilized in the last two decades has resulted tropical legume, Mucuna pruriens var. utilis, J Agric Food in search for indigenous herbs used as remedies Chem, 48 (2000) 6048-6060. against various diseases in the traditional system of 6 Lorenzetti F, Maclsaac S, Arnason JT, Awang DVC & medicine and compounds derived from natural Buckles D, The phytochemistry, toxicology and food sources have been gaining importance. Therefore, potentials of velvet bean (Mucuna adans. spp fabaceae), In: there is a high demand for the Mucuna seeds due to Cover Crops in West Africa: Contributing to Sustainable Agriculture (IDRC, Ottawa, Canada), edited by D Buckles, presence of L-dopa. Presently L-dopa prescribed A Eteka, O Osiname, M Galiba & G Galiba, 1998, 67-84. together with a peripheral dopa-decarboxylase 7 Ingle PK, L-dopa bearing , Ind J Nat Prod Resour, inhibitor (DDCI) remains the most effective 2 (2003) 126-133. drug in the treatment of Parkinson’s disease. 8 Hussain G & Manyam BV, Mucuna pruriens proves more The present work demonstrates that M. monosperma, effective than L-dopa in Parkinson’s disease animal model, M. nigricans and M. gigantea are efficient natural Phytother Res, 11 (1997) 419-423. 9 Manyam BV, Dhanasekaran M & Hare TA, Effect of sources of L-dopa and a good alternate to the antiparkinson drug HP-200 (Mucuna pruriens) on the M. pruriens which is a major current source of central monoaminergic, neurotransmitters, Phytother Res, L-dopa. On the basis of the present studies, it was 18 (2004) 47-48. 154 INDIAN J TRADIT KNOWLE, VOL. 17, NO. 1, JANUARY 2018

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