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GJRMI, Volume 1, Issue 4, April 2012, 101-108

Original Research Article PHYSICOCHEMICAL COMPOSITION OF INDONESIAN VELVET BEAN (MUCUNA PRURIENS L.)
Ratnaningsih Eko Sardjon1, Iqbal Musthapa*1, Hayat Sholihin1, Rizal Pauzan Ramdhani1 Biological Chemistry Group, Chemistry Department, Indonesia University of Education Corresponding Author: *iqbalmust@yahoo.com
1

Received: 10/02/2012;

Revised: 19/03/2012;

Accepted: 22/03/2012;

ABSTRACT
Mucuna pruriens var. utilis or Velvet bean is one of the native plants of tropical regions, including Indonesia. It has been used as a traditional food in some countries. The metal and microbial analysis showed that both seed and shell of Indonesian velvet bean can be consumed safely, because their concentration was below the maximum level required. Phytochemical analysis indicated the presence of alkaloids, tannins, saponins and steroids in both seeds and shells. The high performance liquid chromatography analysis showed that the seeds contained higher amount (7.56%) of 3,4-dihydroxy-L-phenylalanine (L-DOPA) than that of shells (3.89%).

Key words: Mucuna pruriens, physico-chemical, L-DOPA

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GJRMI, Volume 1, Issue 4, April 2012, 101-108

INTRODUCTION Mucuna pruriens (L.) DC. Var. utilis (Wall.ex Wight) Bak. ex Burck.), the Mucuna bean or karabenguk (Indonesia) is also known as velvet bean. Among the wild legumes, the genus Mucuna is widespread in tropical and sub-tropical regions of the world and considered as an alternative protein source. M. pruriens is an under-utilized legume species grown predominantly in Asia, Africa and in some parts of the America (Vadivel and Janardhanan, 2000). Traditionally in Indonesia, the mature seeds of Mucuna bean are consumed by a Central Java tribe, which is boiled repeatly and fermented to prepare a traditional recipe, called tempe. The demand on these seeds

increased after the discovery that Mucuna seeds contain (S)-2-amino-3-(3,4-dihydroxyphenyl)propanoic acid or 3,4-dihydroxy-Lphenylalanine (L-DOPA) an anti-parkinsons disease drug (Farooqi et al., 1999; Mohan and Kala, 2010). Physicochemical properties of the velvet bean from different locations has been studied (Teixeira et al., 2003; Mohan and Kala, 2010; Ujowundu et al. 2010; Raju et.al, 2010) which provided diverse compositions. However, there is no report on the physicochemical properties of velvet bean from Indonesia. In this paper, we have reported the physicochemical composition and determine the level of L-DOPA from shells and seeds of Indonesian velvet bean.

Figure 1. Structure of (S)-2-amino-3-(3, 4-dihydroxyphenyl)-propanoic acid (L-DOPA)

O HO HO NH2 OH

MATERIALS AND METHODS Collection of seed samples The samples of velvet bean, Mucuna pruriens (L.) were collected from Bantul district area, Yogyakarta, Indonesia. After thoroughly drying under sun, the shells and seeds were separated and pulverized with a grinding machine. Determination of metals and microbial contaminant Determination of metal contaminant conducted to determine the level of Lead (Pb), Cadmium (Cd), Tin (Sn), Mercury (Hg) and Arsenic (As) in shells and seeds of velvet bean. Metal contaminant procedure refers to the Indonesian National Standard (SNI) 01-28961998 and 01-4866-1998. On the other hand, determination of microbial contamination was conducted to determine E. coli, Salmonella,

fungi, Yeast, C. perfringens and Bacillus cereus and the procedure refers to ISO 28972008. Those determination were carried out at Balai Besar Industri Agro, Agro Based Industry Calibration and Analytical Laboratories (ABICAL), Bogor, Indonesia. Phytochemical screening Seeds and shells of velvet bean (M. pruriens) were extracted with solution of ethanol:water (1:1) at pH=3 (adjusted with citric acid), using maceration for 3x24 h. This test was performed to determine the secondary metabolites groups contained in the velvet bean extract. Alkaloid test: Alkaloid test was carried out by adding few drops of Mayer's reagent to the solution of 0.5 g in 1 ml of chloroform. Formation of white precipitate indicates presence of alkaloids. Reagent Mayer was prepared by 1 gram KI dissolved in 20 ml of

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distilled water, and 0.271 g HgCl2 was added to a solution of KI Tannin test: Test was done by weighing 0.1 g of extract dissolved in 2 ml of water and then adding a few drops of 1% FeCl3. The appearance of dark blue color indicates the presence of tannin (phenolic). Saponin test: Saponin test was conducted by dissolving 0.1 g of extract in 3 ml of water, then shaken vigorously for 10 minutes. The emergence of foam indicates the presence of saponin. Steroid test: Steroids test was conducted by dissolving 0.1 g of extract in 1 ml of water, then adding 1 ml of CH3COOH and 1 ml of concentrated H2SO4 to it. The emergence of the blue or purple color indicates presence of steroids. Flavonoids test: Flavonoid test was conducted by dissolving 0.1 g of extract in 3 ml of water and then adding 0.1 g of Mg powder and 1 ml of concentrated HCl. Yellow color indicates the presence of flavonoids. Determination of L-DOPA levels using HPLC method The HPLC system consisted of peak series Shimadzu and isocratic pump with UV visible detector. Analysis of L-DOPA was carried out at 280 nm by using a chromosil C18 reverse phase column of 250 x 4.6 mm. The mobile phase consisted of water, methanol and phosphoric acid in the ratio of 97 ml : 20 ml : 1 ml or 82.20:16.95:0.85 and flow rate of 1 ml/min. Standard and Sample Solutions A stock solution containing 0.1 mg/ml of LDOPA was prepared in the mobile phase. Using appropriate aliquots, different dilutions of standard solution in the range of 2550 g/ml were prepared. A sample solution was

prepared by dissolving 10 mg extract in 10 ml of mobile phase. The mixture was homogenized with ultrasonic for 10 minutes and filtered through 0.45 m membrane. 20 l the solution was injected into HPLC. RESULTS AND DISCUSSION Morphological Characteristics The morphological characteristics of seeds and shells of Indonesian velvet bean are shown in Figure 2. The beans were sorted, cleaned, separated between seeds and shells and dried. Grinding the dried bean gave seed and shells powder in ratio 86.2:13.8. The separation between seeds and shells was carried out to determine the physicochemical characteristics of each part of the beans. The morphological characteristics of Indonesian velvet bean are shown in Table 1. The beans were homogenous in shape, diameter, length, weight, smell, and taste, with various colours, i.e. white, black or white with black dots. Colour variation was characteristic of velvet beans, and it would not be an difficult to standardize the sample. Moisture and Ash Content The amount of water content in the material is associated with the purity and the presence of contaminants in the simplicia. Determination of ash content provides an overview of internal and external mineral content derived from the initial process to obtain good simplicia and extracts derived from natural plants and contaminants during the process. The number of maximum allowable ash content is associated with purity and contamination. Water and ash content contained in shells and seeds of Indonesian velvet beans are shown in Table 2. The results indicated that seed and shell contained moisture and ash contents below the maximum level. Therefore, the seed and shell of Indonesian velvet are safe to be used as a drug or food.

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GJRMI, Volume 1, Issue 4, April 2012, 101-108

Figure 2. The physical performance: (a) Indonesian velvet beans (b) Seeds (c) shells

(a)

(b)

(C)

Table 1. The morphology characteristics of Indonesian velvet bean The morphology parameter Shape Diameter Lenght Weight Colour Smell Taste Indonesian velvet beans Oval and flat 0.682 cm 1.491 cm 0.963 g White, black, white with black dots Characteristic of velvet bean bitter

Table 2. Moisture and ash content of shells and seeds of Indonesian velvet bean Parameter Shell Seed Standard* (Maximum Level) 10 10

Moisture content (%) 9.14 10.80 Ash content (%) 2.18 3.04 *Indonesia Ministry of Health Regulations no 661/MENKES/SK/VII/1994

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Metals contaminants Metal measurements were performed to determine how many metal contaminants were contained in the sample. The measurements includes that of Lead (Pb), Cadmium (Cd), Tin (Sn), Mercury (Hg) and Arsenic (As). The analysis was based on SNI (Standard National Indonesia) 7387:2009 about maximum limit of metal contaminants in food (Table 3). Table 3 shows that shells and seeds of Indonesian velvet bean contain metal contamination of lead, cadmium, tin, mercury and arsenic below the safe limit for consumption, except the lead content of shells. Therefore, the velvet beans could be considered safe to be used as a food or medicine after the shells are removed.

Table 3. Metal contaminat on shells and seeds of Indonesian velvet bean (mg/kg) Parameters Lead (Pb) Cadmium (Cd) Tin (Sn) Mercury (Hg) Arsen (As)
*

seeds < 0.048 < 0.003 < 0.8 < 0.005 < 0.003

shells 1.66 < 0.003 < 0.8 < 0.005 < 0.003

Standard* (Maximum level) 0.5 0.2 0.03 1.00

SNI 7387:2009 = Maximum level of metal contaminants on food in Indonesia

Microbial Contaminants Microbial contamination measurement was performed to quantify the microbial contaminants contained in the sample. The microbial contamination is a crucial parameter for food and medicinal products. The microbe determination includes, E. coli, Salmonella, fungi, yeast, C. perfringens and Bacillus

cereus. The test results of microbial contamination are enlisted in Table 4. Based on the results comparison with standards, contaminations in the shell karabenguk were within the consumption as food or for purposes. obtained and the microbial and seeds of safe limits for pharmaceutical

Table 4. Microbial contaminantion of shells and seeds of Indonesian velvet bean Parameters seeds shells 5.4 x 104 <3 0 < 10 < 10 0 0 Standard1 (Maximum level) 1 x 106 10 Negative 1 x 104 1 x 104 Negative 1 x 104

Total Plate Count (30oC, 72 h) 4.5 x 104 (coloni/g) E. coli (MPN2/g) <3 Salmonella (25g-1) 0 fungi (coloni/g) < 10 yeast (coloni/g) < 10 C. perfringens (g-1) 0 Bacillus cereus (coloni/g) 0
1 2

SNI 7388:2009 about Maximum level of microbial contaminant on food MPN (The Most Probable Number) = the estimate number of microbes present in samples

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Phytochemical screening This test was performed to determine the class of compounds present in the extracts. The extracts were examined for the presence of alkaloid, tannins, saponins, steroids, and flavonoids. The phytochemical test results have been summarized in Table 5, which indicates that velvet bean contained alkaloids, tannins, saponins, and steroids, while flavonoid was absent.

Table 5. Phytochemical screening results for shell and seed of Indonesian velvet bean Parameter Seed Alkaloids Tannins Saponins Steroids Flavonoids Note: () = present; (-) = absent L-DOPA Content Determination of L-DOPA was carried out using HPLC. L-DOPA has a chromophoric group, so it can be detected with the aid of UV detector in HPLC at = 250 nm. Mobile phase used was a mixture of water : methanol : phosphoric acid pH 2.5 with a ratio of 97:20:1 or 82.20:16.95:0.85. Analysis was carried out using C18 column and UV detection at = 250 nm with a flow rate of 1 ml/min, isocratic separation, at a temperature of 27oC. The LDOPA standard chromatograms are shown in Figure 3. Figure 3 shows that the peak of L-DOPA standard appears at the retention time (RT) 2.47 minutes. The same procedures were also applied into sample of seed and shell of Indonesian velvet bean extracts. Their chromatogram can be shown in Figure 4a and 4b. Figures 4a and 4b shows that both the seeds and shells of Indonesian velvet bean gave each peak at retention time of 2.47. It is meant that both the sample contain L-DOPA. The intensity of peak in the chromatogram of seed was higher than the shell. The concentration of LShell -

DOPA was calculated by comparing with a standard calibration curve. The results are shown in Table 6. The observation made in the present study shows that both seed and shell of Indonesian velvet bean were still safe for consumption as the metal and microbial contaminants were below the maximum level. The alkaloids, tannins, saponins and steroids were also present in both the samples, nevertheless the L-DOPA levels were different. CONCLUSION The present study indicated that Indonesian velvet bean could be used as a source of LDOPA which is used for the preparation of antiparkinson drugs. The metal and microbial analysis showed that both seed and shell of Indonesian velvet bean were still safe for consumption, they are below the maximum level. Phytochemical analysis showed that both the samples contain alkaloid, tannin, saponin and steroid. The data indicated that seed extract of velvet bean contained higher L-DOPA (7.56%) while shells contain only 3.89%.

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Table 6. L-DOPA Content Sample Seeds Shells L-DOPA Content (%) 7.56 3.89

Figure 3. Standard L-DOPA Chromatogram (Column Length: 25 cm; concentration: 1000 ppm)

Figure 4 Chromatogram of seeds (a) and shell (b) of Indonesian velvet beans extract

(a)

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(b) ACKNOWLEDGEMENTS The authors acknowledge Rector of Universitas Pendidikan Indonesia for funding the study. They also acknowledge the Balai Besar Industry Agro, Agro Based Industry Calibration and Analytical Laboratories (ABICAL), Bogor for providing metal and microbial contaminant evaluation. REFERENCES Farooqi AA, Khan MM, Asundhara M. (1999). Production technology of medicinal and aromatic crops, Natural Remedies Pvt. Ltd., Bangalore, pp 2628. Mohan MR, Kala BK (2010). Chemical Composition and Nutritional Evaluation of Lesser Known Pulse of the Genus, Mucuna. Adv. Biores. 1(2):105106. Raju RR, Babu NB, Rao PS (2010) RP-HPLC Method Development and Validation of

Levodopa. International Journal of Research in Pharmaceutical and Biomedical Sciences.1:2326 Teixeira AA, Rich EC, Szabo NJ (2003). Water Extraction of L-Dopa from Mucuna Bean. Tropical and Subtropical Agroecosystem. 1:159171. Ujowundu CO, Kalu FN, Emejulu AA, Okafor OE, Nkowonta CG, Nwonsunjoku E.C. (2010). Evaluation of The Chemical Composition of Mucuna utils Leaves used in Herbal Medicine in Southeastern Nigeria. Afr. J. Pharm. Pharmacol. 4(11):811816. Vadivel V, Janardhanan K (2000). Nutritional and anti-nutritional composition of velvet bean: An under-utilized food legume in South India. Inter. J. Food Sci. Nutri. 51: 279287

Source of Support: Nil

Conflict of Interest: None Declared

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