Assessment of Essential and Toxic Mineral Elements in Bitter Gourd (Momordica Charantia) Fruit

Abstract

In the United Arab Emirates (UAE), overall diabetes prevalence in adult population is around 20%. Along with, or instead of oral hypoglycemic drugs and insulin, some patients use bitter gourd (Momrodica charantia) because of its hypoglycemic properties. The aim of this study was to assess the essential (Ca, Mg, V, Cr, Mn, Co, Cu, Zn, and Mo) and toxic (As, Cd, Hg, and Pb) trace elements of bitter gourd using inductively coupled plasma-mass spectrometry (ICP-MS). Mean concentrations of each essential element were compared with literature data. Toxic As and Cd could not be detected, while Hg and Pb concentrations in our samples were much lower compared to values reported previously from contaminated areas. Results obtained in our study after ICP-MS/MW analysis, indicate that bitter gourd can be a good source of essential elements.

Keywords:

• Bitter gourd
• Trace elements
• Antidiabetic
• ICP-MS

INTRODUCTION

Diabetes mellitus is a major world health problem affecting about 180 million people worldwide.[1] In the United Arab Emirates (UAE), overall diabetes prevalence in adult population is around 20%.[2] Glucose autooxidation and protein glycation in diabetes lead to enhanced production of reactive oxygen species (ROS); therefore, oxidative stress and associated tissue damage represent a common end-point of diabetes.[3] Along with oral hypoglycemic drugs and insulin, many patients use vegetables, fruits and other natural products with assumption that they have hypoglycemic properties.[4] Rahman and Zaman[5] and Ivorra, et al.[6] published lists of several hundred spices which had antidiabetic properties. In the region of the UAE, bitter gourd (Momrodica charantia) has been traditionally used as a natural remedy for diabetes. Hypoglycemic property of this fruit has been proven in many animal and human studies.[7,8,9] Older studies were focused more on effects of hepatic glucose mechanism, while recent ones discuss antioxidant activity and glucose uptake in muscle cells.[10]

Fruit and leaves of the plant contain Gurmarin, a polypeptide considered to be similar to bovine insulin and rich in essential vitamins and minerals.[11] Although the data on the content of major elements (calcium, magnesium, potassium, sodium, and phosphorus) are abundant, only limited information concerning both essential and toxic trace element content in Momrodica charantia is available.

Essential and toxic elements in an agroecosystem can be inherited from soil parent material or introduced through human activities such as industrialization or fuel combustion. The contribution of metals and their ions toward pollution problems is of serious concern because their physiological effects can be substantial even at extremely low concentrations. Some plants have the ability to concentrate both essential and toxic trace elements, compared to concentration found in the soil and water.[12] The actual role of trace metals in human health is complex and by no means fully understood which makes the determinations of safe levels very difficult.[13] Marginal or severe trace element imbalances can be considered as risk factors for several diseases. Also, antioxidant trace element intakes have been shown to be particularly important in the prevention of aging and various diseases.[14]

Some trace elements have been postulated as essential for glucose tolerance and utilization. Vanadium-containing compounds have been assessed clinically for use in treatment of human diabetic patients for almost hundred years. Although the mechanism of action of different V compounds still remains unknown, the therapeutic potential is clearly present.[15] Chromium, manganese, cobalt, copper, zinc, and molybdenum have been recognized as essential trace elements since they act as catalytic or structural components of larger molecules such as enzymes, vitamins and proteins. They also participate in different reactions of biotransformation and have specific functions indispensable for life such as normal skeletal growth and development, glucose utilization, lipid synthesis and lipid metabolism, cholesterol metabolism, pancreatic function and development, prevention of sterility, protein, and nucleic acid metabolism, activation of enzyme functions.[16] Arsenic, cadmium, mercury, and lead are toxic metals that can be assimilated, stored and concentrated by organisms through food chain.[17],18] Arsenic and cadmium are ubiquitous and dangerous environmental toxins, known to cause cancer in humans.[19,20] The environmental hazards of mercury are well documented through consumption of contaminated food.[21] Mercury is very toxic, cumulative poison having its chief effects on nervous system. The toxicity of lead in the environment has caused extensive concern in recent decades. Like other heavy metals strongly bound by biological complexing agents, lead is a cumulative poison and can act through long term ingestion of relatively small quantities.

Recent studies have shown that metals cause oxidative stress by inducing the generation of reactive oxygen species, reduction of the capacity of antioxidant defense system of cells via glutathione depletion, inhibition of sulfhydryl dependent enzymes, interference with some essential metals necessary for the activity of antioxidant enzyme and increasing cell susceptibility to oxidative attack by altering the membrane integrity and fatty acid.[22,23]

Efforts to compare levels of specific trace elements in fruits and vegetables across time and place are limited by many obstacles. A range of diverse issues such as species selection, time and place of sampling, conditions of sample preservation (temperature, etc.), can significantly affect study results and make obtained analytical values noncomparable. Several studies on trace elements in vegetables and medicinal plants which have been conducted in past decade have shown that levels of some trace elements are extremely variable around the world.[24] The goal of our study was to assess the levels of essential and toxic trace elements of fresh bitter gourd fruit.

EXPERIMENTAL

Sampling

Green bitter gourd was purchased in the month of June 2006 from a local UAE market. The country of origin was Pakistan. Before analysis, whole fruits (n = 10) were washed to avoid potential contamination.

Reagents

Nitric acid 70%, trace metal certified, analytical grade (redistilled 99.999% purity, Aldrich, USA), acetone (HPLC grade, BDH, England) and hydrogen peroxide concentrate (Sigma- Aldrich, USA) were used without additional purification. Deionized Milli-Q water (Millipore, Milli-Q, Bedford, USA) resistivity 18 MΩcm⁻¹ was used.

Analytical standards used for calibration were prepared from single analytical standard solutions (1gL⁻¹ SPEX Plasma Standards, Edison, USA, and Promochem, Boras, AB, Sweden). Stock solutions of multielement calibrators and single standard dilutions as well as all subsequent dilutions were prepared with 2% nitric acid. All lab-ware used was polystyrene made and acid-washed by soaking in 30% nitric acid over night, rinsed thoroughly with distilled and milli-Q water and kept in PVC bags. All dissection equipment was plastic made and treated like other labware. Non powdered gloves were used for sample handling.

Sample Preparation

For analysis, samples were accurately weighted into vessels for microwave digestion. Nitric acid, milli-Q water, hydrogen peroxide and germanium (Ge), indium (In), and thallium (Tl) as internal standards were added. Digested residues were quantitatively transferred to volumetric flasks, and volumes set up with milli-Q water, thus the final dilution of digested sample was approximately 50 times. Reagent blanks were prepared for each series of samples in order to measure the level of contamination. Standard reference material (SRM) NIST 1572 and NIST 1640 were also processed using same analytical conditions. Each sample was analyzed in triplicate.

Statistical Analysis

Statistical package software for social sciences (SPSS) version 14 was used for the statistical analysis.

Calibration

Both external and internal calibration procedures were applied. External calibrator solutions consisted of mixtures of calibrators in 2% nitric acid in three different levels supposed to cover the range of expected concentrations. Germanium (Ge), indium (In), and thallium (Tl) as internal standards were added to all blanks, external calibrators and samples in a final concentration of 25 μg/L each.

Instrumentation

Ethos D (Milestone, Sorisole, Italy) closed vessel microwave digestion system was used for sample preparation. The ICP-MS instrument Plasma Quad 3 manufactured by VG Elemental UK, equipped with Cetac 500 autosampler and controlled by VG PQ ExCell software was used. A conical nebulizer, a standard torch and spray chamber, sampling and skimmer cones were used. A peristaltic pump controlled solution uptake. For an optimization of the whole mass range in order to obtain optimum signal intensity for multielement analysis a 10 μg/L multielement tuning solution was used: ⁹Be at low mass, ¹¹⁵In at medium mass and ²³⁸U at high mass.

RESULTS AND DISCUSSION

In this study, life essential and toxic elements in fresh bitter gourd fruit were analyzed. The accuracy of the method was assessed by analyzing appropriate SRM. Analytical methodology choices can also introduce biases and variability, which can result from volatilization losses during digestion, contamination during digestion or analysis and background or spectral interferences.[25]

Validity of the analytical data was established through the repeated preparation procedure and analysis of reference material. Table 1 also shows good agreement of measured values for all elements when compared to the certified ones. Measured values for SRM expressed as mean ± SD are result of thirty measurements during five executive days. For all selected elements the most abundant isotope were chosen. Interference equations, which are part of VG Plasma Lab software, were applied. Calibration data for each analyte were forced through the blank, which comprised 2% nitric acid containing 25 μg/L of Ge, In, and Tl, respectively. All calibration curves were linear in the examined range with correlation coefficients of 0.993 or better. Calculation of the detection limit of the method (MDL) was based on 3σ criterion of digested sample blank obtained from thirty measurements during five consecutive days. For all examined elements MDL values were at least ten times lower than expected in SRM.

Table 1 Method detection limits (MDL), accuracy and precision of the method and measured concentrations in bitter gourd samples ^a

Element	Mg	Ca	V	Cr	Mn	Co	Cu	Zn	As	Mo	Cd	Hg	Pb
MDL (μg/L)	0.235	0.658	0.009	0.147	0.05	0.03	0.024	0.024	0.07	0.09	0.04	0.009	0.005
NIST 1572 Certified value (mg/kg)	5800 ± 300	31500 ± 1000	N/A	0.8 ± 0.2	23 ± 2	N/A	16.5 ± 1	29 ± 2	3.1 ± 0.3	0.17 ± 0.09	0.03 ± 0.01	0.08 ± 0.02	13.3 ± 2.4
Measured value	5672 ± 271	31338 ± 986		0.77 ± 0.17	24 ± 1.52		15.8 ± 1.24	30 ± 1.87	2.8 ± 0.24	0.16 ± 0.1	0.027 ± .0009	0.078 ±  0.017	12.8 ± 1.97
Accuracy (%)	98	99		96	93	109	104	102	98	99	98	90	96
Intraday precision (CV%)	3.3	3		2.5	2.6	2.9	2.6	4	3.3	3.1	3.4	1.3	2.7
Interday precision(CV%)	5.6	5.5		4.7	6.2	5.9	5.7	6.7	4.7	3.9	4.4	4.3	4.2
NIST 1640 Certified value (μg/kg)	5819 ± 56	7045 ± 89	12.99 ± 0.37	38.6 ± 1.6	121.5 ± 1.1	20.28 ± 0.31	85.2 ± 1.2	53.2 ± 1.1	26.67 ± 0.41	46.75 ± 0.26	22.79 ± 0.96	N/A	27.89 ± 0.14
Measured value in 10 times diluted sample	587.5 ± 20	702 ± 0.84	1.22 ± 0.19	4.08 ± 0.33	12.56  ± 0.3	2.02 ± 0.15	8.61 ± 0.19	5.45 ± 0.3	2.06 ± 0.16	4.56 ± 0.095	2.28 ±  0.05		2.73 ± 0.11
Accuracy (%)	101	99	94	105	103	100	101	103	77	97	100		98
Intraday precision (CV%)	3.6	2.8	3.1	2.7	1.3	1.1	1.24	4.9	2	2.4	0.8		0.6
Interday precision (CV%)	4.9	3.9	5.6	5	2.4	2	2.3	5.5	3.4	3.2	2.3		3.9
Bitter gourd flesh (μg/kg)	163078 ± 37219	39027 ± 10299	5.36 ± 0.79	33.3 ± 7.3	2540 ± 929	0.16 ± 0.07	790341 ±	5035 ± 1239	n.d.	193 ± 64	n.d.	1.74 ± 0.84	10.95 ± 2.92
(mg/kg)	163 ± 37.2	39 ± 10.3			2.54 ± 0.93			5 ± 1.2
^aWet mass of flesh.

Not all elements that are present in the plant tissues are required for plant growth. Although plants can, to some extent, selectively absorb elements necessary for their growth, they also take up toxic metals from the environment.[26] Large diversity of responsiveness among individuals to environmental exposure to metals makes it difficult to determine actual risks, particularly at the low doses to which most people are exposed in case of consumption of contaminated food.

Our results for Mg and Ca concentrations confirm previously published data, and are in line with values reported by Sanchez-Castillo et al. [27] who found 140 ± 110 mg/kg of Mg, and 17 ± 3 mg/kg of Ca (wet weight) in different gourd samples. In our samples vanadium value corresponds with data published by British Food Standards Agency[28] for vanadium content in fresh vegetables and fruits (5–30 μg/kg). According to the same source, spinach, parsley, and some sorts of mushrooms contain >100 μg /kg of vanadium. It has been known for more than hundred years that vanadium has glucose enhancing potential. In numerous animal and human studies, doses, and chemical forms of vanadium widely varied, but in all cases they were in milligram range.[15]. Chromium values obtained in our study were in line with those reported by Lendinez et al.[29] who found Cr in the range 0.135–0.456 μg/kg in sweet corn and 0.327–0.341 μg/kg in wheat. Tinggi et al.,[30] however, reported chromium values in the range 0.05–0.3 μg/kg wet weight for different vegetables, which is much lower than our finding. Manganese values in bitter gourd samples analyzed in our laboratory correspond with findings of Sanchez-Castillo et al.[27] and Tinggi et al.[30] who reported 800–2600 μg/kg in different gourd samples. Co and Cu values measured in our samples are in agreement with those published by other authors,[27] who reported around 0.2 μg/kg and 1100 μg/kg for Co and Cu. Zn concentration measured in our samples is around five times higher than reported by same authors. Zn values obtained in our study are in good agreement with zinc contents of raw vegetables from different regions, as published by Scherz and Kirchhoff,[31] who reported a range between 1000 μg/kg and 10000 μg/kg of edible fresh product. Molybdenum content in plants and vegetables strongly depends on its soil concentration and pH. Mo value in bitter gourd samples measured in our laboratory was 193.6 ± 64 μg/kg, is in line with values reported by British Food Standards Agency,[28] ranging between 230 μg/kg in cereals and 960 μg/kg of fresh nuts. In bitter gourd samples toxic elements such as As and Cd could not be detected. Bakare, et al.[32] reported Cd values ranging from 200 μg/kg–300 μg/kg in fresh vegetable grown on farms close and far from highways in Nigeria. Hg concentration in our samples was 1.74 ± 0.84 μg/kg, which is below 500 μg/kg, the FDA safety guideline for Hg in food. Although some authors[27] could not detect Pb in fresh bitter gourd samples, our measurements have shown the value of almost 11 μg/kg. Compared to data presented by Bakare et al.,[32] where Pb concentration ranged between 1800 μg/kg and 16900 μg/kg, depending on vegetable samples and farm location, our value is for three orders of magnitude lower.

CONCLUSION

On the basis of results presented in this study, it could be concluded that Momordica charantia is a good source of essential trace elements, particularly zinc, copper, molybdenum, chromium, and vanadium. Insulin potentiating activity, however, could not be related to the concentration of essential elements since obtained values are not significantly higher compared to literature data for other vegetables. On the other hand, concentration of heavy metals and potentially toxic elements was within the safe margins recommended by international bodies such as FDA and British Food Standards Agency. Since the beneficial effects of trace elements are related to their chemical form and consequently bioavailability,[32] further research is needed to establish the chemical form of trace elements in this plant.

ACKNOWLEDGMENTS

This research was supported by General Authority for Health Services-Abu Dhabi Emirate (funding for student's summer research 2006).