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Mal J Nutr 9(1): 31-39, 2003
Determination of Vitamin C,
β
-carotene and Riboflavin Contents in Five
Green Vegetables Organically and Conventionally Grown
Amin Ismail & Cheah Sook Fun
Department of Nutrition and Health Sciences, Faculty of Medicine and Health Sciences,
Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia
ABSTRACT
As consumer interest in organically grown vegetables is increasing in Malaysia, there is a need to
answer whether the vegetables are more nutritious than those conventionally grown. This study
investigates commercially available vegetables grown organically and conventionally, purchased
from retailers to analyse β-carotene, vitamin C and riboflavin contents. Five types of green
vegetables were selected, namely Chinese mustard (sawi) (Brassica juncea), Chinese kale (kai-
lan) (Brassica alboglabra), lettuce (daun salad) (Lactuca sativa), spinach (bayam putih)
(Amaranthus viridis) and swamp cabbage (kangkung) (Ipomoea aquatica). For vitamin analysis,
a reverse-phase high performance liquid chromatography was used to identify and quantify
β -carotene, vitamin C and riboflavin. The findings showed that not all of the organically grown
vegetables were higher in vitamins than that conventionally grown. This study found that only
swamp cabbage grown organically was highest in β -carotene, vitamin C and riboflavin contents
among the entire samples studied. The various nutrients in organically grown vegetables need to
be analysed for the generation of a database on nutritional value which is important for future
research.
INTRODUCTION
A diet rich in vegetables (more than 5 servings per day) is recommended along with fruits and
whole grains; an epidemiological study found that a diet of this composition has a negative
association with the risk of chronic diseases. Antioxidant vitamins in vegetables are some of the
important nutrients besides other vitamins, minerals, flavonoids and phytochemicals, which have
been reported to contribute to health. Our local markets offer a variety of vegetables ranging
from leafy to tubers for consumption. Malaysians mostly consume green vegetables such as
Chinese mustard, Chinese kale, lettuce, spinach and swamp cabbage. According to the Nutrient
Composition of Malaysian Foods, these green vegetables have been found to contain about 1825
– 4760 µg of β -carotene /100 g edible portion, 27.6 – 107 mg of vitamin C/100 g edible portion
and 0.15 – 0.55 mg of riboflavin/100 g edible portion (Tee et al., 1997).
Beside the conventionally grown vegetables, currently, organically grown foods are gaining
popularity among consumers, health educators, farmers and food retailers. Many consumers
believe that organically grown vegetables are of better quality, healthier and more nutritious than
conventionally grown ones. Organically grown vegetables are presently available in the
Malaysian market, although the organic market is still new and is not as yet firmly established
compared to the more developed countries. In these countries, consumer demand for organic
Amin Ismail & Cheah Sook Fun
foods has increased tremendously. This is due to a number of reasons which may vary from
country to country such as safety, effect of environment, flavour, freshness, health benefits and
nutritional value (Bourn & Prescott, 2002).
In general, organic foods are products produced by organic farming practices, grown without the
use of chemical fertilizers, pesticides, fungicides and herbicides. They are usually fertilized
solely with organic fertilizers such as animal waste, crop residues, green manures or off-farm
organic wastes. Organically grown vegetables are believed to contain higher vitamins and
minerals compared to conventionally grown vegetables. Smith (1993) reported that organic foods
are more nutritious in terms of mineral content than conventional ones. Leclerc et al. (1991)
found that carrots and celeriac roots grown organically were higher in ascorbic acid and β-
carotene contents. In addition, they reported that the ascorbic acid content in potatoes grown
organically was significantly higher than those grown under the conventional method. On the
other hand, Svec, Thoroughgood & Hyp Chung (1976) reported no significant difference in the
ascorbic acid content in potato, tomato and pepper grown using these two methods. Eggert and
Kahrmann (1984) reported no differences in β-carotene content of carrots grown under organic
and conventional fertilization.
Few studies have been conducted to investigate nutritional composition of organic vegetables
purchased from retailers. Owing to the rapid growth in our country, research should be initiated
to analyse the nutrient values of these vegetables for inclusion into the Malaysian Food
Composition Database. In addition, the study should also be useful for consumers to know
whether organic vegetables purchased from a supermarket are more nutritious than the
conventional ones. Therefore, our first attempt is to analyse selected vitamins such as β-carotene,
vitamin C and riboflavin, in five organically and conventionally grown commercially available
green vegetables.
MATERIALS AND METHODS
Vegetables
Five types of green vegetables grown organically and conventionally were selected based on
popular consumption among Malaysian. Conventionally grown vegetables (400 g) were
purchased from a local wet market at Seri Kembangan, Selangor, while organically grown
vegetables (400 - 500 g) were purchased from Abad Hijau Organik Enterprise at Seri
Kembangan, Selangor. Convenience sampling was used to obtain the samples.
Preparation of samples
Upon arrival at the Department of Nutrition and Health Sciences laboratory, the fresh and
healthy vegetables were immediately washed under tap water and excessive water dripped off.
Edible portions (100 g) of the vegetables were cut into small pieces and homogenised using a
blender (National; model MX-291N) for 2 min. The homogenised sample was transferred into an
air-tight container and kept at -20°C before vitamin analysis. The above procedure was applied
to both organically and conventionally grown vegetables. All procedures were carried out
Determination of Vitamin C, β-carotene and Riboflavin in Green Vegetables
carefully without much exposure to light. All the chemicals and reagents used were of analytical
grades or as otherwise stated.
Extraction of vitamins
Vitamin C
Vitamin C was extracted according to the modified method of Abdulnabi et al. (1997). The
sample (10 g) was homogenised with an extracting solution containing meta-phosphoric acid
(0.3 M) and acetic acid (1.4 M). The mixture was placed in a conical flask (wrapped with
aluminum foil) and agitated at 100 rpm with the aid of an orbital shaker for 15 min at room
temperature. The mixture was then filtered through a Whatman No. 4 filter paper to obtain a
clear extract. The ratio of the sample to extraction solution was 1 to 1. All samples were
extracted in triplicates.
β-Carotene
The β-carotene in the sample was extracted according to the method described by Tee et al.
(1996) with slight modifications. The sample (10 g) was added with 40 ml of 99.8% ethanol and
10 ml of 100% (w/v) potassium hydroxide, and homogenised for 3 min using a blender. The
mixture was saponified by means of a refluxing apparatus, and heated using a heating mantle for
30 min, and then cooled to room temperature. The mixture was frequently agitated to avoid any
aggregation. For the extraction step, the mixture was transferred into a separation funnel and 50
ml of n-hexane was added. The funnel was inverted, vented and then shaken vigorously for a few
seconds, and the layers were allowed to separate. The upper layer (hexane extract) was pipetted
out, and the aqueous layer was re-extracted twice, each time with 50 ml of n-hexane. Then, the
upper layer was pooled and washed with distilled water until free of alkali. Phenolphthalein
solution (1%) was used to check for any alkali. The presence of alkali turns this indicator to pink.
The extract was then filtered through anhydrous sodium sulphate to remove any water residue.
The hexane residue was removed under reduced pressure at 45°C using a rotary evaporator
(Laborata 4000, Heidolph Instruments GmbH & Co. KG, Germany). The resulting extract was
diluted to 10 ml with n-hexane. All samples were carried out in triplicates.
Riboflavin
Riboflavin was extracted according to the method described in AOAC International (1990). One
gram of sample was weighed and transferred into a 50 ml graduated polypropylene centrifuge
tube. Then 17.5 ml of 0.1 N sulphuric acid was added to it. The mixture was shaken vigorously
for 1 min, and then placed in boiling water for 30 min and shaken at 10 min intervals. The
mixture was cooled in an ice bath before the addition of 2.5 ml of 2% a-amylase (Sigma
Chemical Co., St. Louis, MO, USA). After a gentle mixing, the mixture was incubated at 55°C
for 1 hr in a shaking water bath. The mixture was cooled and then diluted to 25 ml with
deionised water. The resulting mixture was spun at 2500 rpm for 15 min at room temperature
using a bench top centrifuge (Janetzki; model T32c). The supernatant was filtered through a 0.45
µm nylon filter disc before HPLC analysis. All samples were carried out in triplicates.
Amin Ismail & Cheah Sook Fun
Determination of vitamins
The vitamins were determined by a reverse-phase HPLC technique. A Hewlett Packard HPLC
Series 1100, USA equipped with degasser, quaternary pump, autosampler and diode array
detector was used. A Ultrasphere octadecylsilyl (ODS) Hypersil C18, 5 mm particle size, in a
250 mm length x 4.0 mm I.D stainless steel column (Hewlett Packard) was used to determine the
vitamins. The separation conditions for the antioxidant vitamins and riboflavin are tabulated in
Table 1.
Identification and quantification of vitamins
Vitamin C
Two techniques were used to identify the peak of vitamin C on the chromatogram: comparing
the retention time and spiking test with that of L-ascorbic acid (Sigma, Co. Chemical, St. Louis,
USA). Ascorbic acid standard was prepared by dissolving 100 mg of L-ascorbic acid in a
metaphosphoric acid (0.3 M) – acetic acid (1.4 M) solution at the final concentration of 1 mg/ml.
Table 1. HPLC conditions for separation and identification of vitamin C, β-carotene and riboflavin.
Conditions
Parameters Vitamin C β-Carotene Riboflavin
Mobile phases (1) 0.1 M potassium acetate, pH 4.9
(2) Acetonitrile-water (50:50) Acetonitrile-methanol-
ethyl acetate (88:10:2) Methanol-water-acetic
acid glacial (65:35:0.1)
Flow rate 1.5 ml/min 1.0 ml/min 1.0 ml/min
Detection 254 nm 250 nm 270 nm
β-Carotene
The peak of β-carotene was identified based on two techniques: comparing the retention time and
spiking test with that of trans-β-carotene (Sigma, Co. Chemical, St. Louis, USA). Ten milligram
of trans-β-carotene was weighed and dissolved in pure n-hexane to give a stock solution of 100
µg/ml. The solution was stored in a brown bottle and kept as stock in the fridge (4 – 5°C). The
standard solution of 1µg/ml was prepared daily from the stock solution.
Riboflavin
For the identification of the riboflavin peak, comparison of the retention time and spiking test
with that of riboflavin standard (Sigma, Co. Chemical, St. Louis, USA) was applied. The
riboflavin was prepared by adding 20 mg riboflavin in deionised water with the addition of three
drops of pure acetic acid glacial. It was warmed at 80°C in a water bath in order to dissolve the
riboflavin. The final concentration of the standard was 100 µg/ml.
Statistical analysis
Determination of Vitamin C, β-carotene and Riboflavin in Green Vegetables
Data were expressed as mean value ± standard deviation. Independent t-test was applied to
determine the significant difference at the level of p<0.05. A Statistical Package for Social
Science (SPSS) for Windows version 10.01 was used to analyse the data.
RESULTS
Vitamin quantification was calculated from the curve generated by plotting the peak area of each
authentic standard versus concentration.
Ascorbic acid
Organically (124.8 mg/100 g of fresh weight; CV = 15 %) and conventionally (114.7 mg/100 g
of fresh weight; CV=16 %) grown Chinese mustard had the highest ascorbic acid content among
the vegetables sampled (Figure 1). The lowest ascorbic acid content, as determined in
conventionally grown lettuce, was 15.3 mg/100 g of fresh weight. There were significant
differences (p<0.05) for Chinese kale, lettuce and swamp cabbage grown organically and
conventionally. The results showed no significant difference (p>0.05) in ascorbic acid content of
Chinese mustard and spinach grown conventionally and organically. Compared to the Nutritional
Composition of Malaysian Foods and ASEAN Food Composition Tables, the results obtained on
vitamin C content of conventionally grown vegetables were lower in Chinese kale, lettuce and
swamp cabbage (Tee et al., 1997; Puwastien et al., 2000).
β-Carotene
The organic swamp cabbage had the highest content of β-carotene of 3503 µg/100 g fresh
weight, while Chinese mustard (1994 µg/100 g fresh weight) and lettuce (2006 µg/100 g fresh
weight) grown conventionally had the lowest β-carotene content among the vegetables (Figure
2). Organically grown swamp cabbage was highly significant (p<0.01) in β-carotene content
compared to the conventionally grown ones. There was also significant difference (p<0.05) in β-
carotene content of Chinese mustard grown using the two methods. Besides, lettuce grown
conventionally and organically showed a similar β-carotene content. On the other hand, there
were no significant differences (p>0.05) for Chinese kale, lettuce and spinach grown using the
two different techniques. The β-carotene content in organically grown Chinese mustard and
swamp cabbage was significantly higher (p<0.05) than that of the conventionally grown ones.
Except for lettuce, the results obtained on β-carotene content were lower than that of Tee et al.
(1997).
Riboflavin
Only three types of B vitamins (riboflavin, thiamin and niacin) have been analysed and compiled
in Nutrient Composition of Malaysian Foods Table (Tee et al., 1997). Riboflavin was selected in
this study due to the established procedures in the laboratory. Furthermore, the present findings
on this vitamin could be compared to a previous database generated by Tee et al. (1997).
Amin Ismail & Cheah Sook Fun
Figure 1. Ascorbic acid content in five types of green vegetables grown organically and
conventionally. Asterisk (*) indicates a significant difference at the level p<0.05 between
organically and conventionally grown vegetables.
Figure 2. β-Carotene content in five types of green vegetables grown organically and conventionally.
Asterisk (*) indicates a significant difference at the level p<0.05 between organically and
conventionally grown vegetables.
Determination of Vitamin C, β-carotene and Riboflavin in Green Vegetables
Figure 3. Riboflavin content in five types of green vegetables grown organically and conventionally.
Asterisk (*) indicates a significant difference at the level p<0.05 between organically and
conventionally grown vegetables.
The conventional Chinese mustard had the highest riboflavin content of 0.26 mg/100 g of fresh
weight, while the lowest content of riboflavin was found in organically grown Chinese kale, that
is 0.22 mg/100 g of fresh weight (Figure 3). In this study, riboflavin content in conventionally
and organically grown lettuce was not detected. In addition, riboflavin content in conventionally
grown Chinese kale and spinach was also not detected compared to the organically grown
vegetables. The conventionally grown Chinese mustard was found to be significantly higher
(p<0.01) in riboflavin content compared to that of organically grown ones. However, riboflavin
was found to be significantly higher in organically grown swamp cabbage compared to
conventionally grown ones. There were significant differences (p<0.05) in riboflavin and β-
carotene contents between organically and conventionally grown swamp cabbage and Chinese
mustard.
DISCUSSION
Three main factors can influence the nutritional composition of vegetables grown organically or
conventionally, namely genetics, environment and post-harvest practices (Salunkhe & Desai,
1988). These factors are not controlled in vegetables purchased from retailers. In this study,
although little or nothing is known about the origin of the vegetables analysed, any differences in
vitamin values from both techniques of growing would be useful information to the consumers.
No significant difference (p>0.05) was found in vitamins C and β-carotene contents between
Chinese mustard and spinach grown organically and conventionally. This finding was similar to
Amin Ismail & Cheah Sook Fun
a report published by Organic Retailers and Growers Association of Australia (ORGAA) which
found no major differences in vitamin C and β-carotene contents of vegetables produced
organically or conventionally (Anon, 2000). Schuphan (1974) found that spinach and lettuce
grown organically were higher in ascorbic acid compared to those grown conventionally, using
composted manure over organic fertilizers. Through high nitrogen fertilization, Hornick (1989)
reported a lower content of ascorbic acid in kale grown organically compared to those grown
conventionally. Based on a farming comparison study, Lairon et al. (1984) observed no
difference in vitamin C content of kale and lettuce grown organically and conventionally.
As reported by Mercadante & Rodriguez-Amayua (1991), kale grown on farms using herbicides
was found to contain lower β-carotene compared to organically grown kale. Schuphan (1974)
found that inorganic fertilization slightly reduced the β-carotene content in spinach. Leclerc et al.
(1990) reported no significant differences in β-carotene between lettuce grown with
or without organic fertilization. Due to variation in the study designs by other researchers, it is
extremely difficult to compare with our findings. In addition, post-harvest factors which may
introduce variables such as maturity at harvest could well confound any apparent differences in
vitamin content.
Furthermore, our study used the HPLC method to quantify the vitamins content compared to Tee
et al. (1997). Probably some of the values obtained in the study were underestimated. For
instance, the content of vitamin C in Chinese mustard grown conventionally was found to be
higher compared to Tee et al. (1997). This could be due to an insufficient number of samples
or/and experimental error during HPLC analysis. In the present study, we were not able to detect
the presence of riboflavin in Chinese kale, lettuce and spinach grown conventionally, and lettuce
grown organically. In food analysis, determination of riboflavin often uses HPLC equipped with
a fluorescence detector. Riboflavin has a strong inherent fluorescence which allows it to be
detected very exclusively and sensitively (Van Niekerk, 1988). This probably explains why our
HPLC using UV detector could detect the riboflavin in some vegetables.
CONCLUSION
It was found that not all of the organically grown vegetables were higher in vitamin contents
compared to those grown conventionally. Vitamin C content was found to be significantly higher
in Chinese kale, lettuce and swamp cabbage grown organically compared to the conventionally
grown ones. Organically grown Chinese mustard and swamp cabbage were significantly higher
in β-carotene and riboflavin content. In this study, factors such as environment and post-harvest
practices that could influence vitamin content were not controlled. Thus it is extremely difficult
to make a true comparison between the two techniques of growing. Nevertheless, this study is
useful as a step towards further work on the generation of a vitamin content database for
vegetables grown organically.
ACKNOWLEDGEMENTS
Determination of Vitamin C, β-carotene and Riboflavin in Green Vegetables
The authors would like to acknowledge the assistance rendered by the laboratory staff from the
Department of Nutrition and Health Sciences throughout the research project and use of the
laboratory facilities at Universiti Putra Malaysia.
REFERENCES
Abdulnabi AA, Emhemed AH, Hussein GD & Biacs PA (1997). Determination of antioxidant
vitamins in tomatoes. Food Chem 60 : 207-212.
Anon (2000) Organic food is far more nutritious. Newsletter of the National Association of
Sustainable Agriculture Australia (NASAA). 10 February.
AOAC International (1990). Official methods of analysis. 15th ed. Arlington, VA, USA.
Bourn D & Prescott J (2002). A comparison of the nutritional value, sensory qualities, and food
safety of organically and conventionally produced foods. Critical Rev in Food Sci and Nutr
42 : 1-34.
Eggert FP & Kahrmann CL (1984). Response of three vegetable crops to organic and inorganic
nutrient sources. In : Organic farming: current technology and its role in sustainable
agriculture. Kral DM (ed). Madison, USA.
Hornick S (1989). Nitrogen overload may shrivel vitamin content. Agric Res 37 : 10-11.
Lairon D, Termine E, Gautier S, Trouilloud M, Lafont H & Hauton JC (1984). Effects of organic
and mineral ferlizations on the contents of vegetables in minerals, vitamin C and nitrates. In:
The importance of biological agriculture in a world of diminishing resources. H Vogtmann, E
Boehncke & I Fricke (eds). Witzenhausen, Germany.
Leclerc J, Reuille MJ, Miller ML, Lefebvre JM, Joliet E, Autissier N, Martinez Y & Perret A
(1990). Effect of climatic conditions and soil fertilization on nutrient composition of salad
vegetables in Burgundy. Sci Aliments 10 : 633.
Leclerc J, Miller ML, Joliet E & Rocquelin G (1991). Vitamin and mineral contents of carrot and
celeriac grown under mineral or organic fertilization. Biol Agr and Horticulture 7 : 339-348.
Mercadante AZ & Rodriguez-Amayua DB (1991). Carotenoid composition of a leafy vegetable
in relation to some agricultural variables. J Agric Food Chem 39 : 1094.
Puwastein P, Burlingame B, Raroeengwichit M & Sungpuang P (2000). ASEAN Food
Composition Tables. ASEAN Network of Food Data System (ASEANFOODS): Institute of
Nutrition, Mahidol University Thailand.
Amin Ismail & Cheah Sook Fun
Salunkhe DK & Desai BB (1988). Effects of agricultural practices, handling, processing, and
storage on vegetables. In: Nutritional evaluation of food processing, 3rd ed. Karmas E & RS
Harris (eds). New York: Van Nostrand Reinhold.
Schuphan W (1974). Nutritinal value of crops as influenced by organic and inorganic fertilizer
treatments. Plant Foods Hum Nutr 23 : 333-358.
Smith BL (1993). Organic foods vs supermarket foods: elemental levels. J. Appl Nutr 45 : 35-39.
Svec LF, Thoroughgood CA & Hyp Chung SM (1976). Chemical evaluation of vegetables
grown with conventional or organic soil amendments. Communication Soil Science Plant
Analysis 7 : 213.
Tee ES, Kuladevan R, Young SI, Khor SC & Zakiyah HO (1996). Nutrient analysis of foods.
Kuala Lumpur: Institute Medical for Research.
Tee ES, Mohd Ismail N, Mohd Nasir A & Khatijah I (1997). Nutrient composition of Malaysian
foods. Kuala Lumpur: Institute Medical for Research.
Van Niekerk PJ (1988). Determination of vitamins. In : HPLC in food analysis. R Macrae (ed).
London.
