evaluation of the effects of storage condition and processing...

JOURNAL OF HORTICULTURE AND POSTHARVEST RESEARCH 2018, VOL. 1(2), 147-162

Journal homepage: www.jhpr.birjand.ac.ir

University of Birjand

Evaluation of the effects of storage condition and processing on

carotenoids, chlorophyll, and micronutrients in Gnetum

africanum leaf Okpalanma Felix Emeka1 and Ojimelukwe Philippa Chinyere2* 1, Department of Food Science and Technology, Abia State University, Uturu, Nigeria 2, Department of Food Science and Technology, Michael Okpara University of Agriculture, Umudike, PMB 7267, Umuahia, Abia State, Nigeria

A R T I C L E I N F O

A B S T R A C T

Article history:

Received 1 May 2018

Revised 16 June 2018

Accepted 22 June 2018

Available online 29 June 2018

Keywords:

beta carotene

carotenoid profile

Gnetum africanum leaves

minerals

vitamins

DOI: 10.22077/jhpr.2018.1520.1021

P-ISSN: 2588-4883

E-ISSN: 2588-6169

*Corresponding author:

Department of Food Science and Technology, Michael Okpara University of Agriculture, Umudike, PMB 7267, Umuahia, Abia State, Nigeria. Email: [email protected]

© This article is open access and licensed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/4.0/) which permits unrestricted, use, distribution and reproduction in any medium, or format for any purpose, even commercially provided the work is properly cited.

Purpose: The aim of this study was to investigate the effects of ambient temperature storage condition (27-29 °C) and domestic cooking on contents of carotenoids, chlorophylls, vitamins and minerals in the leaves of Gnetum africanum. Research method: Carotenoids were separated and analyzed by HPLC. Total-carotene content, vitamins and minerals were assessed spectrophotometrically. Main findings: Results indicated that G. africanum was rich in lutein (528.87 µg g-1 (dry weight basis) and total β-carotene (248.10 µg g-1). There was no statistical increase in total β-carotene content due to cooking, but there was a decrease due to storage. Total β-carotene isomerized more during thermal processing than in storage. Cooking decreased (p>0.05) the contents of chlorophylls, water soluble vitamins and minerals. Cooking and storage of G. africanum resulted in significant losses in ascorbic acid, riboflavin and niacin. Cooking also reduced the potassium, calcium, magnesium and zinc. Iron contents of cooked and stored samples were higher than that of the raw sample. Research limitations: We had to travel over 600 km to use facilities for carotenoid analysis. Originality/value: The concentrations of lutein, β-carotene and certain micronutrients in G. africanum are much higher than typical contents in conventional edible leafy vegetables. The results of this study therefore provide evidence that G. africanum leaf could be an important contributor for improving the nutritional status of rural and urban people.

mailto:[email protected]

Felix Emeka and Philippa Chinyere

148 JOURNAL OF HORTICULTURE AND POSTHARVEST RESEARCH VOL. 1(2) SEPTEMBER 2018

INTRODUCTION

Gnetum africanum also called “Eru” belongs to the family Gnetaceae. It is a dioecious plant,

up to 10m long, but sometimes longer. Its branches are somewhat thickened at the nodes. The

fresh leaves of Eru is widely used as a vegetable. It is usually cooked and occasionally

consumed as a salad. Eru is mainly collected from the wild (Shiembo, 1997).

The scientific base supporting the unique health benefits derived from eating vegetables

is growing rapidly. The leaf is believed to possess antioxidant, anti-inflammatory and anti-

carcinogenic properties. It may be traditionally used as a remedy for some ailments.

Epidemiological researchers find strong link between increased vegetable consumption and

decreased risk of chronic diseases such as cancer, heart disease (Akwaowo et al., 2000).

Evidence is also emerging about a positive role for vegetable consumption in reducing the

risk of cataracts, diverticulosis, chronic obstructive pulmonary disease and hypertension (Ali

et al., 2011).

Gnetum africanum is a greenish climbing plant with thick papery leaves prevalent in the

tropical regions of Nigeria, Congo, Gabon, Angola, Asia and South America. The two main

species found in Africa are Gnetum buchholzianum and Gnetum africanum. The “okazi” vine

is a non-seasonal perennial plant that can grow as new shoots from the section where the stem

has been cut out or as a rhizome. Okazi, eru or afang vine usually produces tiny flowers on

maturity and the seeds resemble a drupe fruit, which is approximately 4–8 mm × 10–15 mm

in size. It is referred to as wild spinach or wild vegetable; okazi, afang, ukazi leaf (Nigeria)

fumbua (Congo), KoKo; Cameroonians call it okok, m’fumbua, or fumbua, eru (Fig. 1).

Vegetables contain a wide range of compounds including the antioxidant, vitamins C and

E, minerals, phenolics and carotenoids. Carotenoids possess a range of important and well

documented biological activities. They are potent antioxidants and free radical scavengers

(Grassmann et al., 2002) and can modulate the pathogenesis of cancer (Van Poppel &

Goldbohm, 1995). Many carotenoids including α-carotene, β-carotene and β-cryptoxanthin

have provitamin A activity, since they are converted to retinol by mammals. This role is of

particular importance, especially in developing countries where the dietary deficiency of

vitamin A can lead to blindness and premature childhood mortality (Mayne, 1996). The

xanthophylls, lutein and zeaxanthin are also known to provide protection against age-related

macular degeneration, mediated by their ability to quench singlet oxygen and blue light in the

retina (Landrum & Bone, 2001). Vegetables are therefore one of the most cost-effective and

sustainable solutions to micronutrient deficiencies, which affect far more people than hunger

alone, and this is crucial in most of sub-Saharan Africa region (IPGRI, 2008).

Fig. 1. Gnetum africanum leaves

Effects of storage condition and processing on Gnetum africanum leaf

JOURNAL OF HORTICULTURE AND POSTHARVEST RESEARCH VOL. 1(2) SEPTEMBER 2018 149

Development of appropriate postharvest handling techniques for many of these vegetable

species is a critical component in promoting utilization and commercialization of these crops

(Brown et al., 2005). A review by Rickman et al. (2007) also concluded that very few studies

have monitored changes in nutritional parameters in the same commodity from harvest

through storage and domestic cooking.

The purpose of this research is to undertake in-depth analysis based on HPLC on the

carotenoids of G. africanum (lutein, β-carotene, and β-crypotoxanthin). This research reports

on the effects of post-harvest ambient temperature storage condition on nutritional quality of

Gnetum africanum. Leafy vegetables are typically cooked prior to consumption in Africa, so

the other aspect of this study evaluated quality and nutrient retention after cooking. Overall,

the results of this study evaluate potentials of the local leafy vegetable, Gnetum africanum

(Eru) for contributing to the alleviation of micronutrient deficiencies among impoverished

rural populace in Nigeria. This will help to adequately establish its importance in human

nutrition and provide basis for maximum utilization of the crop.

MATERIALS AND METHODS

Selection and collection of sample

Gnetum africanum, a green leafy vegetable that is most commonly consumed by both rural

and urban communities in South-eastern Nigeria was used for research work. The leaves were

collected from the wild around (Amansea town in Anambra State and Udi in Enugu State) in

December 2011. It was selected at random from the plant area and picked by hand mid-

morning during the harmattan season (dry and windy season). A minimum of 1 kg of the crop

was collected randomly from different plants within the field. The leaves were placed in black

polyethylene bags and transported to the Biochemistry department of the University of

Nigeria, Nsukka for processing on the same day. Plant analyses were carried out at the

Biochemistry Department of University of Nigeria, Nsukka and at IITA (International

Institute of Tropical Agriculture), Ibadan, Nigeria.

Processing of samples In the laboratory, the inedible portions of the sample were separated and discarded. The edible

portions were washed with tap water. The edible portions of all the vegetables were divided in

three equal sub-samples. The first sub-sample was cooked for 5 min in boiling water with the

lid on. The second sub-sample was wrapped in a newspaper and stored in the dark for 5 days

at room temperature (29±2 °C) while the third sub-sample was kept raw.

Sample preparation for carotenoid analysis

All the raw, cooked and stored samples were oven dried in glass trays at 50 °C for about 48 h

until there was no further moisture loss. The dried leaves were milled and sieved through a

1mm stainless steel sieve to obtain a homogenized sample. Approximately 30 g of each of the

sieved powdered sample was stored in a sealed polyethylene bags and coded. The samples

were stored at -20 °C until they were delivered to International Institute of Tropical

Agriculture (IITA), Ibadan, Nigeria for carotenoid analysis.

Sample preparation for Analysis of chlorophylls

The leaves were analyzed raw, cooked and stored without the drying, milling and sieving

stages as earlier described. Chlorophyll analysis was carried out in the Biochemistry



laboratory of the University of Nigeria, Nsukka. Raw and cooked leaf samples were analyzed

same day, while stored leaves were analyzed five days later.

Sample preparation for analysis of vitamins

Vitamin analyses were also carried out on raw, cooked and stored leaves. Ascorbic acid,

riboflavin, thiamin, vitamin K and Niacin were analyzed. Raw, cooked and stored samples

were refrigerated (4 °C) before analysis in order to slow down physiological reactions that

lead to deterioration.

Sample preparation for analysis of mineral content

The mineral elements analyzed were potassium, calcium, magnesium, zinc and iron. They

were analyzed on raw, cooked and stored samples. The samples were subjected to wet

digestion before mineral.

HPLC Determination of total β-carotene Content The method of Howe and Tanumihardjo (2006) was used. The extraction of carotenoid from

dried, milled leaf samples (0.6 g) was done by adding ethanol (6 ml) containing 0.1% BHT,

mixing by Vortex, and placing in a reciprocal shaking water bath (model 25, precision

scientific, 373 Illinois 60647-4793) at 85 °C for 5min. Potassium hydroxide (500 l, 80% w/v)

was added to the mixture to saponify the interfering oil and chlorophyll. Samples were

vortexed and placed in a water bath (85 °C) for 5min, vortexed again and returned to the

water-bath for additional 5min. Upon removal, they were immediately placed in an ice bath

(AF 200PSC 50R, Serial No. A 9601 BF 0981) where 3 ml of cold deionized water was

added. Carotenoids were separated 3 times with addition of 3 ml of hexane, vortexed and then

centrifuged (1200 g) for 5 min.The combined hexane fractions were washed with deionized

water 3 times, vortexed and centrifuged for 5 min at 1200 g. The hexane fractions were dried

down in a vacuum rotary evaporator.

Samples were reconstituted in methanol/dichloromethane (1 ml, 50:50 v/v) and 100 l

were injected into the HPLC. A Waters HPLC system (water corporation, Milford, MA)

consisting of a guard-column, C30 YMC carotenoid column (4.6 X 250 mm, 3 l) water 626

binary HPLC pump, 717 auto sampler and a 2996 photodiode array detector was used for

carotenoids quantification. Solvent A consisted of methanol/water (92:8 v/v) with 10mm

ammonium acetate. Solvent B consisted of 100% methyl tert-butyl ether. Sample purification

was performed by gradient elution at 1 ml/min with the following conditions: 29min linear

gradient from 83% to 59%, 6 min linear gradient from 59% to 30% 1 min hold at 30%, 4 min

linear gradient from 30% to 83%, and a 4 min hold at 83%.

Chromatograms were generated at 450 nm. Identification of lutein, -cryptoxathin, and

-carotene were carried out using standards and with verification of absorption spectrum.

Standard curves for lutein, -cryptoxanthin and -carotene standards are already established

in the crop utilization laboratory of the International Institute of Tropical Agriculture (IITA),

Ibadan, Nigeria.

Spectrophotometric determination of total carotene content

Determination of total carotene content of the leaf samples was according to the method of

Rodriguez-Amaya and Kimura (2004). About 0.2-0.3 g of the homogenous and representative

sample was weighed into a mortar and a small amount (0.5 g) of sea sand of size -50+70 mesh

(Sigma-Aldrich) was added to it. Pestle was used to grind the mixture with 20 ml of cold

acetone to extract the carotenoids. The resulting solution was filtered under suction through a



Buchner funnel with filter paper (Whatman No. 2 filter paper, Middlesex, U.K). After that,

the mortar, pestle, funnel and residue were washed with small amounts of acetone, and the

washings were received in the suction flask through the funnel. Thereafter, the residue was

returned to the mortar and fresh cold acetone was added and it was macerated again. The

resulting solution was filtered and washed as before.

The acetone extract was further partitioned to petroleum ether. Five milliliters (5 ml) of

distilled water, followed by 20 ml of petroleum ether was put into a 500 ml separating funnel.

After that, the acetone extract was transferred into the separating funnel in batches. Also

distilled water (about 300 ml) was slowly added by allowing it to flow through the walls of

the funnel and prevent the formation of emulsion. The washing was completed with 250 ml

brine solution to break any emulsion formed. During the last washing, care was taken to

discard the lower phase as completely as possible without interfering with the upper phase.

The upper phase was collected in a 25 ml volumetric flask. After that, 20 ml aliquot was

transferred to a 50 ml round-bottomed flask and concentrated in a rotary evaporator (Buchi

Waterbath, B-481 Switzerland) to about 1 ml volume.

Furthermore, chromatographic separation of the Petroleum ether (P.E.) carotenoid

solution was also carried out. The column was prepared by putting some quantity of neutral

alumina (of activity III) (about 1 g). After that, the side of the column was tapped several

times to better accommodate the adsorbent in the column. Also the column was topped with

0.5 cm layer of anhydrous sodium sulphate. An aliquot of the P.E. carotenoid solution was

added to the column with a dropper. Thereafter, the column was eluted with petroleum ether.

Also, the round-bottomed flask was rinsed two times with about one ml of P.E. and the

rinsing was added to the column. Addition of P.E. was continued after which the first fraction

was collected into 5 ml volumetric flask. The volume was made up with P.E. and the

absorbance was later read at 450 nm, using Jenway Spectrophotometer (1).

Total carotene content (g g-1) =𝐴𝑓𝑟1 × 𝑣𝑜𝑙𝑢𝑚𝑒 (𝑚𝑙) × 104×(𝐷𝐹)

𝐴1%

1𝑐𝑚 × 𝑠𝑎𝑚𝑝𝑙𝑒 𝑤𝑒𝑖𝑔ℎ𝑡

× 1.25 (1)

Where,

𝐴𝑓𝑟1 = Absorbance at 450nm

Volume (ml) = volume of fraction 1 (5 ml)

𝐴1%1𝑐𝑚

= 2592 (absorption coefficient of 𝛽-carotene in petroleum ether (P.E)

Determination of chlorophylls a, b, and total chlorophyll Chlorophylls a, b, and total chlorophyll content of the leaf samples were determined

according to the method of AOAC (2005). Two grams (2 g) of each leaf sample was

macerated with 0.1 g CaCO3 and extracted repeatedly with 20 ml of 85% acetone until the

pigment was completely extracted. The extract was made up to 100 ml with 85% acetone.

Twenty five milliliter (25 ml) of ether and 25 ml of water were added to the filtrate and

shaken vigorously in a separating funnel. The lower layer was discarded and another 50 ml

was added and shaken.

The mixture was allowed to settle and the lower layer discarded. Ten milliliter (10 ml) of

the upper layer was washed 5 times with 10 ml portion of water until all the acetone was

removed. The ether layer was transferred and made up to 10 ml with ether. A teaspoonful of

anhydrous Na2SO4 was added to remove residual water and the absorbance taken at 663 nm

and 645 nm against ether as the blank, the concentrations of the chlorophylls were calculated

from the following equations (2, 3 and 4).

Total chlorophyll (mg/g) = (7.12𝐴663+16.8𝐴643) ×𝑣

∝ ×1000 ×𝑤 (2)



Chlorophyll a (mg/g) = (9.93𝐴663−0.777𝐴643) ×𝑣1

∝ ×1000 ×𝑤 (3)

Chlorophyll b (mg/g) = (17.6𝐴643− 2.81𝐴663) ×𝑣

∝ ×1000 ×𝑤 (4)

Where V1 = volume in ml

W = weight of sample

= the length of light path in the cell (usually 1 cm)

Determination of Vitamins

The vitamin content of Gnetum africanum leaf was determined by standard methods

described by AOAC (2005).

Determination of minerals

A quantity of 0.5 g of each of the leaf samples was weighed into Kjeldhal flask. Twenty-five

(25) ml of a mixture of concentrated Hydrochloric and Nitric acid at the ratio of 3:1 (v/v) was

added to the flask. The Flask and its content were heated until the color of the mixture

cleared. The colorless liquid was then transferred to a 250 ml volumetric flask and was made

up to the mark with distilled water. This solution was evaluated for its content of Potassium,

Calcium, Magnesium, Zinc and Iron using Atomic Absorption Spectrophotometer (AAS)

(Buck Scientific 210VGP) was used for the detection of the minerals.

Statistical analysis

The means, standard deviations and analysis of variance (ANOVA) of all the data obtained

from the study were computed using the Statistical Package for Social Science (SPSS) version

17. Analysis of Variance was specifically performed to detect significant differences (p

0.05) among the sample means followed by the application of Least Significant Difference

test (LSD) for the separation of significant means.

The experiment has a randomized complete block design having vegetable type x 1 and

processing (treatments) x 3 as some of the variations giving 1 x 3 = 3 observations. Each

observation was repeated three times giving 3 x 3 = 9 observations for each parameter tested.

RESULTS AND DISCUSSION

Chromatographic profiles of carotenoids

The chromatogram of the most common carotenoids pattern presented in investigated leafy

vegetable is shown in Figure 2. Two classes of carotenoids namely; xanthophylls and

carotenes were identified and quantified under the HPLC conditions used in our research.

Carotenoids were eluted in the following order: Lutein, β- cryptoxanthin, 13-cis β-carotene,

15-Cis- β-carotene, trans- β-carotene and 9-cis- β-carotene. The chromatographic peaks of

standard were used to identify carotenoids from samples. Carotenoid peaks of samples given

different treatments (raw leaves; leaves cooked for 5 minutes before extraction and leaves

stored for 5 days) are shown in the Figures given as follows.

Total β-carotene Content

Table 1 shows the carotenoids concentration of Gnetum africanum leaf as a result of effects of

storage and cooking treatments. There were no significant changes in the levels of cooked leaf

Total β-carotene (Tβ-c) (248.10 µg g-1) and its raw leaf (246.93 µg g-1). Cooking for 5 min

did not soften the tissues of G, African leaf that would have resulted in higher extractability

(Rodriguez-Amaya, 2002). This could be explained by hard and tough attribute of freshly



harvested eru leaves. However, cooking resulted in 100.47% apparent retention when

compared with the raw leaf (Table 1). Dietz and Erdman (1989) reported that cooking resulted

in greater than 100% retention of β-carotene in vegetables, because denaturation of carotene

binding proteins releases the carotenoids so that they can be extracted more easily. Apparent

retention was calculated according to Bergström (1994) since our leaf samples were analyzed

on dry weight basis.

As expected, the percentage trans- β-carotene (Table 1) was lower in the cooked than in

raw leaves. During the cooking, some of the tans-β-carotene could have been converted to

Cis-isomers or other oxidation products (Nyambaka & Riley, 1996). The levels of Cis-

isomers of β-carotene are therefore higher in cooked leaves than in raw leaves (Kao et al.,

2012). Literature suggests that the consequences of trans/Cis-isomerization are changes in

bioavailability and physiological activity (Casternmiller & West, 1998). However, only trans-

β-carotene is preferentially converted to retinol in the enterocyte (Faulks & Southian, 2005).

Table 1 also shows a significant decrease in the content of Tβ-c (122.6 µg g-1) due to

storage. This could be attributed to an increase in the degree of liquefaction of the tissue.

Immature tissues with high respiratory rates often exhibit hardening and lignification during

storage (Vina & Chaves, 2003). Meanwhile, the decrease in Tβ-c during storage resulted in an

apparent retention of 49.66%. Comparing the Tβ-c content of G. africanum with previous

reports, Schönfeldt and Pretorius (2011) recorded similar trend. They reported that Tβ-c

content of cooked leaves can range from 23.43 µg g-1 Amaranthus tricolor to 61.53 µg g-1,

(Corchorus tridens) and of raw leaves from 16.01 µg g-1 (Amaranthus tricolor) to 36.63 µg/g

(Corchorus tridens). ŽnidarČiČ et al. (2011) recorded 70.1 µg g-1 and 79.6 µg g-1 in wild

rocket and Garden rocket, respectively. It seems therefore, that the eru leaves are very rich

dietary source of β-carotene.

The data shown on Table 1 indicates that the most abundant cis-isomers of β-carotene in

the raw, cooked and stored of samples was 15-cis- β-carotene. There was no significant

change in concentrations of cooked leaf β-carotene isomers when compared with raw leaves.

The reverse was the case in stored leaf β-carotene isomers. Thermal processing of foods

results in trans-/-cis-isomerization (Rodriguez-Amaya, 2002). Available information from

literature suggests that the consequences of trans/cis- isomerization are changes in

bioavailability and physiological activity (Casteinmiller & West, 1998). However, only trans

β-carotene is preferentially converted to retinol in the entorocyte (Faulks & Southian, 2005).

Several different geometric isomers of β-carotene trans-, 9-cis, 13-cis-, and 15-cis isometric

farms exist in food and human tissues (Chandler & Schwartz, 1987). The major β-carotene

isomers in the circulation of humans are trans- β-carotene, with small amount of 13-cis- and

9-cis- β-carotene. However, circulating levels of the cis-isomers of β-carotene are not

responsive to increased consumption of their isomers (Stahl et al., 1993). Besides, literature

suggests that each carotenoid shows an individual pattern of absorption, plasma transport and

metabolism (Deming & Erdman, 1999). The levels of cis-isomers of β-carotene are much

higher in leafy vegetables (Marx et al., 2010). Therefore, isomer separation is needed for the

accurate determination of the Vitamin A activity of leaf meals.

Xanthophyll Content

The β-crytoxanthin content of the leaf was relatively small (Table 1) β-cryptoxanthin is a

minor provitamin A constituent of leaves (Rodriguez-Amaya, 2002). There was no significant

change in the concentration of cooked leaf β-cryptoxanthin (6.22 µg g-1) and raw leaf β-

cryptoxanthin content (5.77 µg g-1). However, stored leaf β-cryptoxanthin content was

significantly lower (4.79 µg g-1).



Fig. 2. Carotenoid profile of Gnetum africanum leaf samples by HPLC (a) raw (b) cooked (c) stored

Lutein content of stored leaves (394.31 µg g-1) was significantly lower than both the

cooked leaves (504.92 µg g-1) and raw leaf sample (526.87 µg g-1 dwt). Cooking of the leaves

for 5min did not result in any significant change in the concentration of lutein when compared

with the raw leaves. (Table 1). Several previous reports on lutein contents of leafy vegetables

had been recorded. Kopsell and Kopsell (2009) had showed that lutein concentration can

range from 4.8 to 13.4 mg 100g-1 for kale and from 6.5 to 13.0 mg 100g-1 for spinach. A

similar trend was reported by Dias et al. (2009) who have shown values from 0.52 to 6.4 mg

100g-1 for kale and from 3.6 to 5.6 mg 100g-1 fwt for leaf beat and turnip greens. Lefsrud et al.

(2009) reported 11.0 ± 0.70 mg 100g-1 lutein in win terbor Kale. Comparing the lutein

concentration in raw G. africanum with the values reported, G. africanum is therefore

considered a rich source of dietary lutein. According to Wisniewska and Subczynski (2006),

the presence of lutein and/or zeaxanthin in the diet may be beneficial for reducing the

incidence of the two common eye diseases of ageing, age related macular degeneration and

cataracts formation.

C

b

a



Pro-vitamin A Content

The Pro-vitamin A concentration in the leaf sample was calculated (Table 2) by adding the

value of total β-carotene to one-half the value of the corresponding β-cryptoxanthin

(Rodriguez-Amaya & Kimura, 2004). The principal pro-vitamin A carotenoids in leaves is β-

carotene, while the β-cryptoxanthin is a minor constituent (Rodriguez-Amaya & Kimura,

2004). The pro-vitamin A content of the raw, cooked and stored leaf sample are 251.21,

249.82, and 125.03 µg, respectively. Several different geometric isomers of β-carotene (all-

trans, 9-cis, 13-cis and 15-cis isomeric forms) exist in food and human tissues. The major β-

carotene isomer in the circulation of humans is all-trans- β-carotene with small amounts of

13-cis- and 9-cis- β-carotene (Chandler & Schwartz, 1987). The FAO/WHO joint expert

consultation on Vitamin A requirements has assumed that the Vitamin A activity of other

provitamin A carotenoids, including cis-isomers of β-carotene is 50% that of β-carotene. Also

studies conducted by Bauernfeind (1972) and Zechmeister (1949) have generally supported

these data: All-trans- β-carotene, 100%, 9-cis- β-carotene, 38%, 13-cis- β-carotene 53% and

β-cryptoxanthin 55%.

Table 1. Effects of storage and processing methods on carotenoids content of Gnetum africanum leafy vegetable

Treatment Lutein β-Crypt. 13-cis 15-cis Trans 9-cis Total BC

Raw 528.87a±1.24 5.77a±0.25 34.35a±0.52 91.84a±1.95 96.53a±1.45 24.21a±1.68 246.93a±6.68

Cooked 504.92a±1.85 6.22a±0.25 36.90a±0.45 89.14a±2.94 95.56a±0.86 26.51a±0.76 248.10a±0.35

Stored 394.31b±1.27 4.79b±0.29 8.10b±0.19 27.56b±2.34 65.41b±2.02 21.56b±1.35 122.63b±5.50

Values are means ± standard deviations of duplicate determinations on dry weight basis. Means with different superscripts

within the same column are significantly different (p≤ 0.05). Carotenoid Content (𝜇𝑔/𝑔 edible portion, dry weight basis)a 9-

cis: 9-cis-β-carotene, 13-cis: 13-cis-β-carotene, 15-cis: 15-cis-β-carotene, Trans-: Trans-β-carotene: β-crypt = β-

Cryptoxathin.

Table 2. Change in pro-vitamin A content of leaves of G. africanum due to cooking and storage

Pro-vitamin A % trans % Retention

(µg/g) of total β-Carotene (Total β-Carotene)

251.21 39.09

249.82 38.51 100.47 (after cooking)

125.03 53.33 49.66 (after storage)

Table 3. Effects of storage and processing methods on chlorophyll content of Gnetum africanum leafy vegetable

Treatment Chl a Chl b Tchl Chl 𝑎 𝑏⁄ ratio

Gnetum africanum Raw 2.18b±0.002 0.83b±0.008 3.01b±0.008 2.62

Cooked 0.82a±0.006 0.59a±0.007 1.41a±0.005

Stored 1.26a±0.004 0.65a±0.003 1.91a±0.002

Values are means ± standard deviations of duplicate determinations on fresh weight basis. Means with different superscripts

within the same (Specie) column are significantly different (P≤ 0.05). Chl a: Chlorolphyll a, Chl b: Chlorophyll b, Tchl:

Total Chlorophyll. Chlorophyll content (mg/g edible portion, dry weight basis).

Table 4. Effects of storage and processing methods on selected vitamins of Gnetum africanum leafy vegetable

Treatment Ascorbic acid

(mg)

Riboflavin

(mg)

Thiamin

(mg)

Niacin

(mg)

Vit. K

(µg)

Gnetum africanum Raw 57.20a±0.14 0.63a±0.02 0.18a±0.01 3.05a±0.01 120b.44±0.21

Cooked 27.91b±0.02 0.33b±0.01 0.14b±0.01 1.59b±0.01 124a.04±0.20

Stored 35.45b±0.07 0.47b±0.01 0.16a±0.014 2.05b±0.01 119b.21±0.23

Values are means ± standard deviations of duplicate determinations on fresh weight basis. Means with different superscripts

within the same column are significantly different (p≤ 0.05). Vitamin content (mg per 100g edible portion), fresh weight

basis.



Vitamin A is expressed as β-carotene, retinol, retinol equivalent (RE) or retinol activity

equivalent (RAE). Hence 1 retinol equivalent = 1 µg of retinol = 12 µg of β-carotene = 24 µg

of other pro-vitamin A and cis-isomers of β-carotene (FBN, 2003). Using the above

conversion factors and excluding the 15-cis- β-carotene isomer values, the calculated vitamin

A activity RE/100g of the raw, cooked and stored leaf samples were 1072.74, 1086.45 and

688.62 respectively. It follows therefore, that the consumption of about 100 g/day of the

cooked leaves analyzed in our study would meet the recommended daily allowance (RDA) of

900 RE/day for men and 700 RE/day for women, 19-30 years old (FBN, 2003).

Total–Carotene Content

Postharvest storage and cooking effects on total-carotene content of G. africanum leaf was

presented in Fig 3. The total-carotene content in cooked leaf (768.35 µg g-1) was significantly

(p>0.05) higher than in raw (694.30 µg g-1) and stored (608.56 µg g-1) leaf. This could be

explained by higher extractability of carotenoids in cooked leaves. There were no statistical

differences in total-carotene content in raw leaf sample when compared with the stored leaf

sample. This implies that the conditions under which the leaves were stored did not degrade

the carotenoid. However, there was numerical increase of total-carotenes in stored leaf

samples when compared with raw leaf. Total-carotene value was higher than the

corresponding total- β-carotene value. The differences were attributed to the assay method.

The spectrophotometric method (AOAC) gives higher values for samples that contain a

complex mixture of carotenoids, because the AOAC method measures total-carotene

(Simpson et al., 1983). Granado et al. (1997) pointed out the HPLC methods now available

are more specific than other spectrophotometric assays. The HPLC we used was specific to

Tβ-c.

Fig. 3. Total carotene content of raw, cooked, and stored leaf samples of Gnetum africanum

Table 5. Effects of storage and processing methods on selected mineral content of Gnetum africanum leafy vegetable

Treatment K Ca Mg Zn Fe

Gnetum africanum Raw 2.45a±0.07 0.90a±0.14 0.95a±0.07 1.50a±0.14 5.00b±1.27

Cooked 1.70b±0.14 0.55b±0.07 0.60b±0.14 0.95b±0.07 6.70a±0.14

Stored 2.10a±0.14 0.80b±0.14 0.85a±0.07 1.35a±0.07 6.05a±0.07

Values are mean±standard deviation of triplicate determination on fresh weight basis means with different superscripts

within the same column are significantly different (p=0.05). Mineral content (mg/100g edible portion fresh weight basis).

0

100

200

300

400

500

600

700

800

900

Raw Cooked Stored

)1-

To

tal

caro

ten

e co

nte

nt

(µg g

a b

b



Chlorophyll Content

Chlorophyll a (chl a), chlorophyll b (chl b) and total chlorophyll (tchl) contents of Gnetum

africanum are shown in Table 3. Initial contents of chl a, chl b and tchl in the raw leaf were

2.18, 0.83 and 3.01mg/g respectively. Chlorophyll degradation is accompanied by the loss of

colour during processing and storage. Colour change during the selling period is very

important because it affects produce attractiveness to consumers (Biswall, 1995). The loss of

chl a, chl b and tchl after cooking the leaves for 5mins at 100 °C were 62.38%, 28.91% and

53.15%, respectively. While the loss at the end of storage period (5days) were 42.20%,

21.68% and 36.54% respectively. The data showed that chl a degraded faster than chl b in all

cases. These results were in accordance with previous experiments (Ferrente et al, 2004; Negi

& Roy, 2000; ŽnidarČiČ et al., 2011). The ratio chl a/b (2.62) was similar to the values

reported for other dark green leafy vegetables (Schwartz & Von Elbe, 1983).

We can conclude that G. africanum had relatively high content of chlorophyll similar to

that in Kale (Kospell et al., 2004), Garden rocket (ŽnidarČiČ et al., 2011) and exceeding that

in Spinach (Jaworska & Kmiecik, 1999).

Vitamin content

The vitamin content of Gnetum africanum leaf is listed in Table 4. Vitamin concentrations (in

mg 100 g-1) were 57.20, 0.63, 0.18 and 3.05 in Ascorbic acid, riboflavin, thiamin and Niacin

respectively, while vitamin K1 concentration was 120.44 µg 100 g-1. Cooking decreased

significantly (p>0.05) the contents of the water-soluble vitamins. Water soluble vitamins

leach into cooking water and some portion of it may actually be destroyed by heating

(Usuwazo & Tanulo, 2006). However, cooking increased significantly (p>0.05) the content of

vitamin K1 in the leaf. Vitamin K1 is located in chloroplasts in plants, cooking by boiling may

disrupt the cell wall, thereby releasing vitamin K1 available for measurements (Damon et al.,

2005). The loss of Ascorbic acid observed in this study was 51.35%. The reported cases of

ascorbic acid loss during blanching or cooking are enormous and may vary between 40 and

70% in some cooked vegetables when processed at 100 °C for 10 min. (Mepba et al., 2007).

Cooking is often responsible for the greatest loss of vitamin C and the effect of the loss

depends upon variations in cooking methods and periods (Oboh, 2005). Also at the end of

postharvest storage, the contents of the vitamins decreased significantly (p>0.05). Vitamin C

degradation is due to auto-oxidation and also enzymatic degradation. Therefore Vitamin C

losses continue through postharvest handling, processing, cooking, and storage of fruit and

vegetables (Moreira et al., 2006). The loss in ascorbic acid after storage in this study was

35.75%. Other researchers have also reported postharvest losses in ascorbic acid. A 29-50 and

34-38% losses were recorded by Prabhu and Barett (2009) in Cassia tora and Corchorus

tridens leaves stored at 20 °C for 8 days. The fact that losses of ascorbic acid from vegetables

are large during blanching procedures and relatively small during storage suggests that losses

during blanching occur primarily by leaching rather than by chemical degradation (Fennema,

1988). Ascorbic acid is easily oxidized, so it will gradually decrease during storage.

Initial contents of some of the vitamins were in agreement with previous reports. Mepba

et al. (2007) reported 56.8 mg 100 g-1 ascorbic acid in G. africanum. However, lower values

of some of these vitamins were reported for various dark green leafy vegetables by Agtel et al.

(2009) and Uusiku et al. (2010). Variations in nutrient content in processed vegetables’,

concentrations can vary depending on vegetable type, maturity at harvest, genetic variations,

preharvest conditions, postharvest handling, storage conditions, processing and preparation

(Gregory, 1996; Selman, 1994). It is therefore evident that concentrations of ascorbic acid,

Riboflavin and vitamin K1 in G. africanum are high and adequate to meet the RDAs of 90



mg/day ascorbic acid, 1.3 mg/day, riboflavin and 90 µg/day vitamin K1 in children and adults,

respectively (FNB, 2003). Eru leaf is therefore crucial in micronutrient malnutrition (MNM)

intervention in most developing countries of Sub-Sahara Africa.

Mineral Content

The effects of storage and processing on the selected mineral contents of Gnetum africanum is

presented on Table 5. The mineral contents of the raw leaf were (in mg 100g-1) Potassium

2.45, calcium 0.90, magnesium 0.95, zinc 1.50 and Fe 5.00, respectively. Cooking of the

leaves for 5min at 100 °C significantly (p>0.05) decreased the levels of the elements.

Oladunmanye et al. (2005) observed significant (p>0.05) reductions in K, Na, Ca and Fe

contents of cooked tender and matured cassava leaves. During the cooking process, these

minerals leached into cooking water (Souzan et al., 2007). The Potassium, Calcium and Iron

in G. africanum correlated well with the findings in the reports of Ekpedeme et al., (2000),

Mepba et al. (2007) and Sobowale et al. (2010). However, higher values for calcium and

potassium were reported by Luciane et al. (2003) in watercress Kale and Cabbage and in the

review of Uusiku et al. (2010) in Amaranths spp and Solanum nigrum leaves, respectively.

This variability was expected since the Fresh Green Leafy Vegetable (FGLV) belonged to

different locations, where the soil and climatic conditions including light and temperature are

dissimilar (Federico et al., 2004). The addition of fertilizers, the age of tissue, chemical

composition of the medium in which the food was grown are also important.

Postharvest storage conditions in our study resulted in a non-significant reductions in the

mineral contents when compared with the raw leaves. The reductions could be due to effects

of oxidizing agents, exposure to heat, light, and extremes of pH and other factors that affect

organic nutrients (Damodaran et al., 2008).

The results for mineral analysis of G. africanum suggest the consumption of large

quantities to meet the recommended daily allowance (RDAs) for minerals.

CONCLUSION

The potential of locally consumed leafy vegetable, G. africanum in alleviating micronutrient

deficiencies, especially Vitamin A deficiency prevalence was evaluated. The results showed

that the consumption of about 50g per pay of cooked G. africanum leaf would meet the

required daily allowance (RDA) of Vitamin A in children and adults. Also the result indicated

that consumption of about 100g of G. africanum leaf per day would meet the RDAs of

Riboflavin, Ascorbic acid and Vitamin K1 in children and adults respectively.

Storage and cooking of the leaves after harvest resulted in several changes in quality and

nutritional parameters. There were significant changes in carotenoids, chlorophylls, and

vitamins and minerals during cooking and significant changes in ascorbic acid during storage

and cooking of the leaves. Therefore the data generated on the composition of carotenoids,

chlorophylls, vitamins and minerals in this leafy vegetable could be the basis for suggesting

the inclusion of this leaf in a daily diet to overcome health problems such as vitamin A

deficiency, iron deficiency anemia and age-related macular degeneration. Gnetum africanum

is presently underutilized and mainly found in the wild. There is need to domesticate and

diversify the utilization of this nutritious leafy vegetable.



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هایکارتنوئیدها، کلروفیل و ریز مغذیبر شرایط نگهداری و فرآوری بررسی تأثیر

)اسفناج وحشی( افریقاییوم تموجود در برگ گ

اوکیاالنما فلیکس امیکا و اوجیملوکوه فیلیپه چینیری

چکیده

بر داخلی( و پختن گراددرجه سانتی 92-92) ی محیطدمادر هدف از این مطالعه، بررسی تاثیر شرایط نگهداری

ها با کاروتنوئیدبود. Gnetum africanum هایها و مواد معدنی در برگمیزان کاروتنوئیدها، کلروفیل، ویتامین

ها و مواد معدنی به وسیله اسپکتروفتومتری ، ویتامینروتنمقدار کل کا .شدند آنالیزو جداسازی HPLC استفاده از

کل کاروتن -بتا و میکرو گرم بر گرم( 72/897) غنی از لوتئین G. africanum نتایج نشان داد که ند.شد رزیابیا

وجود ه شدنپخت واسطهبه کل کاروتن -بود. هیچگونه افزایش آماری در مقدار بتامیکرو گرم بر گرم( 01/947)

حرارتی آوری فر یدر طکل کاروتن -بتانگهداری وجود داشت. واسطهبه کل کاروتن -بتادر کاهش اما شت،ندا

های محلول در آب و مواد معدنی را کاهش داد. محتوای کلروفیل، ویتامینپختن، . نگهداری، ایزومری شدبیشتر از

ن . پختشد در اسید اسکوربیک، ریبوفالوین و نیاسینداری افت معنیموجب G. africanum و نگهداری نپخت

شده نگهداریهای پخته شده و نمونهدر مقدار آهن باعث کاهش پتاسیم، کلسیم، منیزیم و روی شد. همچنین

بسیار باالتر از G. africanum کاروتن و بعضی از مواد مغذی در-بتالوتئین، هایلظتغ بود.بیشتر از نمونه خام

د که برگدانتایج این مطالعه نشان ،است. بنابراینمتداول خوراکی یسبزیجات برگدر موجودمقادیر

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evaluation of the effects of storage condition and processing...

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