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Populations suffering from food shortages

Abstract

Prior to commercialization of wild food plants their nutritional and safety status requires investigation. Currently a few plants from the family Amaranthaceae are used by indigenous communities as sources of nutrition worldwide. This study focused on the nutritional analysis, bioactivity and safety of three Amaranthaceae plants, namely Achyranthes aspera, Alternanthera sessilis and Guilleminea densa. A. aspera and A. sessilis met the RDA levels for energy, protein, dietary fibre and carbohydrates however G. densa met approximately 50% of the RDA. A.aspera contained Vitamin B1, B2 and C, A.sessilis contained Vitamin A and B3 and G. densa contained only Vitamin B1 and B2. A. sesslis met the RDA for Mg, Mn and Fe, G. densa contained Fe and A. aspera did not contain sufficient quantities of minerals to meet the RDA. All three plants showed antioxidant activity ranging from 54% to 87%. The aqueous and methanolic extracts of the three plants were generally effective against the Gram negative bacteria except in the case of Proteus mirabilis. The extracts were not effective against the Gram positive bacteria except Staphylococcus aureus and Staphylococcus epidermidis. All extracts did not show antifungal activity although A. aspera and A. sessilis had activity against the yeasts Sacchyromyces cerevisae and Candida albicans. The plants were considered safe because they showed no toxicity against the brine shrimp or mutagenicity against the Salmonella typhi strains TA 98 and TA 100 or cytotoxicity against the K562 cell line. It is thus recommended that the three plants be considered as safe choices of famine food plants.

Keywords

Famine food plants, Amaranthaceae, Nutrition, Antimicrobial, Antioxidant, Anti-inflammatory, Safety

Corresponding Author

Bharthi Odhav. Mailing address: Department of Biotechnology and Food Technology, ML Sultan Campus, Durban University of Technology. Telephone: 27 31 373 5330, Fax: 27 31 373 5351, Email: odhavb[at]dut.ac.za; reddyla[at]dut.ac.za and alveeras[at]yahoo.com.

Introduction

During times of natural and man made disasters populations suffering from severe food shortages become heavily reliant on wild food plants for their survival. This gives rise to the notion of famine food plants (Leborgne et al., 2002). Famine plants have been eaten and utilized for centuries. These may be reintroduced as standard crops to stabilize the land and mitigate the cycles of famine if they prove to be suitable as nutritional sources. Improved strains of native African "famine plants" that thrive through adverse weather cycles of drought and excess rainfall, should be reintroduced and cultivated in place of foreign cash crops as these crops are more nutritious than foods introduced from abroad. Imported cash crops that have been introduced into African agricultural systems may be detrimental to the sustainability of African farming. Publications (Maundu et al., 1999, Freedman, 2006) showed that wild plants are essential components of many Africans' diets, especially in periods of seasonal food shortage and ethnobotanic studies deal with their medicinal properties.

One of the plant families frequently consumed as leafy vegetable in Africa belongs to the family Amaranthaceae. Data pertaining to amaranths is limited to Amaranthus dubius, Amaranthus spinosus and Amaranthus hybriduswhich are commercially cultivated. The other amaranth species used as famine plants are not well studied. Plants from this family are used in indigenous system of medicine for their antiarthritic, antifertility, laxative, ecbolic, abortifacient, anthelmintic, aphrodisiac, antiviral, antispasmodic, antihypertensive, anticoagulant, diuretic and antitumour activities (Naples, 2005). They are used to treat cough, renal dropsy, fistula, scrofula, skin rash, nasal infection, chronic malaria, impotence, fever, asthma, amennorrhoea, piles, abdominal cramps and snake bites (Manandhar, 2002). Furthermore, some of the members from this family have important phytochemicals such as rutin which is a strong antioxidant compound and saponins (Odhav et al., 2007). The nutritional, antibacterial, antifungal, anthelmintic, anti-amoebic, antischistosomal, antimalarial, anti-inflammatory and antioxidant activity, as well as psychotropic and neurotropic activity using appropriate in vitro tests forms basis for validating the usefulness of famine plants. Final commercialization potential is then often provided through the isolation of active compounds. It is also important to investigate how safe, these plants are for consumption, since some are potentially toxic and can produce toxic constituents during stressful environmental conditions. A review of literature showed that many of the medicinal properties are attributed to the common amaranths i.e. A. dubius, A. spinosus and A. hybridus (Odhav et al., 2007). Little information is available about the other amaranths. This study determined the nutritional and biological activities as well as safety of Achyranthes aspera, Alternanthera sessilis and Guilleminea densa because there is little information available.

Methodology

Plant Material:

A.aspera, A. sessilis and G. densa were collected from the greater Durban area, Kwa-Zulu Natal in 2006. The plants were collected and identified by botanist Prof. Baijnath (UKZN) using taxonomic keys. Upon receipt, the leaves were separated and washed several times with distilled water until no foreign material remained and air dried for 24 h. Thereafter, they were dried in an oven at 25°C for 7 days. The dried leaves were powdered using an industrial grinder (Retsch Gmbh, West Germany) and stored in Schott bottles until use. All analyses were conducted in duplicate and the reagents and solvents used were that of analytical grade (Merck, Germany). Results are based on fresh weight per 100 g of sample.

Preparation of Plant Extracts:

Aqueous extracts from dried plant powder was prepared by shaking 20 g in 200 ml of distilled water for 24 h. This was then filtered using Whatman No. 1 filter paper and the filtrate was collected and concentrated by freeze drying using the Virtis Benchtop Freeze Dryer. Methanolic extracts of the dried plant powder were prepared as above but the sample was extracted in 80% methanol. The filtrate was concentrated using a rotary evaporator. The extract concentrate was prepared as above. Aliquots were prepared from the dried crude extracts and dissolved in methanol, acetone, distilled water or dimethyl sulfoxide (DMSO) depending on the experimental protocol.

Proximate Analysis:

Energy was calculated using the Atwater system as described by the World Health Organization in 1985. The moisture, ash, protein and fat were analyzed by methods as outlined by the AOAC in 1990. Moisture was determined using the drying oven method. The ash content was determined by the incineration of a sample. The total protein content was evaluated by the Kjeldahl method. The Soxhlet method was used to determine total fat and this method is based on acid hydrolysis that liberates the bound fat followed by solvent extraction of the fat. Dietary fibre analysiswas carried out according to Scheizer and Wuersch (1979) with some modifications. The carbohydrate analysis was performed using the phenol sulphuric acid carbohydrate assay method as outlined by Dubois in 1956.

Mineral Analysis:

The mineral metallic elements calcium, copper, iron, magnesium, manganese, zinc, sodium and phosphorus were determined on dried samples that were digested in a microwave digester using the method of Milestone Microwave Lab Systems (MMLS, 1999). The concentrations of the minerals were determined with an Inductively Coupled Plasma (ICP) spectrometer (Perkin-Elmer). Sample solutions were quantified against standard solutions of known concentrations that were analyzed concurrently according to Perkin Elmer (Perkin, 1996). All assays were carried out in duplicate. Mean values and standard deviations were based on these results.

Vitamin Analysis

Vitamin A, B1, B2, B3 and C were analyzed according to methods outlined by Hoffman in 1969. Vitamin A analysis was carried out using the Carr Price Method. Vitamin B1 analysis was carried out using a fluorescence method described as the Thiochrome Method. Vitamin B2 analysis was carried out using the Fluorimetric method. Vitamin B3 analysis was carried out using a method described by Horowitz in 2000.

Antioxidant Activity

The anti-oxidative properties of the crude extracts were tested using the DPPH (1.1-diphenyl-2-picrylhydrazyl radical) photometric assay as outlined by Choi and coworkers in 2002. The freeze dried aqueous and methanolic plant material was diluted in ethanol to the desired concentrations. Rutin (Sigma) was used as a comparative standard. 1 ml of 0.3 mM DPPH in ethanol, was added to 2.5 ml of the sample solution of different concentrations and were allowed to react at room temperature for 30 min. 1.0 ml ethanol plus plant extract solution (2.5 ml) was used as a blank, while DPPH solution and 2.5 ml ethanol was used as a negative control. The positive control was DPPH solution (1 ml) plus 2.5 ml 1mM Rutin. Each test was carried out in triplicate and results are expressed as the mean and standard deviation of the mean. The absorbance values were measured in a Varian Cary 1E UV-visible spectrophotometer at 518 nm and the average absorbance values was converted into the percentage antioxidant activity,using the following equation:

Scavenging capacity % = 100- (Abs of sample-Abs of blank) × 100

Abs of negative control

Anti-inflammatory properties

Lipoxygenase is known to catalyse the oxidation of unsaturated fatty acids containing 1-4 diene structures. The conversion of linoleic acid to 13-hydroperoxy linoleic acid was followed spectrophotometrically by the appearance of a conjugate diene at 234 nm on a UV/Visible spectrophotometer (Varion Cary 1E UV- Visible spectrophotometer). Nordihydroguaiaretic acid (NDGA) was used as a control. The reaction was initiated by the addition of aliquots (50 μl) of a soybean lipoxygenase solution (potassium phosphate buffer 1M pH 9.0) in a sufficient concentration to give an easily measurable initial rate of reaction to 2.0 ml of sodium linoleate (100 μM) in phosphate buffer. The enzymatic reactions were performed in absence or in presence of inhibitor and their kinetics were compared. The inhibitors were dissolved in DMSO in such a manner that an aliquot of each (30 μl) yielded a final concentration of maximum 100 ppm in each assay. The initial reaction rate was determined from the slope of the straight line portion of the curve and the percentage inhibition of the enzyme activity was calculated by comparing with the control (using 30 μl of phosphate buffer (pH 9.0) instead of 30 μl of the inhibitor solution). Each inhibitor concentration was tested in triplicate and the results averaged; the concentration that gave 50% inhibition (IC50) was calculated from the outline of the inhibition percentages as a function of the inhibitor concentration (Njenga and Viljoen, 2006). All the analysis were carried out in triplicate and the results were expressed the mean ±SD. Regression analysis was used to calculate IC50, defined as the concentration of inhibitor necessary for 50% inhibition of the enzyme reaction.

Antimicrobial Activity

The antimicrobial activity of methanolic and aqueous plant extracts were carried out on selected bacteria and fungi by evaluating the bactericidal and anti fungal effect and the minimum inhibitory concentration on selected bacteria and fungi in a petri dish using the agar disk diffusion method (Vlotman, 2003). The ten bacteria used as test organisms were as follows Bacillus cereus (DBT*_F), B. stearothermophilus (DBT*_Q), Escherichia coli (DBT*_L), Klebsiella oxytoca (DBT*_AM), Micrococcus sp. (DBT*_AR), Pseudomonas aeruginosa (DBT*_D), Proteus mirabilis (DBT*_O), Salmonella typhimurium (DBT*_AF), Staphylococcus aureus (DBT*_E) and S. epidermis (DBT*_ Q). * The two yeasts and seven fungi used as test organisms were: Candida albicans (DBT*_AB)and Saccharomyces cerevisiae (DBT*_R), Aspergillus flavus (DBT*_AR), Cladosporium sp (DBT*_AS), Fusarium verticilloides (DBT*_AT), Geotrichum sp(DBT*_AA), Penicillium sp (DBT*_AC), Rhizopus sp (DBT*_Y) and Trichoderma sp (DBT*_AU). DBT is a reference for the Durban University of Technolgy Culture Collection which is based at the Department of Biotechnology and Food Technology. Control disks with ethanol (5 ml) served as the negative control, whilst Ciprofloxacin (5 mg per disk) and 5 μg/ml Amphotericin B (Fluka, Biochemika) were the positive control. All tests were carried out in triplicate. The minimum inhibitory concentration (MIC) was taken as the lowest concentration of plant extract that inhibited growth after incubation.

Brine Shrimp Lethality Assay

The brine shrimp lethality assay was used with minor modifications (Meyer et al., 1982). Plant extracts were dissolved in DMSO and impregnated on filter paper disks. Control disks were prepared using only DMSO. Three replicates of each dose and the control were tested. 25 mg of Class C Artemia salina eggs (Natures Petland, Durban, South Africa) was added to artificial salt water and kept at room temperature. This was incubated in a hatching chamber at room temperature. After 24 h, 15 ml of yeast solution was added to the chamber for every litre of salt water in order to feed the larvae, 48 h after the eggs were incubated, the larvae were extracted by picking up the moving larvae and visibly counted. Every vial with 100 µl of plant sample at different concentration contained 10 larvae of brine shrimp, including the control group, and was filled to 5 ml total volume with artificial salt water. A drop of yeast suspension (3 mg in 5 ml sea water) was added to each vial. The vials were then incubated at 27°C for 24 h. After 24 h, dead larvae were counted and percentage death determined.

MTT assay

Cytotoxicity was assessed using the MTT assay (Hanelt et al., 1994) which indicates the degree of loss of mitochondrial activity of the cells. Human chronic myelogenous leukaemia, K562 (Highveld Biological was cultured according to the method outlined by Reddy et al. (2006). The cells were grown in Dulbecco's modified Eagle's medium (DMEM), containing 10% fetal calf serum and supplemented with antibiotics (10 000 U/ml penicillin and 10 000 U/ml streptomycin sulphate) and 1 mM sodium pyruvate (Highveld Biological, Modderfontein, South Africa). The cells were trypsinized with 0.1% EDTA and 0.25% trypsin (1:1). A 96 well plate of 200 µl per well cell culture (3x104 of cells/ml) was prepared and incubated for 24 h. Thereafter 2 µl of aqueous and methanolic plant extracts were added to the wells and incubated for 24 h. Each well was then treated with 20 µl of MTT reagent and incubated for 3 h at 37ºC. The formazon crystals produced were then dissolved with DMSO for 30 min. The absorbance was read at 578 nm (reference wavelength 630 nm). The percentage cytotoxicity was then calculated.

Ames test

The Ames test (Maron and Ames, 1983) was used to evaluate the mutagenic potential of the plant extracts by studying its effect on a histidine-requiring strain of Salmonella typhimurium. When the cultures are exposed to a mutagen some of the bacteria undergo genetic changes due to chemical interactions resulting in reversion of the bacteria to a non-histidine-requiring state. The reverted bacteria are then grown in the absence of exogenous histidine thus providing an indication of the potential of a chemical to cause mutation. Salmonella typhimurium TA 98 and TA 100 tester strains were used in this assay (ATCC, USA). Dilutions of the plant extracts were made in soft agar. Sodium azide (NaN3) was used as the positive control and sterile dH2O and DMSO were used as the negative controls. Nutrient broth cultures of S. typhimurium (100 µl) were added to test tubes, followed by 50 µl of plant extract and 2.9 ml of top agar. This was mixed and poured onto minimal glucose agar plates. Upon solidification of agar the plates were inverted and incubated for 48 h at 37°C, after which the number of revertant colonies were counted and the mutant frequency determined. The mutant frequency is calculated as follows:

Mutant frequency = Revertant number of colonies / Negative control.

Results and Discussion

Table 1. The proximate values, mineral values and vitamin values of famine foods A. aspera, A. sessilis and G. densa

A. aspera

A. sesslis

G. densa

Proximate (mg/ml)

Energy

513±9.88

636.11±9.88

341.37±2.63

Protein

21.87±0.10

27.70±0.12

12.95±0.08

Dietary Fibre

40.85

38.44±0.47

48.84±1.43

Fat

2.06±0.1

2.8±0.06

1.95±0.05

Carbohydrate

4.29±0.03

4.01±0.25

3.07±0.03

Moisture

83.24±2.5

65.5±4.9

54±1.4

Ash

13.5±1.03

15.6±1.81

12.51±1.05

Mineral (mg/ml)

Calcium

142.97±10

83.97±0.5

140.84±2.5

Copper

0.22

0.20

0.17

Iron

4.70±1.6

8.57±3

8.71±0.5

Magnesium

73.03±2.06

53.23±0.2

45.27±2.9

Manganese

5±0.2

4.47±0.1

0.31

Phosphorus

26.41±`0.6

53.58±2.4

35.08±1.0

Sodium

114.72±6.7

72.94±2.1

12.76±0.7

Zinc

1.85±0.1

2.65±1.9

1.01±

Vitamin (mg/ml)

Vitamin A

806.55±27.34

982.32±33.07

929.46±11.60

Vitamin B1

20.02±0.12

10.98±0.24

11.08±0.12

Vitamin B2

22.2±0.57

9.7±0.42

41.75±0.35

Vitamin B3

5.98±0.16

9.05±0.08

8.96±0.10

Vitamin C

43.57

27.19

16.27

Data are mean± S D (n=2).

The carbohydrate, protein, moisture, energy, fat, dietary fibre and ash levels of the famine food plants from the family Amaranthaceae are shown in Table 1. A. sessilis had the highest energy value (636.11±8.56 g/100 g), followed closely by A. aspera (513.99±9.88 g/100 g) and G. densa (341.37±2.63 g/100 g). The moisture levels in A. aspera, A. sessilis and G. densa were 83.24±2.5 g/100g, 65.5±5.0 g/100g and 54±1.4 g/100g respectively. The high moisture content of A. aspera is keeping with its succulent nature and the low moisture content in G. densa is due its woody nature. The carbohydrate values for A. aspera. A. sessilis and G. densa were 4.29±0.03 g/100 g, 4.01±0.25 g/100 g and 3.07±0.03 g/100 g respectively. The highest protein levels were found inA. sessilis (27.70±0.12 g/100 g) when compared to A. aspera (21.87±0.10g/100g) and G. densa (12.95±0.08g/100g).The fat content of A. aspera was marginally lower than A. sessilis followed by G. densa (2.06±0.16 g/100 g, 2.8±0.06 g/100 g and 1.95±0.05 g/100 g respectively). G. densa (48.84±1.43) contained the highest amount of dietary fibre in comparison to the other famine food plants A. aspera and A. sessilis. Odhav et al. (2007) reported low dietary fibre contents for A. spinosus, A. hybridus and A. dubius. The famine food plants analyzed in this study contained higher dietary fibre contents than the leafy vegetables from the Amaranthaceae family (Odhav et al., 2007). These results correlate well with the recently published results of Freedman (2006) for A. sessilis where the values for moisture, fat and protein are similar. For A. aspera and G.densa we did not find any published data pertaining to nutritional analysis. A comparison of the energy values with cultivated species in the Amaranthaceae family viz. A. spinosus, A. hybridus, A. dubius and A. hypochondriacus indicate that the famine species A. aspera and A. sessilis in this study have twice as much energy and also higher values than Maundu et al. (1999) on indigenous leafy vegetables as well as those reported by Odhav et al. (2007). Furthermore, in A. sessilis the protein quantity is sufficient to make up 50% of the RDA.

The calcium, copper, Iron, Magnesium, Manganese, Phosphorus, Sodium and Zinc levels of the famine food plants from the family Amaranthaceae are shown in Table 1. A. aspera and G. densa had similar levels of calcium present (142.97±10 mg/100 g and 140.84±2.5 mg/100 g respectively) and A. sessilis had a lower value (83.97±0.5 mg/100 g) (Table 1). The RDA of calcium for adults is 800 mg per day (NRC, 1989). Thus 100g/day of the famine plants in this study do not meet the RDA. These values reported in this study are low. Reports of calcium levels in other famine plants Ximenia americana, A. viridus, and the leaves of the baobab tree (Adansonia digitata) contained higher quantities of calcium (Glew et al., 2005). A. aspera and A. sessilis had similar amounts of copper present with values of 0.22 mg/100 g and 0.20 mg/100 g respectively (Table 1). G. densa had a slightly lower amount of copper present than the other two famine food plants with a concentration of 0.17 mg/100 g (Table 1). According to the National Research Council, the daily requirement for copper is 2 mg per day (NRC, 1989). A. aspera and A. sessilis meet about 10 % of the RDA for adults. In comparison to the leafy vegetables (A. hybridus, A. dubius and A. spinosus) in the Amaranthaceaefamily reported by Odhav et al. (2007), the famine food plants in this study had low copper concentrations. A. sessilis and G. densa had iron concentrations of 8.57±3 mg/100 g and 8.71±0.5 mg/100 g respectively and A. aspera had 4.70±1.6 mg/100 g(Table 3). The RDA for iron is 10 mg per day adults (NRC, 1989). A. sessilis and G. densa satisfied more than 85% and 87% respectively of the iron requirement for the RDA for adults. Odhav et al. (2007) reported very high levels of iron in the leafy vegetables in the Amaranthaceae family. In G. densa we found low levels of magnesium (45.27±2.9 mg/100 g), however in A. aspera and A. sessilis the magnesium concentration were 73.03±2.6 mg/100 g and 53.23±0.2 mg/100 g respectively (Table 1). The daily consumption of magnesium should amount to 120 mg (NRC, 1989). The famine food plants A. aspera satisfies 60% and A. sessilis satisfies 53% of the RDA for magnesium. In G. densa we found very low levels of manganese (0.31 mg/100 g) and A. aspera and A. sessilis had amounts of 5.00±0.2 mg/100 g and 4.47±0.1 mg/100 g respectively (Table 1). The RDA for manganese is 7 mg (NRC, 1989). A. aspera and A. sessilis meet more than 50% of the daily recommended and have a greater manganese concentration than the food plant A. spinosus (Table 1). A. sessilis had the highest amount (53.58±2.4 mg/100 g) of phosphorus present, followed by G. densa (35.08±1.0 mg/100 g) and thereafter A. aspera (26.41±0.6 mg/100 g) (Table 3). The RDA for phosphorus is 800 mg for adults. According to Odhav et al. (2007) the leafy vegetables analyzed from the Amaranthaceae family satisfied more than half the RDA for phosphorus, however the plants analyzed in this study had very low phosphorous concentrations (Table 1). A. aspera (114.72±6.7 mg/100 g) had a very high concentration of sodium in comparison to G. densa which only had 12.76±0.7 mg/100 g (Table 1).The RDA for sodium is 300 mg. A. sessilis had the highest concentration of zinc (2.65±1.9 mg/100 g) compared to the other two famine food plants which had values of 1.85±0.1 mg/100g and 1.01 mg/100g for A. aspera and G. densa respectively (Table 1). The RDA for zinc is 10 mg per day for adults (NRC, 1989). Thus, the famine plants from this study do not meet the RDA.

The Vitamin A, B1, B2, B3 and C levels of the famine food plants from the family Amaranthaceae are shown in Table 1. The famine food plant A. sessilis had the highest amount of Vitamin A (982.32±33.07 mg/ml) followed by G. densa (929.46±11.60 mg/ml) and A. aspera (806.55±27.34 mg/ml). According to the Food and Nutrition Board, the recommended daily allowance is 800 micrograms for adult females and 1000 micrograms for adult males (NRC, 1989). A. aspera had the highest content of Vitamin B1 compared to the other famine food plants tested; almost twice the amount of A. sessilis and G. densa had 11.08±0.12 mg (Table 1). The RDA for Vitamin B1 is 1.1-1.5 mg. The Vitamin B2 concentrations in this study ranged from 9.7±0.4 mg in A. sessilis to 41.75±0.35 mg in G. densa (Table 1). This meets more than the recommended daily allowance for Vitamin B2. The recommended daily allowance is 1.2-1.7 mg (Bender, 2003). A. sessilis and G. densa had similar Vitamin B3 levels with values ranging between 8.96±0.10 mg and 9.05±0.08 mg respectively (Table 1). A. aspera's Vitamin B3 level was only 5.98±0.16 mg. A. sessilis and G .densa meet only 60 % of the RDA. According to the Food and Nutrition Board, the recommended daily allowance is 15-19 mg (NRC, 1989). Vitamin C levels range from 16.27 mg in G. densa to 43.57 mg in A. aspera (Table 1). According to the Food and Nutrition Board, the RDA for Vitamin C is 60 mg. The famine food plants studied do not meet the daily allowance standard set for Vitamin C by the Food and Nutrition Board (NRC, 1989). The vitamins of relevance to plant foods are the fat soluble vitamins (A, D, E and K) and the water soluble vitamins (B1, B2, B6 and B12, biotin, C, folic acid, niacin and pantothenic acid). The recommended daily allowance is the quantity which is required to prevent disease. Micronutrient deficiencies and infectious diseases often coexist and exhibit complex interactions leading to the vicious cycle of malnutrition and infections among underprivileged populations of the developing countries, particularly in preschool children. Several micronutrients such as Vitamin A, β-carotene, folic acid, Vitamin B12, Vitamin C, riboflavin, iron, zinc, and selenium, have immunomodulating functions and thus influence the susceptibility of a host to infectious diseases and the course and outcome of such diseases (Bhaskaram, 2002).

Rutin, the positive control used for free radical scavenging capacity reduced 92.43 ±0.22 % whilst the famine food plants A. aspera, A. sessilis and G. densa reduced 83.86±2.63%, 86.51±0.37 % and 54±55 respectively. The minimum concentration at which these A. aspera and A. sessilisexhibited free radical scavenging activity was 100 µg/ml. The aqueous extracts of the three plants showed no antioxidant activity. Akula and Odhav (2008) reported high antioxidant activities from plants in the Amaranthaceae family. A. hybridus was reported to have 90.5±0.24%, A. spinosus had 88.2±0.22% and A. dubius had 78.4±0.22% (Akula and Odhav, 2008). The recognized dietary antioxidants are vitamin C, vitamin E, selenium, and carotenoids. In our study A. sessilis and A. aspera had vitamin C present. This may explain the high level of anti-oxidative properties displayed. Furthermore, there is a connection between plant stress levels and the production of secondary metabolites, including many polyphenols and antioxidants. There is substantial agreement among plant pathologists, physiologists, and entomologists that relatively higher levels of antioxidant secondary plant metabolites are produced by plants in response to biotic and abiotic stress. Famine plants grow in stress conditions and hence it is plausible that they have better anti-oxidative capacity than commercially cultivated amaranth species.

The in vitro inhibition of soybean lipoxygenase constitutes a good model for the screening of plants with anti-inflammatory potential (Abad et al., 1995). In our study, we used NDGA as a standard for the comparison of anti-inflammatory potential of the three famine plants. The IC50 value of NDGA was 2.48 µg/ml and the famine food plants A. aspera, A. sessilis and G. densa were 246 µg/ml, 341.1 µg/ml and 582.1 µg/ml respectively. These results indicate that the famine food plants analyzed had low anti-inflammatory activity. Reduced IC50 values suggest better inhibitory action on 5 LOX. A negative result in the lipoxygenase assay does not necessarily mean that the plant is without anti-inflammatory activity. The active compounds could work at other sites in the complex process of inflammation (Jäger et al., 1995). Previous work in our laboratory on anti-inflammatory activity with methanolic extracts among leafy vegetables from commonly consumed leafy Amaranthaceae species showed that A. dubius had IC50 value of 69.4 µg/ml and A. spinosus had 57.3 µg/ml (Akula and Odhav, 2008). These results suggest that the plants of Amarantheceae family do not have chemicals that can induce anti-inflammatory activity in this specific site. It has been reported by Gokhale et al. (2002) that the ethanolic extract of A. aspera possessed anti-inflammatory and anti-arthritic activity and he also reported that A. aspera is used in the indigenous system of medicine for the treatment of inflammatory conditions; however, there is no reported literature on detailed investigation for rationality behind their use in inflammation.

Table 2. Antibacterial minimum inhibitory concentration of A. aspera, A. sessilis and G. densa

Minimum Inhibitory Concentration

(µg/ml)

Plant Extracts

Ec

Pa

Ko

Pm

St

Se

Bs

Bc

M

Sa

A. aspera (aqueous)

250

100

Na

na

100

1000

na

Na

na

1000

A. aspera (methanolic)

250

100

1000

na

10

Na

na

Na

na

500

A. sessilis (aqueous)

na

500

Na

na

na

1000

na

Na

na

na

A. sessilis (methanolic)

na

250

Na

na

na

Na

na

Na

na

1000

G. densa (aqueous)

250

250

1000

na

na

Na

500

na

na

na

G. densa (methanolic)

1000

1000

Na

na

na

Na

na

na

na

500

Data are mean± S D (n=3); na: no activity Ec- Escherichia coli, Pa- Pseudomonas aeroginosus, Ko- Klebsiella oxytoca, Pm- Proteus mirabilis, St- Salmonella typhi, Se-Staphylococcus epidermis, Bs- Bacillus stereathermophilus, Bc- Bacillus cereus, M-Micrococcus, Sa- Staphylococcus aureus

Table 3: Antifungal minimum inhibitory concentration of A. aspera, A. sessilis and G. densa

Minimum Inhibitory Concentration (µg/ml)

Plant Extract

Sc

F

P

Ca

R

T

C

G

Af

A. aspera (aqueous)

na

Na

na

na

na

na

Na

na

na

A. aspera (methanolic)

500

Na

na

na

na

Na

Na

na

Na

A. sesslis (aqueous)

500

Na

na

na

na

Na

Na

na

Na

A. sessilis (methanolic)

250

Na

na

500

na

Na

Na

na

na

G. densa (aqueous)

Na

Na

na

250

na

Na

Na

na

na

G. densa (methanolic)

Na

Na

na

500

na

Na

Na

na

na

Data are mean±SD (n=3); na: no activity; F- Fusarium, P- Penicillium, Ca- Candida albicans, R- Rhizopus, T- Trichoderma, C- Cladosporium, G- Geotrichum, Af- Aspergillus flavus

The aqueous extract of A. aspera showed zones of inhibition against the Gram negative bacteria E. coli, P. aeruginosa and S. typhi with MIC values of 250 µg/ml, 100 µg/ml and 100 µg/ml respectively (Table 2) and against two Gram positive bacteria S. epidermis and S. aureus with MIC values of 1000 µg/ml and 1000 µg/ml respectively (Table 2). In the studies of Perumal Samy et al. (1998) the aqueous leaf extract of A. aspera did not show any activity against E. coli, K. oxytoca and P. aeruginosa at lower doses whereas, it exhibited activity against P. bulgaricus at higher doses of 4000 and 5000 ppm. Similar results related to Perumal Samy et al. (1998) were reported by Belachew Desta (1993). The extracts of A. aspera analyzed in our study showed activity against E. coli, P. aeruginosa, K. oxytoca and S. typhi which is contradictory to Perumal Samy et al. (1998) and Belachew Desta (1993). Lamikarna (1999) reported that Gram negative bacteria have an impervious cell envelope which makes them resistant to many antibacterial agents. Although the A. aspera plant extracts did not inhibit fungal growth, the methanolic extract showed inhibition against the yeast S. cerevisiae at a minimum inhibitory concentration of 500 µg/ml (Table 3). The aqueous extracts of A. sessilis showed activity against P. aeruginosa and S. epidermis and the methanolic extracts showed activity against P. aeroginosa and S. aureus. The aqueous extract of A. sessilis also showedthe best inhibition against C. albicansat a minimum inhibitory concentration of 500 µg/ml (Table 3) in comparison to the rest of the plant extracts when determining the zone of inhibition. The aqueous and methanolic extract of A. sessilis showed antifungal activity against S. cerevisiae at a minimum inhibitory concentration of 500 µg/ml and 250 µg/ml respectively (Table 3). G. densa extracts showed activity against the Gram negative E. coli, P. aeroginosa and K. oxytoca (Table 2) as well as the Gram positive B. sterathermophilus and S. aureus Table 2. The methanolic and aqueous extracts of G. densa displayed antifungal activity against C. albicans at minimum inhibitory concentrations of 500 µg/ml and 250 µg/ml respectively (Table 3).

The aqueous extract of A. aspera stimulated the growth of the K562 cell line. As the concentration of the extract increased, there was a slight decrease in cell viability. The methanolic extract of A. aspera showed some differentiation where at concentrations of 10 and 1000 µg/ml there was stimulation of cells but at a concentration of 100 µg/ml there was a decrease in cell viability. The same trend was observed for A. sessilis. The aqueous extract of G. densa stimulated the growth of the cell at low concentration but was toxic to the cells at a high concentration of 1000 µg/ml. The methanolic extracts stimulated the growth of cells at low concentrations and as the concentration increased there was a decrease in the cell viability. Peer et al. (2005) reported that A. spinosus and A. hybridus from the Amaranthaceae family were cytotoxic to the HepG2 cell line.

None of the plants showed any level of toxicity with all the plants showing 100% of shrimp survival whereas the positive control which was an organophosphate showed 100 % mortality. A study performed by Peer et al., (2005) showed that other plants from the Amaranthaceae family such as A.dubius, A spinosus and A. hybridus has some toxic effects. Gayathri et al. (2006) reported that histopathological testing revealed degenerative and necrotic changes in the liver and kidney in Swiss mice, caused by oral administration of water extract of A. sessilis in high doses. He suggested that this could be due to the effects of cytotoxic substances in A. sessilis, however, in our study no toxic activity was noted.

None of the plant extracts up to concentrations of 1000 µg/ml showed any mutagenic potential. Sodium azide, the chosen mutagen used in this experiment showed a mutagenic potential; as the concentration increased so did the number of revertant colonies. Thus the extracts of A. aspera, A. sessilis and G. densa do not have compounds that may a potential to be a carcinogenic agent. Peer et al. (2005) reported that none of the leafy vegetables (A. dubius, A. spinosus and A. hybridus) had any mutagenic activity.

The study provided evidence for the nutritional analysis, bioactivity and safety for the three chosen famine amaranths (A. aspera, A. sessilis and G. densa) being valuable sources of nutrients under famine conditions or normal conditions. The high levels of some vitamins, minerals and antioxidants can be used to prevent diseases. These plants may help eradicate malnutrition in areas rife where people are starving. A. aspera and A.sessilis should be cultivated on a large scale because the plants have antimicrobial effects against a range of bacteria and yeasts so it can prevent diarrhea, vomiting, bronchitis and candidiasis. The plants are also considered safe as they showed no toxicity or mutagenicity or cytotoxicity.

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