Phytochemical Constituents and Antioxidant and Antimicrobial Activity of Selected Plants Used Traditionally as a Source of Food

Abstract

Many indigenous plants have also been used as a source of food and medicine in many African rural communities in the past. The study investigated the antimicrobial activity, phytochemical constituent, and antioxidant activity of selected traditional plants used traditionally as a source of food and medicine. The methanol and water extracts of different plant parts were analyzed for phytochemicals using standard phytochemical screening reagents while the broth microdilution assays were used to analyze antimicrobial activities. Alkaloids, phenols, flavonoids, saponins, tannins, and terpenes were found in one or more of the plant extracts, and all the plant extracts demonstrated scavenging activities. The back extracts of Sclerocarya birrea and the leaf extracts of Garcinia livingstonei exhibit the best antioxidant activities, while the water and methanol back extracts of S. birrea and G. livingstonei were the most active against all the tested foodborne bacteria.

Introduction

A dansonia digitata (baobab) is a multipurpose tree whose fruit pulp, seeds, leaves, flowers, roots, and bark are used as food.^1,2 The fruits of Garcinia livingstonei (mangosteen or imbe) and Sclerocarya birrea (marula) are also edible and are used for the making of many traditional food products.^3,4 A. digitata, G. livingstonei, and S. birrea have also been used as a source of medicine for thousands of years by many African communities.⁵ The seeds, pulp, and leaves of A. digitata and bark of G. livingstonei and S. birrea have been implicated traditionally in the treatment of digestive problems and diarrhea-related diseases.^6

–9 In recent years, the interest of many pharmaceutical industries has been switched to the natural world, prompting investigations of medicinal plants for their potential biological and health benefits, which include antioxidant activity, anticancer, antiaging, antiatherosclerotic, antimicrobial, and anti-inflammatory activities.¹⁰ In Mexico, many indigenous plants have been tested to have important antimicrobial properties, and hence, the possibilities to research further for the purpose of drug discovery.¹¹ Studies conducted in Nigeria also found the methanol extracts of different plant parts to possess significant antioxidant and radical scavenging activities that may be due to their phytochemical content, which makes them potential natural chemoprophylactic agents.¹² The objective of this study is to determine the phytochemical constituents, antioxidant activity, and antimicrobial activities against selected foodborne bacteria, of extracts of the seed, pulp, and leaf of A. digitata and extracts from the back of G. livingstonei and S. birrea.

Materials and Methods

Preparation of plant extract

The leaves, fruit pulps, and seeds of A. digitata and bark of G. livingstonei and S. birrea were collected from Venda, South Africa, and voucher specimens were prepared and identified at the H.G.W.J Schweickerdt Herbarium, University of Pretoria. Plant parts were air-dried and ground to fine powder. Twenty grams of each powdered material was macerated in 200 mL of each solvent (100% methanol and water) in extraction pots. The extracts were then filtered out by vacuum filtration. The methanol extracts were concentrated in a Buchi Rotavapor R-200, while the water extracts were concentrated using a freeze dryer.¹³

Phytochemical screening

To test for alkaloid using the Wagner's test, a fraction of extract was treated with Wagner's test reagent and observed for the formation of reddish brown color precipitate.¹⁴ To test for alkaloid using the Dragendorff's Test, 1 mL of extract and few drops of dragendorff's reagent were added in a test tube and color change was observed. Appearance of orange color is an indication of the presence of alkaloids.¹⁵ To test for flavonoids using the sodium hydroxide test, plant extracts were treated with dilute NaOH, followed by addition of dilute HCl. A yellow solution of NaOH, which turned colorless with added dilute HCl, indicated the presence of flavonoids.¹⁶ A conclusive test was further carried out using the aluminum chloride test, in which 0.2 g of each extract was heated with 10 mL of ethyl acetate in boiling water for 3 min. Four milliliters of the filtrate was shaken with 1 mL of 1% aluminum chloride solution. A yellow colored solution indicated the presence of flavonoids.¹⁷

To test for Phenols, the plant extracts were spotted on a filter paper and a drop of phosphomolybdic acid reagent was added and exposed to ammonia vapors. Blue coloration of the spot indicated the presence of phenols.¹⁸ To test for Tannins, 10% alcoholic ferric chloride was added to 2–3 mL of methanolic extract and a dark blue or greenish gray coloration of the solution indicated the presence of tannins.^18,19 To test for terpenoids, 5 mL of plant extract was added to 2 mL of chloroform and 3 mL of concentrated sulfuric acid. The presence of terpenoids was indicated by a reddish brown color of interface.²⁰ To test for saponins, 0.5 mL of plant extract was added to 5 mL of distilled water and shaken well. The persistence of frothing after shaking indicated of the presence of saponins.¹⁹

Determination of antioxidant activity

The antioxidant activity was determined as described by Du Toit et al. ²¹ with some modification. Two milligrams of both plant extract and vitamin C (positive control) was dissolved in 200 μL of ethanol to give stocks of 10 mg/mL. 1,1-diphenyl-2-picrylhydrazyl (DPPH) was prepared by dissolving 20 mg in 500 mL ethanol to give a stock of 0.04 mg/mL. The start-up concentrations were 500 μg/mL each for all plant extracts and 100 μg/mL for vitamin C. The samples were serially diluted from the first row to the last row, and DPPH was added. The plates were covered in foil and incubated for 30 min, and absorbance was read with the ELISA plate reader (KC junior) at 515 nm.

Preparation of test bacteria

Foodborne pathogens, Staphylococcus aureus ATCC^®11632, Escherichia coli 015:H7 ATCC^®43888, Klebsiella oxytoca ATCC^®43086, Salmonella enterica ATCC^®51741, and Shigella sonnei ATCC^® 25931, were grown on nutrient agar at 37°C for 24 h after which colonies were suspended in a saline solution (0.85% NaCl) and adjusted to the turbidity of the 0.5 MacFarland's standard (∼10⁶ colony forming units per mL) to form the inoculum.²²

Broth microdilution assays

To determine the minimum inhibitory concentrations (MICs) of extracts, 100 μL of nutrient broth was added to all the wells of a 96-well microtiter plate. One hundred microliters of each dissolved plant extract (50 mg/mL) and 100 μL of 2.5 mg/mL of dissolved ciprofloxacin (positive control) were then added to each bacterial well on the first row of the plate. The mixtures in the first row were then serially diluted row by row, and 100 μL of the mixture was discarded from the last row. One hundred microliters of each bacterial suspension in nutrient broth was then added to the wells, except the negative control wells. MIC values were determined after 20 μL of PrestoBlue was added per well and incubating at 37°C for 30 min.²³

To determine the maximum bactericidal concentrations (MBCs) of extracts, 150 μL of nutrient broth was pipetted into every well of a 96-well microtiter plate and 50 μL was taken from the 24-h-old bacteria culture without PrestoBlue in the MIC plates and added to it. The plates were incubated at 37°C for 24 h, and 20 μL of PrestoBlue was added to determine MBC values.²⁴

Analysis of cytotoxicity

One hundred microliters of HEK cell suspension (1 × 10⁵ cells/mL) was added to the inner wells of a 96-well plate incubated at 37°C in a humidified atmosphere (5% CO₂, 95% air) for 24 h. Serial dilutions of the plant extracts dissolved in DMSO; Actinomycin D (positive control) and DMSO with the complete medium (negative control) were transferred in a 24-well plate to give 1 mL of eight different concentrations of each sample per well. One hundred microliter solutions from each sample were added to 50 μL of XTT in a new plate and incubated for 2 h and 30 min absorbance was measured at 450 and 690 nm to analyze cell proliferation and viability.²⁵

Results

Phytochemical screening

Alkaloids were absent in both the water and methanol extracts of A. digitata seeds, pulps, and leaves. Flavonoids and phenol were present in both the water and methanol extracts of A. digitata seeds, pulps, and leaves. Tannin was only detected in the leaf extract of A. digitata, but was absent in the seed and pulp extracts. In addition, terpenoid was only detected in the pulp extract of A. digitata, but not in its seed or leaf extracts. Saponin was present only in the pulp and leaf extract of A. digitata. Alkaloids, flavonoids, phenol, tannins, terpenoids, and saponins were present in the bark extracts of G. livingstonei and S. birrea (Table 1).

Table 1.

Phytochemical Analysis of Extracts of Selected Traditional Plants

	Ads ^a	Adp ^a	Adl ^a	Gab ^a	Scb ^a,b
Alkaloids^c	−	−	−	+	+
Flavonoid	+	+	+	+	+
Phenol	+	+	+	+	+
Tannins	−	−	+	+	+
Terpenoids	−	+	−	+	+
Saponins	−	+	+	+	+

Similar test results for both water and methanol extracts of plant parts.

Only the methanol extract was positive for Flavonoids.

Same alkaloid test results for Wagner's & Dragendorff's test.

+, positive test; −, negative test; Adl, Adansonia digitata leaf extracts; Adp, A. digitata pulp extracts; Ads, A. digitata seed extracts; Gab, Garcinia livingstonei bark extracts; Scb, Sclerocarya birrea bark extract.

Antioxidant activity

G. livingstonei and S. birrea bark extracts had the lowest IC₅₀ values. The water extract of A. digitata pulp had the highest IC₅₀ value followed by the water extract of A. digitata seed. There was a little difference in the IC₅₀ values between the water and methanol extract of G. livingstonei bark, but the IC₅₀ value of the water extract of S. birrea was slightly higher than the methanol extract. The IC₅₀ of the water extracts of A. digitata seed and pulp was greater than the methanol extracts (Table 2).

Table 2.

The Free Radical Activity of Extracts of Selected Traditional Plants

Plant extracts	Solvent used	IC₅₀ (μg/mL)
Ads	Water	23.11 ± 1.37
	Methanol	6.65 ± 1.51
Adp	Water	28.58 ± 2.07
	Methanol	9.65 ± 2.19
Adl	Water	2.79 ± 0.07
	Methanol	2.82 ± 0.05
Gab	Water	0.35 ± 0.06
	Methanol	0.39 ± 0.04
Scb	Water	0.40 ± 0.02
	Methanol	0.28 ± 0.02
Vit C	Water	10.62 ± 0.87

Values are expressed as mean ± SD (n = 3). The IC₅₀ of Vitamin C; the positive control was 10.62 ± 0.87.

Minimum inhibitory concentration

The water and methanol extracts of A. digitata seeds and pulp had MICs greater than 12.5 mg/mL. The water extract of A. digitata leaf had MIC of 1.56 mg/mL for all the test bacteria, except for S. sonnei, with an MIC of 6.25 mg/mL (Table 2). Methanol extract of A. digitata leaf had MIC of 1.56 mg/mL for all the test bacteria, except for S. aureus with an MIC of 0.78 mg/mL and K. oxytoca with an MIC of 0.39 mg/mL. Water extract of G. livingstonei bark had an MIC of 1.56 mg/mL for all the test organisms except for E. coli, and S. sonnei with an MIC of 0.78 mg/mL, while the methanol extract of G. livingstonei bark had an MIC of 0.39 mg/mL for all the test organisms with the exception of E. coli, and S. sonnei which maintained an MIC of 0.78 mg/mL. Water extract of S. birrea bark had an MIC of 0.78 mg/mL for all the test bacteria, except E. coli, and S. enterica that had lower MICs of 0.39 mg/mL, while the methanol extract of S. birrea bark had varied MIC values ranging from 0.39 to 1.56 mg/mL (Table 3).

Table 3.

Minimum Inhibitory Concentration (mg/mL) of Extracts of Selected Traditional Plants on Selected Foodborne Bacteria

	Plant extract
Bacteria	Adlw	Adlm	Gabw	Gabm	Scbw	Scbm	Cipro
Staphylococcus aureus	1.56	0.78	1.56	0.39	0.78	0.39	0.002
Escherichia coli	1.56	1.56	0.78	0.78	0.39	1.56	0.002
Klebsiella oxytoca	1.56	0.39	1.56	0.39	0.78	0.78	0.002
Salmonella enteric	1.56	1.56	1.56	0.39	0.39	0.39	0.002
Shigella sonnei	6.25	1.56	0.78	0.78	0.78	0.78	0.002

Adsw; Adsm; Adpw; and Adpm had MIC values >12.5 mg/mL; figures not presented in the table.

Adlm, methanol extract of A. digitata leaves; Adlw, water extract of A. digitata leaves; Adpm, methanol extract of A. digitata pulp; Adpw, water extract of A. digitata pulp; Adsm, methanol extract of A. digitata seeds; Adsw, water extract of A. digitata seeds; Cipro, ciprofloxacin; Gabm, methanol extract of G. livingstonei bark; Gabw, water extract of G. livingstonei bark; MIC, minimum inhibitory concentration; Scbm, methanol extract of S. birrea bark; Scbw, water extract of S. birrea bark.

Maximum bactericidal concentration

The water and methanol extracts of A. digitata seeds, pulp, and leaves had MBCs greater than 12.5 mg/mL, while the water extract of G. livingstonei bark had an MBC of 12.5 mg/mL for all the test bacteria (Table 4). Similarly, the methanol extract of G. livingstonei bark had an MBC of 12.5 mg/mL for the other test bacteria, except for S. aureus, with MBC of 6.25 mg/mL and S. enterica with MBC greater than 12.5 mg/mL. The methanol extract of S. birrea bark had an MBC of 12.5 mg/mL for all the test bacteria, except S. sonnei, with MBC of 6.25 mg/mL. In contrast, the water extract of S. birrea bark also had an MBC of 6.25 mg/mL for all the test organisms (Table 5).

Table 4.

Minimum Bactericidal Concentration (mg/mL) of Extracts of Selected Traditional Plants on Selected Foodborne Bacteria

	Plant extracts
Bacteria	Gabw	Gabm	Scbw	Scbm	Cipro
S. aureus	12.5	6.25	6.25	12.5	0.002
E. coli	12.5	12.5	6.25	12.5	0.002
K. oxytoca	12.5	12.5	6.25	12.5	0.002
S. enterica	12.5	12.5	6.25	12.5	0.002
S. sonnei	12.5	12.5	6.25	6.25	0.002

Adsw, Adsm, Adpw, Adpm, Adlw, and Adlm had maximum bactericidal concentration values >12.5 mg/mL, figures not presented in the table.

Table 5.

The Half Maximal Effective Concentration of Extracts of Selected Traditional Plants and Actinomycin D on HEK Cell Lines

Plant extract	EC₅₀ (μg/mL)	R² value
Gabw	769.9 ± 36.33	0.35
Gabm	400 ± 36.33	0.89
Scbw	149.3 ± 1.4	0.99
Scbm	105.9 ± 19.50	0.96
Act	0.003 ± 0.000095	0.88

Act, Actinomycin D.

Cytotoxicity

The cytotoxicity results are expressed as EC₅₀, which is the maximal effective concentration needed to kill fifty percent of the HEK cells. The water extract of G. livingstonei bark had the highest EC₅₀ value of 769.9 ± 36.33 μg/mL, followed by the methanol extract while the methanol extract of S. birrea bark had the lowest EC₅₀ value of 105.9 ± 19.50 μg/mL, followed by its water extract (Table 6). The therapeutic index (TI) is determined as the ratio of the cytotoxicity to that of the MIC ranged from 0.19 to 0.98 with the water extract of G. livingstonei having a relative higher index for all the test foodborne bacteria.

Table 6.

Cytotoxicity and Therapeutic Index of Extracts of Selected Traditional Plants on Selected Foodborne Bacteria, Calculated by Dividing Cytotoxicity Values by Minimum Inhibitory Concentration Values

Plant extracts	Cytotoxicity (mg/mL)	S. aureus	E. coli	K. oxytoca	Salmonella enterica	S. sonnei
Therapeutic index (cytotoxicity values/MIC values
Gabw	0.7699	0.49	0.98	0.49	0.49	0.98
Scbw	0.1493	0.19	0.38	0.19	0.38	0.19
Scbm	0.1059	0.27	0.067	0.14	0.27	0.14

Therapeutic index for Gabm could not be determined because its cytotoxic value was greater than 400 μg/mL.

Discussion

Phytochemical composition

Alkaloids were absent in both the water and methanol extracts of A. digitata seeds, pulps, and leaves. Many chemical components that have been characterized from A. digitata usually belong to the classes of terpenoids, flavonoids, steroids, vitamins, amino acids, carbohydrates, and lipids, hence, an explanation for the presence of flavonoids and phenols.²⁶ Tannin was only detected in the leaf extract of A. digitata and was absent in the seed. Other authors have identified soluble and insoluble tannins in the back extract of A. digitate.^27,28

Saponin was present in both the pulp and leaf extract of A. digitata, but was not detected in its seed extracts. However, the slight presence of saponin has been reported in A. digitata seed oil, but this could be due to differences in the extraction solvent.²⁹ The fruit pulp of A. digitata has been found to have anti-inflammatory properties due to the presence of sterols, saponins, and triterpenes in its aqueous extract.³⁰ Terpenoid was present only in the pulp extract of A. digitata, but not in its seed and leaf extracts. This finding is also consistent with those of other authors who found triterpene in A. digitata fruit and seeds.^29,31 The presence of terpenoid in the seed extract of A. digitata might be due to the usage of hexane as the solvent of extraction.

Alkaloids, flavonoids, phenol, tannins, terpenoids, and saponins were present in the bark extracts of G. livingstonei and S. birrea with the exception of water extract of S. birrea, which did not contain flavonoid. The bark of S. birrea in previous work has been reported to contain tannins, flavonoids, alkaloids, and steroids.³² Xanthones and biflavonoids have also been isolated from the root and bark extract of G. livingstonei.³³ Tannins and flavonoids are thought to be responsible for antidiarrhoeal activity by increasing colonic water and electrolyte reabsorption,³⁴ which explains why the bark of these plants are used traditionally in treating diarrhea. Terpenoids have been shown to be active against bacteria, fungi, viruses, and protozoa and their mechanism of action is speculated to involve membrane disruption by the lipophilic compounds. Furthermore, alkaloids have been found to have antimicrobial properties with microbicidal effects against Giardia and Entamoeba species as well as an antidiarrheal effect, which are probably due to their effects on transit time in the small intestine.³⁵ Saponins have several biological effects, some of which are antibacterial, antifungal, antiparasitic, antitumor/cytotoxicity, antiviral, and antioxidant activities.³⁶

Antioxidant activity

Water and methanol extracts of bark of S. birrea and G. livingstonei exhibited excellent antioxidant activities with their 50% inhibitory concentration of DPPH radical ranging from 0.28 ± 0.02 to 0.40 ± 0.02 μg/mL. This is quite impressive when compared to the positive control vitamin C, which had a 50% inhibitory concentration of 10.62 ± 0.87 μg/mL. These results can be attributed to the presence of phenols, flavonoids, tannins, alkaloids, saponins, and terpenoids and this in agreement with studies in which these compounds have been associated with high antioxidant activities.^37

–40 Extracts of A. digitata leaves also exhibited good antioxidant properties with their IC₅₀ values being 2.79 ± 0.07 and 2.82 ± 0.05 μg/mL. These can also be attributed to the presence of phenols, flavonoids, tannins, and saponins.

Antimicrobial activity, cytotoxicity, and therapeutic index of extracts

The broth microdilution method indicated that both the water and methanol extracts of A. digitata leaves exhibited some inhibitory activities against the tested bacteria at concentrations ranging from 0.39 to 6.25 mg/mL. Their bactericidal activity, however, was at concentrations greater than 12.5 mg/mL. The methanol extracts of A. digitata root bark and leaves have been shown to exhibit antibacterial activity against S. aureus, Streptococcus faecalis, Bacillus subtilis, E. coli, and Mycobacterium phlei.⁴¹

The disadvantage of using the agar diffusion method to determine antimicrobial activity is that the antimicrobial effect may be affected by the agar type, salt concentration, incubation temperature, and molecular size of the antimicrobial component. Furthermore, it does not distinguish between bactericidal and bacteriostatic effects.²⁴

The MIC results showed that the G. livingstonei plant extracts displayed antimicrobial activity against all the test bacteria. This was similar to the acetone extracts of G. livingstonei leaves, which were shown to exhibit antimicrobial activity against E. coli, S. aureus, and Enterococcus faecalis with MICs ranging from 8 to 100 μg/mL, while the methanol extract of its root bark showed antiparasitic activity against some selected parasites.^33,42,43

The water and methanol extracts of S. birrea bark showed both significant an inhibitory and a bactericidal effect on all the test organisms with MIC, which ranged from 0.39 to 1.56 mg/mL. This is in line with previous findings in which the acetone extracts of S. birrea bark and leaves showed significant antimicrobial activities with MIC values ranging from 0.15 to 3 mg/mL against S. aureus, Pseudomonas aeruginosa, E. coli, and E. faecalis.⁴⁴

Based on the EC50 levels, these extracts are considered safe, considering that the values greater than 100 μg/mL have been considered safe for extracts of other plant extracts.⁴⁵ Of all the extracts studied, the water extract of G. livingstonei bark had the highest therapeutic index for E. coli and S. sonnei.

In conclusion, the phytochemical phenols, flavonoids, and saponins are present in at least one of the extracts. The bark extracts of S. birrea and G. livingstonei contain all the six phytochemicals analyzed. The leaves of A. digitata contain more phytochemicals than the seed and pulp extract. The back extracts of S. birrea and the leaf extracts of G. livingstonei exhibit the best antioxidant activities. Furthermore, the water and methanol extracts of S. birrea and G. livingstonei were the most active against all the tested foodborne bacteria. Based on these results, there is some justification for the usage of these plants as food and medicinal agents.

Footnotes

Acknowledgment

The authors acknowledge the research department of the University of South Africa for providing the funds required for this research.

Author Disclosure Statement

No competing financial interests exist.

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