Screening of phytochemicals and bioactivities of different parts of Ludwigia erecta (L.) H. Hara

Abstract

The aim of the present study was to investigate various biological activities (antimicrobial, anti-Giardial, antimalarial, antitumor, and antioxidant) as well as the phytochemistry of the root, stem, leaves, flowers and fruit of Ludwigia erecta L. The extracts of different plant parts showed variable activity against the tested microorganisms, with the highest activity (p < 0.05) obtained from the flower chloroform: methanol extract against E. coli (17.7 ± 2.2 mm). The strongest anti-Giardial activity was that of the root extract with a half-maximal inhibitory concentration IC₅₀ value of 41.60 ± 01 μg/mL (p < 0.05). None of the extracts was cytotoxic to Vero cell line (IC₅₀ > 100 µg/mL), whereas the flower extract showed a cytotoxic effect against the MDA-MB231 cell lines, with an IC₅₀ value of 30.5 ± 2.9 µg/mL. Furthermore, the highest antioxidant activity (p < 0.05) was shown by the fruits extract with IC₅₀ 50 µg/mL, whereas the same antioxidant capacity was shown by the stem, leaf, and flowers extracts with IC₅₀ 75 µg/mL. The phytochemical screening of extracts revealed the presence of flavonoids, sterols, tannins, triterpenes and saponins. The total polyphenolic contents, calculated as gallic acid equivalents (mg/L), ranged from 3428.21 ± 12.53 to 3671.50 ± 11.25 mg GAE/L, and the concentration of flavonoids, calculated as quercetin equivalents (mg/L), ranged from 1501.76 ± 16.99 to 3928 ± 219.81 QE mg/L, whereas the tannin content was calculated as the tannic acid equivalent (mg/L) with a value ranging between 9.55 ± 0.11 and 48.66 ± 2.14 TAE mg/L.

Keywords:

• bioactivity
• flavonoids
• Ludwigia erecta
• phytochemical
• polyphenols
• tannins

Introduction

Every year, more than 13 million deaths worldwide are caused by the emergence of new infectious diseases or the reemergence of old pathogens [1]. The traditional applications of medicinal plants have significantly contributed to the development and improvement of health care. It is known that plants produce an array of structurally diverse secondary metabolites. Given the potential health risks, the incidence of multidrug resistance in human pathogenic microorganisms, and the toxicity of synthetic antioxidants, intensive studies are carried out on plants in the search for potential natural therapies and leading biomolecules for the pharmaceutical industry [2,3].

The Onagraceae family consists of 22 genera, divided into two subfamilies, namely, Ludwigioideae (represented by only one genus; Ludwigia) and Onagroideae (the other genera) [4]. The genus Ludwigia is comprised of 82 species and is found in both temperate and tropical regions. Ludwigia spp. are either aquatic or grown in wet places around coastal regions, lakes, lagoons, rivers and water-logged areas [5]. In Sudan, the genus is represented by five species, including L. adscenoens, L. erecta, L. leptocarpa, L. octovalvis and L. perennis.

Some pharmacological properties of Ludwigia spp. were demonstrated by previous reports, including the antimicrobial activity of L. adscendens, L suffruticosa, L. abyssinica, L. decurrens, L. peploides subsp. montevidensis and L. grandiflora subsp. hexapetala [6–10], the anti-inflammatory activity of L. adscendens [6], the antioxidant activity of L. abyssinica [11], as well as the anti-acne, antioxidant and cytotoxic properties of L. peploides [12]. L. octovalvis was characterized with anti-hyperglycemic [13] and antiproliferative [14] activities, and a capacity to inhibit α-glucosidases and pancreatic lipase [15]. Moreover, three new oleanane-type triterpenes isolated from L. octovalvis were found to exhibit cytotoxic activity against two human cancer cell lines [16].

In Sudan, L. erecta (L.) H. Hara (Syn. Jussiaea erecta L., Sp.) is an annual herb found along the riversides of the Nile. Massage lotions are prepared from the whole plant and used in traditional medicine as an antipyretic drug [17]. There is limited data available on the biological properties of L. erecta and only the antimicrobial activity of the plant has been evaluated [18,19]. Therefore, the objective of this study was to evaluate the biological properties including the antimicrobial, anti-Giardial, antimalarial, antiproliferative, and antioxidant activities of the root, stem, leaves, flowers and fruit of L. erecta. Screening for phytochemicals and the determination of the phenolic content were also carried out.

Materials and methods

Plant material

The whole plant of L. erecta was collected from a riverbank in Khartoum State, Sudan, in May 2014. A voucher specimen (No. LE514) was deposited at the Herbarium of the Botany Department, Faculty of Science, University of Khartoum, as a reference material. The root, stem, leaves, flowers and fruit were separated, shade-dried, and coarsely powdered in a hammer mill separately.

Preparation of the extract

Fifty grams of dried powder from the different plant parts was defatted by maceration in hexane and subsequently extracted with chloroform: methanol (CM) (1:1, v/v) using a shaker apparatus at room temperature for 24 h. The filtrate of each extract was concentrated with a rotary evaporator under reduced pressure. Finally, the extracts were weighed and stored in a refrigerator until further usage.

Biological assays

Antimicrobial activity

The bacterial cultures used were Bacillus subtilis NCTC 8236, Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 10145. The used fungi cultures were Aspergillus niger ATCC 9763 and Candida albicans ATCC 7596. Each extract (10 mg/disc) was tested for antimicrobial activity by the disc diffusion method [20]. The standard gentamicin and nystatin at a concentration of 10 μg/disc served as the positive control for evaluation of the antibacterial and antifungal activities, respectively.

Anti-giardial activity

Giardia lamblia was obtained from Ibrahim Malik Hospital (Khartoum, Sudan) and an in vitro susceptibility assay was done according to the method of Cedillo-Rivera et al. [21].

Antimalarial activity

The culture of the malaria parasite, Plasmodium falciparum K1, was obtained from Khartoum Hospital (Khartoum, Sudan), and in vitro antiplasmodial activity was determined using the candle jar method [22].

Cytotoxicity

The extracts were screened for the determination of their cytotoxic effect against normal (Vero cells) and different cancer cell lines, including human breast carcinoma cells (MCF7 and MDA-MB231) and human colon adenocarcinoma cells (HT29 and HCT116) using the MTT assay [23].

Antioxidant activity

The antioxidant activity of the extracts was evaluated using the in vitro DPPH radical scavenging method [24].

Phytochemical analysis

A preliminary phytochemical screening was carried out using standard phytochemical methods [25] to determine the presence of secondary metabolites. The total polyphenolic, flavonoid and tannin content was determined according to the methods described by Wolfe et al. [26], Ordonez et al. [27] and Shivakumar et al. [28], respectively.

Thin-layer chromatography (TLC) autographic assay

Extracts were developed in duplicate on separate TLC silica gel plates using an appropriate solvent system (Toluene: ethyl acetate: formic acid (5:4:1)). The first plate was sprayed with a Vanillin-Sulfuric acid spray reagent, whereas the other with the 2,2-diphenyl-1-picrylhydrazyl (DPPH) spray reagent. Yellow spots against a purple background showed a positive antioxidant activity when sprayed with DPPH [29].

Statistical analysis

Experiments were performed in triplicate and the obtained results were expressed as the mean with standard deviation (±SD). The one-way analysis of variance (ANOVA) was performed for the determination of significant differences between the four extracts and their free-radical–scavenging activity.

Results and discussion

The bioactivity and medicinal aspects of a plant are usually correlated with the presence of various classes of phytochemicals, including phenols, flavonoids, terpenes, among others. Their presence and concentration vary in various parts of the plant which are used in Ethnobotany [30]. In this study, a preliminary phytochemical screening of the different parts of L. erecta, including the root, stem, leaves, flowers, and fruit showed that the plant contained phenols, terpenes, steroids, and saponins and consequently could be a rich source of natural products.

Extraction yield and phytochemical screening

As depicted by the results in Table 1, the highest extraction yield was obtained in the fruit extract (8.67%), followed by the flower (8.07%), leaf (5.43%), stem (3.54%), and root (2.77%) extracts, respectively. Moreover, the presence of flavonoids, sterols, tannins, triterpenes, and saponins was revealed by the phytochemical screening of different extracts. All extracts were devoid of alkaloids and anthraquinones in contrast to Ludwigia octovalvis and Ludwigia hyssopifolia [31–33].

Table 1. Yield and phytochemical screening of different parts of Ludwigia erecta.

Extract (CM)*	Yield (%)	Alkaloids	Flavonoids	Tannins	Terpenes/Steroids	Anthraquinones	Saponins
Root	2.77	–	+	+	+	–	+
Stem	3.54	–	+	+	+	–	+
Leaf	5.43	–	+	+	+	–	+
Flower	8.07	–	+	+	+	–	+
Fruit	8.67	–	+	+	+	–	+

* chloroform: methanol (1:1; v/v) extract.

Quantitative determination of the total polyphenol, flavonoid, and tannin content

The total polyphenolic, flavonoid and tannin content of the CM extracts of the different parts of L. erecta was determined and the results are presented in Table 2. The total polyphenolic content in CM extracts, calculated as the GAE (mg/L), ranged from 3428.21 ± 12.53 to 3671.50 ± 11.25 mg GAE/L. The highest concentration was obtained in the fruit extract, followed by the root, flower, leaf and stem extracts with values of 3671.50 ± 11.25, 3648.79 ± 3.00, 3562.75 ± 11.25, 3466.42 ± 3.33, and 3428.21 ± 12.53 mg GAE mg/L, respectively. The concentration of flavonoids, calculated as the quercetin equivalent (QE, mg/L), ranged from 1501.76 ± 16.99 to 3928 ± 219.81 QE mg/L. Moreover, the fruit extract was identified to contain the highest flavonoid content, followed by the flower, root, leaf, and stem extracts with values of 3928 ± 219.81, 3349.91 ± 55.56, 2946.72 ± 104.25, 1895.38 ± 109.42, and 1501.76 ± 16.99 QE mg/L, respectively. The tannin content was calculated as the tannic acid equivalent (TAE, mg/L) and values ranged between 9.55 ± 0.11 to 48.66 ± 2.14 TAE mg/L. The fruit extract was identified to contain the highest tannin content, followed by the leaf, root, flowers and stem with values of 48.66 ± 2.14, 37.94 ± 2.27, 37.62 ± 1.41, 15.32 ± 0.15, and 9.55 ± 0.11 TAE mg/L, respectively. All the studied plant organs displayed a high polyphenolic and flavonoids content as well as low tannin content with 48.66 ± 2.14 mg TAE/L as the highest concentration detected in the fruit. Furthermore, the plant contained higher phenolic content in comparison with that reported for other Ludwigia spp [12, 34].

Table 2. Total polyphenolic, flavonoid and tannin content of the extracts of different plant parts of Ludwigia erecta.

Extract (CM)	Total polyphenolics (mg GA/L)	Total flavonoids (mg Q/L)	Total tannins (mg TA/L)
Root	3648.79 ± 3.00^a	2946.72 ± 104.25^c	37.62 ± 1.41^b
Stem	3428.21 ± 12.53^c	1501.76 ± 16.99^e	9.55 ± 0.11^d
Leaf	3466.42 ± 3.33^c	1895.38 ± 109.42^d	37.94 ± 2.27^b
Flower	3562.75 ± 11.25^b	3349.91 ± 55.56^b	15.32 ± 0.15^c
Fruit	3671.50 ± 11.25^a	3928 ± 219.81^a	48.66 ± 2.14^a

Antimicrobial activity

The antimicrobial activity of CM extracts from the root, stem, leaves, fruit and flowers of L. erecta was determined against gram-negative and gram-positive bacteria as well as against two species of fungi. The results are presented in Table 3. The extracts of the different parts showed a variable inhibitory activity against the tested microorganisms. Based on the results of inhibition zones, the highest activity was obtained from the flower CM extract against E. coli (17.7 ± 2.2 mm). With fewer inhibition zones (13 ± 1.4-14.7 ± 1.3 mm), B. subtilis and S. aureus were the most susceptible to the root and flower extracts, as well as P. aeruginosa to the flower extract (13.7 ± 0.8 mm). Furthermore, the antifungal activity of all extracts was stronger against C. albicans than A. niger, with the highest inhibition zone (12 ± 1.7 mm) obtained from the root extract.

Table 3. Antimicrobial activity of the extracts of different plant parts of Ludwigia erecta against standard microorganisms.

Extract (MC)/ Antibiotic	Mean diameter of growth inhibition zone (mm)
	B. subtilis	S. aureus	E. coli	P. aeruginosa	C. albicans	A. niger
Root	14.7 ± 1.3^d	13 ± 1.4^d	12.3 ± 3.6^e	10.3 ± 0.8^f	12 ± 1.7^e	–
Stem	13.7 ± 1.2^d	10 ± 1.4^f	9.7 ± 0.8^f	8.7 ± 1.4^g	11 ± 1.7^e	9.3 ± 1.7^f
Leaf	11 ± 2.2^e	10 ± 0.0^f	10.7 ± 1.6^f	10.3 ± 0.8^f	9 ± 1.7^f	–
Fruit	12.3 ± 0.8^e	9.7 ± 1.6^f	13.7 ± 1.6^d	11.7 ± 0.8^e	11 ± 1.7^e	10.7 ± 0.5^f
Flower	14.3 ± 0.8^d	13.3 ± 1.5^d	17.7 ± 2.2^c	13.7 ± 0.8^d	10 ± 0.8^f	7.7 ± 0.9^g
Gentamicin**	25 ± 0.5^a	17 ± 0.8^c	17 ± 1.4^c	16 ± 1.2^c	–	–
Nystatin**	–	–	–	–	24 ± 1.7^a	20 ± 0.5^b

** 10 μg/disc, n = 3.

Except for the flower extract, either moderate or weak antimicrobial activity was revealed by all extracts, with the highest antibacterial activity observed in the flower extract against E. coli. The chloroform extract of the whole plant was active only against S. aureus [18]. Besides, El-Egami et al. [19] demonstrated that the methanolic extract of the whole plant exerted a higher antimicrobial activity than the one that included a chloroform extract, against the four tested bacteria and C. albicans. This observation suggested that the plant as a whole possesses better antimicrobial activity than its separated organs, implying a synergistic interaction between the bioactive constituents present in the various organs of the plant, in contrast to Ludwigia parviflora which showed similar activities but through fruit extract [35] and Ludwigia octovalvis leaf extract [32].

Anti-Giardial activity

The anti-Giardial activity of different CM extracts of different parts of L. erecta against G. lamblia was investigated using three different concentrations (125, 250 and 500 μg/mL) . The anti-Giardial activity of all extracts was both concentration- and time-dependent (Figure 1). The root and fruit extracts were the most effective against G. lamblia with 85% mortality observed at the highest concentration (500 μg/mL) after 72 h. This was followed by the stem and leaf extracts which showed 80% and 77% mortality, respectively, at 500 μg/mL after 72 h. Furthermore, the IC₅₀ values were calculated for the anti-Giardial activity of the extracts after 72 h, and the results are given in Table 4. The ranking order of the extracts with the highest anti-Giardial activity regarding the IC₅₀ values was the root (41.60 ± 01 μg/mL) > fruit (109.89 ± 14 μg/mL) > stem (168.46 ± 20 μg/mL) > leaf (212.26 ± 15 μg/mL).

Figure 1. Anti-Giardial activity of the different parts of the CM extracts of Ludwigia erecta against Giardia lamblia. Note: positive control “ornidazole”

Table 4. Half-maximal inhibitory concentration (IC₅₀) of anti-Giardial, antimalarial, and antioxidant activities of the extracts of the different plant parts of Ludwigia erecta.

Extract CM	IC50 (μg/mL)
	Anti-Giardial activity	Antimalarial activity	Antioxidant activity
Root	41.60 ± 01^b	321 ± 11^b	–
Stem	168.46 ± 20^d	328 ± 21^b	75 ± 0.01^a
Leaf	212.26 ± 15^e	942 ± 19^d	75 ± 0.00^a
Flower	ND	ND	75 ± 0.03^a
Fruit	109.89 ± 14^c	458 ± 11^c	50 ± 0.00^b
Control^a	0.20 ± 0.01^a	0.09 ± 0.02^a	± 0.01^a

^a Anti-Giardia (ornidazol), antimalaria (chloroquine), antioxidant (propyl gallate); ND, not determined.

The results of anti-Giardial activity showed that all extracts displayed considerable anti-Giardial activity depending on concentration and time. The root (IC₅₀ 41.60 ± 01 μg/mL) extract was the most effective against G. lamblia. However, the stems of Fuchsia microphylla, the only species that belonged to the family Onagraceae, were investigated for its anti-Giardia property and the obtained results showed inactivity (IC₅₀ > 500) [36]. In a literature review on a natural chemical molecule with anti-Giardia activity, Amaral et al. [37] reported that flavonoids, especially flavonols, isoflavones, triterpenes, with quassinoids being the most representative alkaloids, including mainly indole alkaloids, sesquiterpenes, steroids, phenolic acids, esters, lignin, and amine were among the classes of metabolites that contained bioactive compounds against giardia.

Antimalarial activity

The antimalarial activity assay of the CM extracts of different parts of L. erecta against P. falciparum showed that the highest activity (IC₅₀ 321 ± 11 ppm) was displayed by the root, followed by the stem (IC₅₀ 328 ± 21 ppm), fruit (IC₅₀ 458 ± 11 ppm) and leaf (IC₅₀ value was 942 ± 19 ppm) extracts (Table 4). The results of the antiplasmodial activity revealed that the different parts of L. erecta exhibited either weak or no antimalarial activity.

Antiproliferative activity

CM extracts from different parts of L. erecta were tested in vitro for their potential cytotoxic effects against the HT29, HCT116, MCF7 and MDA-MB231 cancer cell lines as well as normal Vero cell line (Table 5). None of the extracts were toxic to the Vero cell line (IC₅₀ > 100 µg/mL), and only the flower extract demonstrated cytotoxic activity against the MDA-MB231 cell lines with an IC₅₀ value 30.5 ± 2.9 µg/mL. All other extracts had no anticancer effect on the tested cancer cell lines (IC₅₀ value > 50 µg/mL). Although previous studies on other Ludwigia spp., including L. hyssopifolia [33], L. peploides [12] and L. octovalvis [14, 16], showed a high antiproliferative activity against many cancer cell lines, in this study, the flower extract was the only one to demonstrate moderate antiproliferative activity against MDA-MB231 cell lines with an IC₅₀ value of 30.5 ± 2.9 μg/mL (Table 4). Three oleanane-type triterpenes isolated from L. octovalvis were found to exhibit a cytotoxic effect against two human cancer cell lines [16].

Table 5. Antiproliferative activity of the extracts of the different plant parts of Ludwigia erecta against normal and carcinoma cells.

Extract CM	Cell lines
	Vero	HT-29	HCT-116	MCF-7	MDA-MB231
Root	>100	>50	>50	>50	>50
Stem	>100	>50	>50	>50	>50
Leaf	>100	>50	>50	>50	>50
Flower	>100	>50	>50	>50	30.5 ± 2.9
Fruit	>100	>50	>50	>50	>50

Antioxidant activity

The antioxidant capacity of CM extracts of different parts of L. erecta was first investigated through TLC bioautography. The developed TLC plate was sprayed with DPPH reagent and samples producing yellowish spots on the purple background were considered to possess radical scavenging activity. In addition, the stem and fruit extracts displayed two distinct antioxidant spots, while the leaf and flower gave only one major spot. The root extract did not react with the DPPH reagent (Figure 2). Furthermore, the scavenging effect of the extracts on the DPPH radicals was calculated in regard to the IC₅₀ values. Moreover, the quantitative determination of the DPPH scavenging activity revealed that the fruit extract displayed the highest antioxidant activity with IC₅₀ 50 μg/mL (Table 4), a value lower than that obtained from the standard propyl gallate (IC₅₀ 77 ± 0.01 μg/mL), whereas other organs also displayed good antioxidant activity with IC₅₀ values (75 μg/mL) comparable to the standard, suggesting that the plant is very rich in antioxidant molecules. Moreover, the antioxidant activity bioautograms showed that all extracts except that of the root displayed a distinct antioxidant spot, suggesting the presence of major molecules responsible for the observed scavenging activity. Although the stem displayed an additional antioxidant spot, it did not increase its antioxidant capacity as observed in the fruit, this could be attributable to a lower level of antioxidants. Moreover, the high polyphenolic and flavonoid contents of L. erecta could play a major role in the observed potent antioxidant activity similar to Ludwigia leptocarpa and Ludwigia hyssopifolia [33, 38].

Figure 2. Thin-layer chromatography bioautography of the CM extracts from different parts of Ludwigia erecta. R, root; S, stem; L, leaf; Fl, flower; F, fruit. a: sprayed with vanillin/H₂SO₄; b: sprayed with DPPH reagent.

Conclusions

This was the first study conducted to evaluate the anti-Giardia, antimalarial, cytotoxic and antioxidant potential of L. erecta. The results showed that the different parts of the plant were not toxic and exerted different biological activities. Furthermore, the highest antibacterial activity with the best sensitivity against E. coli and the only organ to possess antiproliferative activity against the MDA-MB231 cell line was the flower. In addition, the root displayed the highest anti-Giardial activity. Besides, all parts exerted potent free-radical–scavenging activity, with the fruit showing the highest capacity. Thus, L. erecta could be a natural source for bioactive agents, although further scientific research is needed to determine the active molecules.

Disclosure statement

The authors declare that they have no conflict of interest.

Funding

The authors extend their appreciation to the Deanship of Scientific Research at King Saud University for funding this work through the research group Project no. RG-1441-472.

Data availability

The data supporting the findings of this study are available from the corresponding author upon reasonable request.

Additional information

Funding

The authors extend their appreciation to the Deanship of Scientific Research at King Saud University for funding this work through the research group Project no. RG-1441-472.