A comparative study on biological activities of different solvent extracts from whole seed, seed coat and cotyledon of two Lathyrus species

Abstract

The present study was conducted to assess the phenolic content, and antibacterial and antioxidant activities of Lathyrus L. species. The extraction of phenolic compounds from whole seeds, seed coat and cotyledon of Lathyrus hierosolymitanus Boiss. and Lathyrus annuus L. seeds was performed employing different solvents. Total phenolic content (TPC) was measured by Folin- Ciocalteau assay, while the antioxidant activity was determined by DPPH radical scavenging activity, and reducing power assay. It was found that TPC of extracts ranged from 0.12 mg to 6.53 mg GAE/gdw. For each solvent, seed coat extracts were generally observed to render higher TPC and antioxidant activities. There was a correlation between TPC and antioxidant activity. In addition, all extracts were also examined for their antimicrobial activity against Bacillus cereus, Escherichia coli and Pseudomonas aeruginosa. Methanol extracts showed the highest antibacterial activity which is consistent with TPC, but there was no correlation between TPC and antibacterial activity. Solvents were observed to have effects on gallic acid, caffeic acid, and epicatechin extractions. HPLC analysis results of extracts confirmed methanol and ethanol as preferred solvents for phenolic extraction from Lathyrus sp. Phenolic content in the extracts could be suggested to contribute to their antioxidant and antibacterial activity.

Keywords:
Antioxidant activity; Antibacterial activity; Lathyrus; Phenolic content; HPLC

INTRODUCTION

Oxidative stress is the disruption of the balance between antioxidants and pro-oxidants. If there is an increase in favor of pro-oxidants, it causes a significant change in DNA biomolecules due to their high reactivity. The body has an endogenous defense system that can reduce the effects of these free radicals. The contribution of exogenous antioxidants may be required if this defense system is insufficient to neutralize free radicals (Sokamte, 2019).

Plants, as suppliers of these exogenous antioxidants, have become important sources of medicine as well as food. Most of the therapeutic effects of plants are attributed to their high antioxidant content and their phenolic composition (Singh et al., 2017a). Phenolic compounds found widely in plants are crucial sources of pharmaceutical products (Khan et al., 2009). They have biological properties including antioxidant, anti- inflammatory, anti-atherogenic and antimicrobial effects (Singh et al., 2017a).

The protective effect of consumption of phenolic-rich grains, and legumes against some chronic diseases was assumed (Zhao et al., 2014; Zhang et al., 2015; Fratianni et al., 2014; Parikh, Patel, 2018). Legumes are essential sources of macronutrients and micronutrients, as well as being rich in proteins, lipids, vitamins, carbohydrates, and polyphenols (Zhang et al., 2015; Fratianni et al., 2014; Zhao et al., 2014). Many studies have demonstrated the phenolic content of legumes and their antioxidant activity (Amarowicz et al., 2004; Pastor-Cavada et al., 2009; Xu, Chang, 2010). The rich phenolic content of legume seeds also makes them the main source of antioxidant activity in food (Zhao et al., 2014).

The legume seed coat, often called hull or testa, is the outer layer of seeds. They act as a protective barrier for the cotyledon which essentially contains proteins and carbohydrates (Zhong et al., 2018; Singh, et al., 2017a; Parikh, Patel, 2018). The seed coats are the major contributors to the phytochemical content of whole seeds (Boudjou et al., 2013; Zhong et al., 2018). They show high antioxidant potential owing to large amounts of phenolic compounds (Singh et al., 2017a; Singh et al., 2017b). For example, lentil seed coat contains a more abundant and diverse type of polyphenols compared to cotyledon (Singh et al., 2017a).

Genus Lathyrus L. is characterized by the presence of leguminous species, belonging to the Fabeae tribe of the Fabaceae family and is represented by more than 200 species worldwide. Genus Lathyrus contains 79 taxa in the species, subspecies, and variety levels in Turkey and 25 of them are endemic (Davis et al., 1970; Genç, Şahin, 2011; Güneş, 2014). Genus Lathyrus is used in different areas including nutrition, agriculture, industry, and ornament (Vaz-Patto, Rubiales, 2014). In addition, Lathyrus species have been used in traditional medical treatments such as analgesic, and anti-rheumatism (Altundag, Ozturk, 2011).

In the literature, several studies have reported that the extracts from Lathyrus species have antioxidant activities, and inhibit enzymes such as cholinesterases, α-amylase, and α-glucosidase (Llorent-Martínez et al., 2017a; Llorent-Martínez et al., 2017b). Lathyrus species have been suggested to be a new source of natural phenolic antioxidants and high-quality proteins for human and animal nutrition (Pastor-Cavada et al., 2009). However, the data on their antioxidant and antimicrobial activities are limited. Furthermore, the differences in phenolic compounds in the seed coat and cotyledon of Lathyrus taxa are not known. As it is known, Lathyrus species have variations in nutrient contents and antinutritional factors in the seeds as well as morphological traits (Grela et al., 2012). Therefore, the studies on wild Lathyrus species are crucial to determine their properties and to benefit from them.

In our previous study, L. hierosolymitanus and L. annuus were found to be excellent protein sources with low β-ODAP (β-N-oxalyl-L-α,β-diaminopropionic) content and to contain natural antioxidants (Yazici Ozbek et al., 2020). These species are very similar in taxonomic terms. Therefore, it is important to thoroughly investigate the biological activities of each of these species.

The aim of this study is to determine the best solvent for the extraction of phenolic compounds from different parts of L. hierosolymitanus and L. annuus seeds growing in Turkey, as well as to compare the distribution of phenolic compounds in seed parts and their antioxidant and antimicrobial activities.

MATERIAL AND METHODS

Material

The chemical materials to be used in this study are Folin-Ciocalteau reagent, gallic acid, DPPH (2,2-diphenyl-1-picryl-hydrazyl), BHA (butylated hydroxyanisole). They were all purchased from Sigma-Adrich.

Plant Material

The seeds of Lathyrus taxa were collected from their natural habitats in 2014. Their taxonomic identification was confirmed by Genç and Yıldırım. Taxa and their localities are given below:

L. annuus Mugla, Yatagan, around Stratonikeia ancient city, Genç-3002, 2015.

L. hierosolymitanus Mugla, Dalyan, around Eskiköy, Genç-3001, 2015.

Extraction procedure

The three samples including whole seed, seed coat, and cotyledon obtained from Lathyrus seeds were extracted to carry out the study. Seed coats were removed manually from their cotyledons. The seeds and their parts were dried at room temperature. After that, the samples were pulverized and were firstly defatted with hexane for 2 h. The defatted samples were shaken at 300 rpm at ambient temperature on an orbital shaker within the solutions of absolute ethanol, absolute methanol, and water for 3 h to extract phenolic compounds. After 3 h, the supernatants were separated by centrifugation at 3000 x g for 10 min. Each supernatant was filtered through filter paper and the extracts were concentrated using a rotary evaporator and stored at + 4 ^ºC in dark until use.

Total Phenolic Content (TPC)

In the extracts, TPC was determined using Folin- Ciocalteu method as previously described (Yazici Ozbek et al., 2020). Gallic acid was taken as standard for comparison and the results were expressed in milligrams of gallic acid equivalent (GAE) per gram dry weight Lathyrus seed (mg GAE/gdw).

Radical Scavenging Assay

The antioxidant activity was determined by the DPPH (2,2-diphenyl-1-picrylhydrazyl) the radical- scavenging method as previously described (Yazici Ozbek et al., 2020). Antioxidant activities of the extracts were expressed as IC₅₀, defined as the concentration of the extract required to cause a 50% decrease in the initial DPPH^• concentration.

Ferric reducing power assay

The modified Oyaizu method (1986) was used to determine the ferric reducing power. Substances which have reduction potential react with potassium ferricyanide to form potassium ferrocyanide, which then reacts with ferric chloride to form a ferric-ferrous complex. Absorbance was measured at 700 nm. The EC₅₀ value (the effective concentration at which the absorbance was 0.5) was calculated from the graph of absorbance at 700 nm against extract concentration.

HPLC-DAD analyses of phenolic compounds

The analytical HPLC system employed comprised of a Shimadzu Prominence high-performance liquid chromatography coupled with a 20A CBM (HPLC System Controller), DAD (diode array detector) (SPD-M20A, Tokyo, Japan), an SIL 20ACHT automatic sampler, a CTO10ASVp column oven, and an LC20 AT pump. LC Solution data processing system was used to evaluate the analytical data. The separation was achieved on Agilent ZORBAX Eclipse plus C18, 4.6 × 250 mm, 5 µm column at 25 C. m. The mobile phase used was 2% formic acid in (A) water and (B) methanol. The elution gradient applied at a flow rate of 1 ml min^-1 was: 95% A/5% B for 3 min, 80% A/20% B in 15 min and isocratic for 2 min, 60%A/40%B in 10 min, 50%A/50%B in 10 min, 100%B in 10 min until the end of the run (Caponio, Alloggio, Gomes, 1999). The quantification was performed by using the standards, and the levels were expressed as µg phenolic compounds/g dry seed weight (gdw).

Antibacterial Activity of Extracts

Antibacterial activity of the extracts of samples was carried out by the standard broth micro-dilution assay against Bacillus cereus ATCC11778, Escherichia coli ATCC25922, and Pseudomonas aeruginosa ATCC35032.

Initially, extracts were solved in the water, and the extract concentrations were adjusted (50, 100 and 2000 µg/ml). The following inocula were produced using bacterial colonies with incubation times, not superior to 24 h, adjusted to the McFarland standard solution of 0.5 of bacterial turbidity, in a concentration of 1.5 × 10⁸ CFU/ml (colonies forming unities for milliliter) and, following, the concentration of 5 × 10⁵ CFU/ml with caso soy broth (CSB).

For the determination of the MIC, 1 ml of test solutions was poured in each test tube (in the specified concentrations) as well as 1 ml of the bacterial suspension, except in the negative control tube. The tubes were incubated at 35 ± 2 ºC for 20 h, and afterward the turbidity (bacterial growth) was evaluated by absorbance measurement at 620 nm. The minimum inhibitory concentration (MIC) value for each sample was determined by testing each concentration in triplicate. The MIC value was the lowest concentration of the extracts (mg/ml) capable of inhibiting the complete growth of the bacterium being tested. CSB without the inoculum was used as negative control tubes. The CSB broth and just bacterial growth was used as positive control tubes.

Statistical analyses

Statistical analyses were carried out with SPSS 20. Data were expressed as the mean ± SD. The independent t-test was used to analyze the differences between the extracts from Lathyrus species while one-way ANOVA was used to analyze the differences among Lathyrus seed parts extracts obtained with different solvents. As the data demonstrated, the differences were significant (p < 0.05). Correlations among variables were performed with Pearson’s correlation test.

RESULTS AND DISCUSSION

Phenolic Content and Antioxidant Activity of Seed Extracts

Natural phenolics show mainly their beneficial effects on health through their antioxidant activities (Xu, Chang, 2007). Responses of phenolic compounds vary in the Folin-Ciocalteu method depending on the phenolic groups in plant samples (Parikh, Patel, 2018). In the present study, total phenolic content, antioxidant activities of the extracts of seeds and their parts were determined and are shown in Table I. All the extracts evaluated in the study were identified to have a significant total phenolic content ranged from 0.12 ± 0.01 to 7.50 ± 0.6 mg GAE/gdw. The total phenolic content of frequently consumed legumes was reported to vary between 0.325 - 6.378 mg GAE/g (Marathe et al., 2011).

TABLE I
Total phenolic content (TPC), DPPH radical scavenging activity (IC₅₀), and Ferric reducing antioxidant activity (EC50) values of extracts from Lathyrus seeds

Species	Parts	Solvents	TPC (mg GAE /gdw)	DPPH IC50 (mg/ml)	Ferric reducing power EC50 (mg/ml)
		Water	1.02 ± 0.12cA	3.57 ± 0.5aB	6.2 ± 0.2aB
	Seed coat	Ethanol	3.05 ± 0.45bA	1.65 ± 0.22cB	1.33 ± 0.11bB
		Methanol	5.95 ± 0.61aA	1.35 ± 0.42bB	1.43 ± 0.1bB
		Water	0.12 ± 0.01bC	6.01 ± 0.9aA	11.7 ± 0.6 aC
L. annuus	Cotyledon	Ethanol	0.84 ± 0.34aB	3.4 ± 0.6bA	4.97 ± 0.67 bB
		Methanol	0.59 ± 0.14aC	4.23 ± 0.52bA	6.19 ± 0.9 bB
		Water	0.53 ± 0.17bB	3.4 ± 0.4aB	3.73 ± 0.19aC
	Whole seed	Ethanol	1.01 ± 0.11bB	2.6 ± 0.6aB	1.55 ± 0.22bB
		Methanol	2.78 ± 0.68aB	1.7 ± 0.7bB	1.55 ± 0.4bB
		Water	1.61 ± 0.19cA	3.0 ± 0.5aB	2.56 ± b.1aC
	Seed coat	Ethanol	4.02 ± 0.38bA	1.3 ± 0.2bB	1.32 ± 0.22bB
		Methanol	7.50 ± 0.6aA	0.73 ± 0.19cB	0.81 ± 0.1cC
		Water	0.24 ± 0.1cC	6.84 ± 0.81aA	10.7 ± 0.2aA
L. hierosolymitanus	Cotyledon	Ethanol	0.56 ± 0.7bC	3.6 ± 0.5bA	2.56 ± 0.25bA
		Methanol	0.94 ± 0.25aC	2.45 ± 0.35bA	2.79 ± 0.49bA
		Water	0.75 ± 0.14cB	3.5 ± 0.34aB	4.34 ± 0.62aB
	Whole seed	Ethanol	1.78 ± 0.15bB	1.44 ± 0.39bB	1.9 ± 0.62bAB
		Methanol	3.81 ± 0.2aB	0.87 ± 0.14bB	1.73 ± 0.13bB

• *Values are mean a standard deviation. Different letter within the same column show differences of means among different solvents for each part of each plant species (p < 0.05). Different caps letter shows differences between extracts from different part of the plant in the same solvent for Lathyrus species (p < 0.05).

The high phenolic content found in the leguminous seeds makes them an important source of antioxidants. However, the total phenolic content varies significantly among legumes (Zhao et al., 2014). This difference in phenolic content of legumes can be explained by environmental factors as well as genetic factors. In this study, TPC in extracts from L. hierosolymitanus was higher than that of L. annuus, but the difference between the total phenolic content of Lathyrus species was not found to be significant (p > 0.05). The result can be attributed to similar environmental conditions. However, there was a significant difference among the TPC of the extracts from different parts of Lathyrus seeds (p < 0.05). In particular, the TPC of seed coat extracts was higher than that of both whole seed and cotyledon extracts (p < 0.05) (Table I). The results imply that the amount of total phenolic content of seed parts (cotyledon, coat, and whole seed) significantly differed. Similarly, mung bean hull extracts were found to have significantly higher TPC than whole seed and cotyledon (Singh et al., 2017b). It is reported that the phenolic compounds in legumes are observed to concentrate in their seed coats due to their protective function in the plants (Marathe et al., 2011; Singh et al., 2017a). The present study also confirmed the availability of high antioxidant content in the legume seed coat.

Some parameters such as the solvent used, the interaction between phenolics and extraction time were observed to affect the extractability of the phenolic compounds (Marathe et al., 2011). The choice of solvents used for extraction is critical because the polarities of the solvents have a direct effect on the amounts and types of recovered phenolic compounds. Several solvents have been used to achieve optimal extraction. Furthermore, the polarity of solvent has an important role in the increasing solubility of phenolic compounds (Haminiuk et al., 2014). In addition, it is known that phenolic compounds are generally associated with biomolecules in the plant, including inorganic compounds, polysaccharides, proteins, terpenes, chlorophyll (Iloki-Assanga et al., 2015).

Methanol and ethanol are some of the most common solvents used for extraction (Gomes, Torres, 2016). However, the use of water for bioactive compounds extraction may be a safer option for pharmaceutical and food applications (Shelembe et al., 2012). Polyphenols are known to be often most soluble in organic solvents which are less polar than water (Haminiuk et al., 2014). For the mentioned reasons, these solvents were preferred in this study. There is a lack of literature to compare the effects of different solvents on the phenolic extraction and activity of antioxidants in Lathyrus materials. Table I shows the effect of different solvents on the extraction of total phenolic from the extracts. These different TPC results can be explained by the polarity of the solvent. In terms of polarity, the solvents used in this research can be put in order according to their dielectric constant as follows: ethanol < methanol < water. In the study, the methanol extracts have mostly higher TPC. The seed coat methanol extract from L. hierosolymitanus also found to contain the highest TPC (p < 0.05) followed by the seed coat methanol extract from L. annuus. ANOVA coupled with post hoc Tukey tests showed significant differences in the TPC of methanol extracts when compared those of extracts obtained with other solvents (p < 0.05). The lowest TPC was obtained by water. In a work with similar solvents, Haminiuk et al. (2014) found that 100% methanol which is not the most or the least polar solvent was the most effective one for the extraction of phenolic compounds.

The antioxidant activity of phenolic compounds is mainly due to their redox properties, which can play an important role in neutralizing and absorbing free radicals (Borra, Gurumurthy, Mahendra, 2013). In this study, the antioxidant activities of all tested extracts were evaluated with reducing power and DPPH assays. The lowest EC₅₀ or IC₅₀ means had the highest antioxidant capacity. Significant differences were detected between antioxidant activities of water extracts and other extracts (p < 0.05). For the IC₅₀ values, the seed coat extracts showed higher values between 0.73 ± 0.19 and 3.57 ± 0.5 mg/ml, while the values in the cotyledon ranged from 2.45 ± 0.35 to 6.84 ± 0.81 mg/ml. The seed coat methanol extract, whole seed methanol extract, and seed coat ethanol extract from L. hierosolymitanus are found to have the highest DPPH radical scavenging activity (p < 0.05). In a previous study, the IC₅₀ values of extracts from L. czeczottianus Bässler and L. nissolia L. were found as 1.42 mg/ml and 3.19 ± 0.11 mg/ml, respectively, for DPPH assay (Llorent-Martínez et al., 2017a). In the present study, EC₅₀ values of ferric reducing power of the extracts also varied from 0.81 ± 0.1 to 11.7 ± 0.6 mg/ml. Similarly, the reducing power of the methanol extracts and seed coat extracts of Lathyrus species was mostly higher. The seed coat methanol extract from L. hierosolymitanus has the highest reducing power (p < 0.05). The extracts from cotyledon and extracts obtained by water exhibited significantly lesser antioxidant activity when compared with the extracts from whole seed and seed coat obtained with both ethanol and methanol (p < 0.05).

Phenolic components contribute to all antioxidant activities of the plant (Xu, Chang, 2007). Previous studies found the correlation between TPC with DPPH scavenging activity and reducing power capacity (Noor Atiqah, Maisarah, Asmah, 2014; Fidrianny, Natalia, Insanu, 2015; Krishnappa et al., 2017). In the present study, Pearson’s correlation coefficient between TPC in the extracts and antioxidant activities of extracts showed that TPC had a negative correlation with IC₅₀ of DPPH scavenging activities (r = -0.733) and EC₅₀ of reducing power (r = -0.594). Phenolic content in the extracts from Lathyrus seed could be suggested to contribute to their DPPH scavenging activity and reducing power activity.

As a conclusion, the TPC, DPPH scavenging activity and reducing power of extracts from seed parts showed high variability, while the variability between the species was lower. The methanol and ethanol extracts from the seed coat and whole seed also showed the strongest antioxidant activity. According to these results, it was found that seed coat has the strongest contribution to the antioxidant capacity of the tested Lathyrus species.

Solvents affect the rate and aggregate quantity of extraction of phenolics (Khan, Bakht, 2016). Therefore, the determination of a suitable solvent provides the stability of extracted phenolic compounds and it is very important for an efficient extraction procedure. The other aim of our study was to investigate the influence of the solvents on some individual phenolic compounds as well as on TPC. In order to identify this, the concentrations of gallic acid, caffeic acid, and epicatechin in the extracts were evaluated by using HPLC. The phenolic compositions of the extracts are shown in Figure 1-3.

FIGURE 1
Distribution of gallic acid in Lathyrus seed extracts. Different letter show differences of means among different solvents for each part of each plant species (p < 0.05). Different caps letter shows differences between extracts from different part of the plant in the same solvent for Lathyrus species (p < 0.05).

FIGURE 2
Distribution of caffeic acid in Lathyrus seed extracts. Different letter show differences of means among different solvents for each part of each plant species (p < 0.05). Different caps letter shows differences between extracts from different part of the plant in the same solvent for Lathyrus species (p < 0.05).

FIGURE 3
Distribution of epicatechin in Lathyrus seed extracts. Different letter show differences of means among different solvents for each part of each plant species (p < 0.05). Different caps letter shows differences between extracts from different part of the plant in the same solvent for Lathyrus species (p < 0.05).

In the present study, the higher total phenolic contents were mostly obtained from methanol extracts compared with ethanol and water extracts, but this is not the case for all the individual phenols. Similar effects of the solvents were observed in a previous study (Jouki, Khazaei, 2010). The highest concentration of gallic acid (1.32 µg/gdw) was extracted from seed coat of L. annuus with ethanol while the lowest from whole seed of L. hierosolymitanus with ethanol (0.1 µg/gdw) as seen in Figure 1. Significant differences were observed when the gallic acid concentrations in the same seed parts were evaluated with respect to solvents. This difference was particularly marked in water extract. The highest gallic acid concentrations were obtained from the bark extracts of plants and showed a similar trend to their total phenolic contents.

Caffeic acid has gained an increasing attention related to its antioxidant, anticancer, and anti-inflammatory properties. Strong antioxidant property of caffeic acid was attributed to the dihydroxylation of the 3,4 position on the phenolic ring (Jafri et al., 2017). In the current study, more caffeic acid was observed in all extracts compared to gallic acid and epicatechin concentrations (Figure 2). The highest concentration of caffeic acid was detected in methanol extract from seed coat of L. annuus (91.11 µg/gdw), whereas the least in water extract from cotyledon of L. annuus (20.11µg/gdw) as seen in the HPLC chromatogram (Figure 4-5). The epicatechin showed a wide range of variation according to Figure 3. The range for epicatechin varied between 0.17 and 32.55 µg/gdw. The highest amount of epicatechin was detected in methanol extract from the seed coat of L. hierosolymitanus while the least in ethanol extract from cotyledon of L. annuus (Figure 3).

FIGURE 4
HPLC chromatogram of L. annuus seed coat methanol extract. The peaks correspond to: 1 gallic acid (280 nm wavelength); 2 epicatechin (260 nm wavelength); 3 caffeic acid (280 nm wavelength).

FIGURE 5
HPLC chromatogram of L. annuus cotyledon water extract. The peaks correspond to: 1 gallic acid (280 nm wavelength); 2 epicatechin (260 nm wavelength); 3 caffeic acid (280 nm wavelength).

At the same time, when phenolic concentration obtained with different solvents were compared statistically, the concentrations of epicatechin and caffeic acid from the methanol extracts was the highest and significantly different from those of the water extracts (p < 0.05), except for cotyledon extracts from L. hierosolymitanus for caffeic acid (Figure 2). Similarly, the gallic acid concentrations from methanol extracts were different from those of the water extracts, but there was no difference among the water extracts from different parts of L. annuus. In addition, the difference between the epicatechin and caffeic acid from extracts was found to be significant in terms of Lathyrus species (p < 0.05). It was observed that caffeic acid was higher in L. annuus, while L. hierosolymitanus was richer in epicatechin. HPLC analysis results of the extracts imply that methanol and ethanol are the appropriate ones as the solvents to be preferred for phenolic extraction from L. annuus and L. hierosolymitanus.

As a result, the amount of individual phenolic components in the extracts significantly varied in relation to different solvents as well as Lathyrus species and their seed parts. The variations in phenolic compounds might attribute to the polarities of solvents and the chemical nature of components extracted from the plant as well as the plant matrix. This emphasizes that different phenolic compounds can individually be extracted by using specific solvent extraction. Therefore, a comparison of different solvents provides valuable information for efficient phenolic extraction from Lathyrus species.

Antimicrobial activity of the extracts from Lathyrus seeds

Antimicrobial properties of plants are being increasingly reported from different parts of the world. The search for new plants with potential antimicrobial properties has intensified owing to the side effects of drugs (Khan et al., 2009). Considering the dominant resistance of B. cereus, E. coli, and P. aeruginosa to a wide range of antibiotics, antibacterial effects of Lathyrus species extracts were investigated to find a suitable alternative to antibiotics. This study is the first to report the extracts from L. annuus and L. hierosolymitanus to be against microorganisms. In Table II, the extracts for antibacterial activity were tested based on minimal inhibition concentration (MIC). According to the results, methanol extracts from all parts of Lathyrus species showed mostly higher antibacterial activity for bacteria tested. The methanol extract from the seed coat of L. hierosolymitanus also showed the highest antibacterial activity against E. coli, while the highest antibacterial activity against B. cereus was obtained with methanol extract from whole seed of L. annuus and methanol extract from the seed coat of L. hierosolymitanus (p < 0.05). The methanol extract from whole seed of L. hierosolymitanus was also observed to have the highest antibacterial activity against P. aeruginosa. When the parts were compared, extracts obtained from cotyledons showed significantly lower antibacterial activity than seed coat and whole seed extracts as seen in Table II (p < 0.05). In particular, the water and ethanol extracts from the cotyledon parts showed no activity at all against the tested microorganisms.

However, no statistically significant difference was found among the extracts in terms of Lathyrus species (p > 0.05). In terms of solvents, similarly, methanol extracts showed better antibacterial activity than other solvents (p < 0.05). These differences can be attributed to the high phenolic content. The phenolic content is suggested to increase the antibacterial activity. Phenolic compounds are the most important antimicrobial agents and bioactive constituents in the plant (Mabrouki et al., 2018).

TABLE II
Minimum inhibitory concentrations of extracts from Lathyrus seeds on tested bacteria

Species	Parts	Solvents	E. coli mg/mL	B. cereus mg/mL	P. aeruginosa mg/mL
		Water	1.25 ± 0.25aB	1.25 ± 0.25aB	1.25 ± 0.2 aC
	Seed coat	Ethanol	0.5 ± 0.1bB	0.5 ± 0.1 bB	0.75 ± 0.05 bC
		Methanol	0.5 ± 0.1 bB	0.5 ± 0.1 bB	0.5 ± 0.1 bC
		Water	nd	nd	nd
L. annuus	Cotyledon	Ethanol	nd	nd	nd
		Methanol	1.5 ± 0.1A	1.5 ± 0.2A	1.5 ± 0.1A
		Water	1.5 ± 0.1 aB	1.5 ± 0.05 aB	1.5 ± 0.15 aB
	Whole seed	Ethanol	1.0 ± 0.1 bB	0.75 ± 0.05bB	1.0 ± 0.1 bB
		Methanol	0.5 ± 0.1 cB	0.25 ± 0.05cB	0.75 ± 0.05 bB
		Water	1.25 ± 0.25 aC	1.25 ± 0.15aB	1.25 ± 0.05aB
	Seed coat	Ethanol	0.5 ± 0.1 bC	0.5 ± 0.25 bB	0.5 ± 0.15bB
		Methanol	0.25 ± 0.05 bC	0.25 ± 0.05bB	0.5 ± 0.1bB
		Water	nd	nd	nd
L. hierosolymitanus	Cotyledon	Ethanol	nd	nd	nd
		Methanol	1.25 ± 0.25A	1.5 ± 0.25A	1.5 ± 0.15A
		Water	1.25 ± 0.1 aB	1.25 ± 0.05aB	1.25 ± 0.15aB
	Whole seed	Ethanol	0.5 ± 0.1 bB	0.75 ± 0.1 bB	0.5 ± 0.05bB
		Methanol	0.75 ± 0.15 bB	0.5 ± 0.1 cB	0.25 ± 0.02cB

• (nd): not determined
• Values are mean ± SD (n =3). Different letter within the same column show differences of means among different solvents for each part of each plant species (p < 0.05). Different caps letter shows differences between extracts from different part of the plant in the same solvent for Lathyrus species (p < 0.05).

There are not many reports of research on the antibacterial screening of Lathyrus species. In another study, MIC values of butanolic extracts from L. aphaca and L. ratan seeds were found to be below 100 μg/ ml against selected bacteria including Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Streptococcus species and Bacillus subtilis (Khan et al., 2009). The methanol and ethanol extracts of the leaf and body of L. karsianus P. H. Davis showed antibacterial activity against K. pneumoniae, P. aeruginosa, S. aureus, S. epidermidis, B. cereus, Salmonella enteritidis, Proteus mirabilis, E. coli, and Enterococcus faecalis (Özkan et al., 2014). Heydari et al. (2019) found that ethyl acetate fraction from L. armenus (Boiss. & Huet) Širj., L. aureus (Stev.) Brandza, L. cilicicus Hayek & Siehe, L. laxiflorus (Desf.) O. Kuntze, and L. pratensis L. were more effective than the methanol extracts, and n-hexane, chloroform, and water fractions against S. aureus, B. subtilis, E. coli, P. aeruginosa, and Candida albicans.

The antimicrobial effect of the plant extracts against the microorganisms may be due to the secondary metabolites like phenolic compounds. Krishnappa et al. (2017) found that the antibacterial activity of green gram seeds, sprouts, husks, and exudate was correlated with TPC, as the extracts showing higher phenolics showed lower MIC values. In the present study, it was found that methanol extracts and extracts from the seed coat and whole seed with higher TPC showed lower MIC values, but there was no correlation between antibacterial activity and TPC in the extracts. Beside the phenolic compounds, the synergistic effects of the variety of major and minor components in the extract constitute another factor to be considered for the antibacterial activity of the extracts. It can be concluded that these extracts, especially ethanol and methanol extracts, have a potential inhibitory effect against pathogenic microorganisms.

CONCLUSIONS

This study provides comprehensive information on the biological activities of Lathyrus species. The results indicated that absolute methanol was the most effective extraction agent on the recovery of phenolic compounds among the tested solvents. Besides, the seed coat extracts showed higher antioxidant activities than cotyledon and the whole seed extracts. The seed coat of L. hierosolymitanus generally contained the maximum phenolic compounds. The correlations between phenolic content and antioxidant activities of the extracts were also found. Therefore, the results are critical to optimize the extraction procedures for the analysis of different classes of phenolic compounds of Lathyrus species which are a rich source of antioxidants. Lathyrus species with biological activities shown in the study can be considered as potential candidates for their use in industrial areas such as pharmaceutical and cosmetic fields.

REFERENCES

• Altundag E, Ozturk M. Ethnomedicinal studies on the plant resources of east Anatolia, Turkey. Procedia - Social Behavioral Sciences. 2011;19:756-777.
• Amarowicz R, Troszynska A, Barylko-Pikielna N, Shahidi F. Polyphenolics extracts from legume seeds: correlations between total antioxidant activity, total phenolics content, tannins content and astringency. J Food Lipids. 2004;11(4):278-286.
• Borra SK, Gurumurthy P, Mahendra J. Antioxidant and free radical scavenging activity of curcumin determined by using different in vitro and ex vivo models. J Med Plants Res. 2013;7(36):2680-2690.
• Boudjou S, Oomah BD, Zaidi F, Hosseinian F. Phenolics content and antioxidant and anti-inflammatory activities of legume fractions. Food Chem. 2013;138(2-3):1543-1550.
• Caponio F, Alloggio V, Gomes T. Phenolic compounds of virgin olive oil: influence of paste preparation techniques. Food Chem . 1999;64(2):203-209.
• Davis PH. Flora of Turkey and the East Aegean Islands. Vol. 3. Edinburgh: Edinburgh University Press; 1970. 628 p.
• Fidrianny I, Natalia S, Insanu M. Antioxidant capacities of various fruit extracts from three varieties of tomato and correlation with total phenolic, flavonoid, carotenoid content. Int J Pharm Clin Res. 2015;7(4):283-289.
• Fratianni F, Cardinale F, Cozzolino A, Granese T, Albanese D, Di Matteo M, et al. Polyphenol composition and antioxidant activity of different grass pea (Lathyrus sativus), lentils (Lens culinaris), and chickpea (Cicer arietinum) ecotypes of the Campania region (Southern Italy). J Funct Food. 2014;7:551-557.
• Genç H, Şahin A. A new species of Lathyrus L. (Fabaceae) from Turkey. J Syst Evol. 2011;49(5):505-508.
• Grela ER, Rybiński W, Matras J, Sobolewska S. Variability of phenotypic and morphological characteristics of some Lathyrus sativus L. and Lathyrus cicera L. accessions and nutritional traits of their seeds. Genet Resour Crop Ev. 2012;59(8):1687-1703.
• Gomes S, Torres AG. Optimized extraction of polyphenolic antioxidant compounds from Brazil nut (Bertholletia excelsa) cake and evaluation of the polyphenol profile by HPLC. J Sci Food Agric. 2016;96(8):2805-2814.
• Güneş F. A new species of Lathyrus (Fabaceae) from Turkey. Pensee J. 2014;76(3):339-350.
• Haminiuk CWI, Plata-Oviedo MSV, de Mattos G, Carpes ST, Branco IG. Extraction and quantification of phenolic acids and flavonols from Eugenia pyriformis using different solvents. J Food Sci Technol. 2014;51(10):2862-2866.
• Heydari H, Işcan GS, Eryılmaz M, Acıkara ÖB, Sarıaltın SY, Tekin M, et al. Antimicrobial and Anti-Inflammatory Activity of Some Lathyrus L.(Fabaceae) Species Growing in Turkey. Turk J Pharm Sci. 2019;16(2):240.
• Iloki-Assanga SB, Lewis-Luján LM, Lara-Espinoza CL, Gil- Salido AA, Fernandez-Angulo D, Rubio-Pino JL, Haines DD.. Solvent effects on phytochemical constituent profiles and antioxidant activities, using four different extraction formulations for analysis of Bucida buceras L. and Phoradendron californicum. BMC Res Notes. 2015;8(1):396.
• Jafri L, Saleem S, Ullah N, Mirza B. In vitro assessment of antioxidant potential and determination of polyphenolic compounds of Hedera nepalensis K. Koch. Arabian J Chem. 2017;10(Supll 2):3699-3706.
• Jouki M, Khazaei N. Compare of extraction of phenolic compounds from Pistacia atlantica in different solvents In: Anninos P, Rossi M, Pham TD, Falugi C, Bussing A, Koukkou M, eds. Advances in Biomedical Research, Proceedings. World Scientific and Engineering Acad and Soc. Athens, Greece; 2010. p. 361-365.
• Khan BM, Bakht J. Antifungal, anti-yeast, anti-oxidant and HPLC analysis of different solvent extracted samples from Calamus aromaticus leaves. Bangladesh J Pharmacol. 2016;11(1):91-100.
• Khan NA, Quereshi S, Pandey A, Srivastava A. Antibacterial Activity of Seed Extracts of Commercial and Wild Lathyrus Species. Turk J Biol. 2009;33(2):165-169.
• Krishnappa NP, Basha SA, Negi PS, Prasada Rao UJS. Phenolic acid composition, antioxidant and antimicrobial activities of green gram (Vigna radiata) exudate, husk, and germinated seed of different stages. J Food Process Preserv. 2017;41(6):e13273.
• Llorent-Martínez EJ, Ortega-Barrales P, Zengin G, Mocan A, Simirgiotis MJ, Ceylan R, et al. Evaluation of antioxidant potential, enzyme inhibition activity and phenolic profile of Lathyrus cicera and Lathyrus digitatus: Potential sources of bioactive compounds for the food industry. Food Chem Toxicol. 2017b;107(Pt B):609-619.
• Llorent-Martínez EJ, Zengin G, Fernández-de Córdova ML, Bender O, Atalay A, Ceylan R, et al. Traditionally Used Lathyrus Species: Phytochemical Composition, Antioxidant Activity, Enzyme Inhibitory Properties, Cytotoxic Effects, and in silico Studies of L. czeczottianus and L. nissolia. Front Pharmacol. 2017a;8(83):1-20.
• Mabrouki H, Duarte CMM, Akretche DE. Estimation of Total Phenolic Contents and In Vitro Antioxidant and Antimicrobial Activities of Various Solvent Extracts of Melissa officinalis L. Arabian J Sci Eng. 2018;43(7):3349- 3357.
• Marathe SA, Rajalakshmi V, Jamdar SN, Sharma A. Comparative study on antioxidant activity of different varieties of commonly consumed legumes in India. Food Chem Toxicol. 2011;49(9):2005-2012.
• Noor Atiqah AAK, Maisarah AM, Asmah R. Comparison of antioxidant properties of tamarillo (Cyphomandra betacea), cherry tomato (Solanum lycopersicum var. cerasiform) and tomato (Lycopersicon esculentum). Int Food Res J. 2014;21(6):2355-2362.
• Oyaizu M. Studies on Product of Browning Reaction: Antioxidative Activities of Products of Browning Reaction Prepared from Glucoseamine. Jpn J Nutr Diet. 1986;44(6):307-315.
• Özkan O, Adıgüzel MC, Erdağ D, Bağcigil AF, Aydın H. In vitro comparison of the antibacterial activity of extracts from endemic plants species. Int J Ayurvedic Herbal Med. 2014;4(5):1608-1614.
• Parikh B, Patel VH. Total phenolic content and total antioxidant capacity of common Indian pulses and split pulses. J Food Sci Technol . 2018;55(4):1499-1507.
• Pastor-Cavada E, Juan R, Pastor JE, Alaiz M, Vioque J. Antioxidant activity of seed polyphenols in fifteen wild Lathyrus species from South Spain. LWT-Food Sci Technol. 2009;42(3):705-709.
• Shelembe JS, Cromarty D, Bester MJ, Minnaar A, Duodu KG. Characterisation of phenolic acids, flavonoids, proanthocyanidins and antioxidant activity of water extracts from seed coats of marama bean [Tylosema esculentum]- an underutilised food legume. Int J Food Sci Tech. 2012;47(3):648-655.
• Singh B, Singh JP, Shevkani K, Singh N, Kaur A. Bioactive constituents in pulses and their health benefits. J Food Sci Technol . 2017a;54(4):858-870.
• Singh B, Singh N, Thakur S, Kaur A. Ultrasound assisted extraction of polyphenols and their distribution in whole mung bean, hull and cotyledon. J Food Sci Technol . 2017b;54(4):921-932.
• Sokamte TA, Mbougueng PD, Tatsadjieu NL, Sachindra NM. Phenolic compounds characterization and antioxidant activities of selected spices from Cameroon. S Afr J Bot. 2019;121:7-15.
• Vaz-Patto MC, Rubiales D. Lathyrus diversity: available resources with relevance to crop improvement - L. sativus and L. cicera as case studies. Ann Bot. 2014;113(6):895-908.
• Xu BJ, Chang SKC. A comparative study on phenolic profiles and antioxidant activities of legumes as affected by extraction solvents. J Food Sci. 2007;72(2):159-166.
• Xu BJ, Chang, SKC. Phenolic substance characterization and chemical and cell-based antioxidant activities of 11 lentils grown in the Northern United States. J Agr Food Chem . 2010;58(3):1509-1517.
• Yazici Ozbek S, Ozmen I, Yildirim B, Genc H, Ozeloglu B, Gülsün M, et al. Biochemical Composition of Lathyrus L. Seeds: Antioxidant Activities, Phenolic Profiles, β-ODAP and Protein Contents. Legume Res. 2020;43(5):723-727.
• Zhang B, Deng Z, Ramdath DD, Tang Y, Chen PX, Liu R, et al. Phenolic profiles of 20 Canadian lentil cultivars and their contribution to antioxidant activity and inhibitory effects on α-glucosidase and pancreatic lipase. Food Chem . 2015;172:862-872.
• Zhao Y, Du SK, Wang H, Cai M. In vitro antioxidant activity of extracts from common legumes. Food Chem . 2014;152:462-466.
• Zhong L, Fang Z, Wahlqvist ML, Wu G, Hodgson JM, Johnson SK. Seed coats of pulses as a food ingredient: Characterization, processing, and applications. Trends Food Sci Tech. 2018;80:35-42.

Publication Dates

• Publication in this collection
  25 Nov 2022
• Date of issue
  2022

History

• Received
  14 Apr 2020
• Accepted
  14 Mar 2021