Phytochemical Investigation and in vitro Antimicrobial and Antioxidant Activities Evaluation of Erianthemum aethiopicum Wiens and Polhill

Introduction

Since time immemorial, natural sources, mainly medicinal plants, have been the guardians of millions of lives. The use of medicinal plants in local medicine is still in its place, especially in the developing world where modern medicine is inaccessible and unaffordable.¹ The wide array of bioactivities of medicinal plants including anticancer, antimicrobial, antioxidant and anti-inflammatory are attributed to the major phytochemical constituents such as polyphenols and flavonoids. Antioxidant agents originating from plants can block the production of oxidants, thereby preventing oxidative stress-causing illnesses including cardiovascular syndromes, diabetes, cancer tumors, hypertension, skin irritation, and neural disorders.²

In Ethiopia, close to 80% of the people rely on local medicine, with 95% plus of the formulation being based on medicinal plants.^3,4 It is believed that approximately 6000–7000 plant sources are estimated to be found in Ethiopian flora of which 1000 of them are used traditionally to alleviate various human and animal diseases.⁵ Even though the reality is this, most of the previous studies conducted on these medicinal plants remained mainly as a survey of ethno-botanical aspects of the plants. Even studies that focused on the bioactivity and phytochemical analysis of plants were few in number and limited to some plant species. The societies of the Eastern and Western parts of Hararghe, particularly those around the Harla and Dengego valleys, have used plenty of plant species directly for human and animal healthcare.⁶

Erianthemum aethiopicum Wiens and Polhill (genus: Erianthemum, family: Loranthaceae), one of the members of African mistletoes, is a parasitic climber plant native to North-Eastern Africa and indigenous to Ethiopia. In Ethiopia, it is known by the vernacular name “Degalo serkema” (Afan Oromo), and has tomentose twigs with white branches and short hairs and grows on Acacia or Commiphora woodland. It is extensively distributed in tropical areas of East African countries, mainly Ethiopia, Kenya, and Somalia. In the prehistoric town of Harla in Eastern Ethiopia, the leaves of E. aethiopicum are used as therapeutic agents to cure breast swelling, mastitis, morning sickness, and vomiting.^6,7 However, to our knowledge, no previous scientific studies have been conducted on the screening and determination of the main classes of phytochemicals and assessment of the antimicrobial and antioxidant action of E. aethiopicum leaves. This is the first study to investigate the major phytochemicals and examine in vitro antimicrobial and antioxidant effects of different E. aethiopicum leaves extracts.

Materials and Methods

Plant Material

Erianthemum aethiopicum leaves (Figure 1) were collected from prehistoric Harla town, Dire Dewa, Eastern Ethiopia (9° 27′, 9° 39′N latitude, 41° 38′, 42° 20′E longitude), in February 2022. The taxonomic identity of the plant was confirmed at the herbarium of Haramaya University, Ethiopia, and a representative sample with voucher number AHU196 was retained. Freshly collected leaves were air-dried at an ambient temperature under illuminated conditions. The dried samples were then coarsely milled using a grinding apparatus, packed in capped glass bottles, and stored at 4°C °C for subsequent experiments.

Figure 1 Photo of Erianthemum aethiopicum plant (By Teshome Gonfa, February, 2022).

Consumable Materials

Methanol (MeOH, 99.8%), ethanol (EtOH, 99.5%), chloroform (99.8% AR), n-hexane (AR), ethyl acetate (EtOAc), hydrochloric acid (HCl, 37%), Folin-Ciocalteu’s phenol reagent (2 N), DPPH (2, 2-Diphenyl picrylhydrazyl), gallic acid, catechin, ascorbic acid, potassium ferric cyanide, sodium nitrite, aluminum chloride (AlCl₃), trichloroacetic acid (Cl₃COOH), sodium hydroxide (NaOH), ferric chloride (FeCl₃), sodium carbonate (Na₂CO₃), vanillin, Mueller-Hinton agar (MHA), Potato Dextrose agar (PDA), and dimethyl sulfoxide (DMSO) were used as chemicals and reagents. Whatman No.1 filter paper (Whatman International Ltd, England), a Rota evaporator (rotary vacuum, Jainsons, India), and a UV-Vis spectrophotometer (Cecil CE4001 UV/Vis, Cambridge, England) were used for extraction filtration, solvent evaporation, and absorbance measurements, respectively.

Microorganism Culture Collection

The Ethiopian Public Health Institute (EPHI) provided us with two bacterial pathogens (Escherichia coli ATCC 12435 and Staphylococcus sciuris sciuri ATCC 25923) and two fungal pathogens (Candida glaebosa ATCC 76484 and Cryptococcus albidus ATCC 34140).

Extraction Process

Powdered leaves samples of E. aethiopicum (40.00 g) were macerated in n-hexane, chloroform, ethyl acetate, ethanol, and methanol (300 mL each) and extracted for two days on an orbital shaker to avoid any temperature under shedding. Each extract solution was filtered followed by solvent evaporation. The extraction efficiency of each solvent was determined in terms of the crude extract yield percentage, calculated using the following formula (1):

(1)

Qualitative Phytochemical Profiling

All leaves extracts were qualitatively screened for the presence or absence of the major classes of phytochemical constituents. The screening test was performed using the commonly applied procedures described previously.^8–10

Quantitative Determination of Phytochemicals

Determination of Total Phenolic Content (TPC)

The TPC of the chloroform, ethanol, and methanol extracts was determined at a wavelength of 765 nm, following the technique described by Siddiqui et al¹¹ and the detailed procedure described by Gonfa et el.¹² The results were calculated from the graph obtained and presented as milligrams of gallic acid equivalent per gram of dried crude extract (mg GAE/g DCE) in triplicates.

Measurement of Total Flavonoid Content (TFC)

The TFC of the three extracts mentioned above were estimated using the aluminum chloride colorimetric method¹³ and the detailed procedure was similar to that described by Gonfa et el.¹² The absorbance value of each tested analyte was recorded at a wavelength of 415 nm. The TFC value, recorded in triplicate, was calculated as milligrams of catechin equivalent per gram of dried crude extract (mg CE/g DCE).

Total Condensed Tannin Content (TCTC) Determination

The TCTC content of each extract was analyzed using the modified Broadhurst method with reference to the standard catechin.^12,14 The content was determined using absorbance values recorded at 515 nm using a UV-Vis spectrophotometer and expressed in terms of milligram equivalents of catechin (mg CE/g DCE) per dried crude extract. The experiments were conducted in triplicates.

In vitro Antimicrobial Activity Evaluation

Prior to evaluation of the antimicrobial activity assay, each tested organism was sub-cultured and the respective inoculum was standardized based on the standard procedure.¹⁵ A paper disc diffusion assay was followed to study the antimicrobial effectiveness of the extracts on selected pathogens as per the clinical and laboratory standards institute (CLSI).¹⁶ Then, an original solution of each analyte was prepared by reconstituting dry extract (g) in DMSO (mL), followed by the preparation of three corresponding dilutions (50, 75, 125 mg/mL) using the same solvent. MHA and PDA media were prepared according to the instructions described for the respective containers. Then, 100 μL of each plant extract solution was infused onto individual filter paper disc (6 mm width) and allowed to dry at room temperature. The impregnated discs were aseptically placed on the surfaces of MHA and PDA Petri plates. The plates were then sealed with Parafilm and incubated at 37°C for 18 h for bacterial pathogens and at 35°C for 24 h for yeast pathogens. Commercial ciprofloxacin (5 μg) and fluconazole (5 μg) discs were used as standard antibacterial and antifungal agents, respectively. DMSO was used as the negative control. After incubation, the diameters of the inhibited areas were recorded using calipers (in mm). The experiment was repeated thrice.

Minimum Inhibitory Concentration (MIC) Test

The MIC against fungal pathogens was determined using the broth microdilution technique.¹⁷ Two-fold serial dilutions of the extracts showing antifungal activity up to the lowest concentration (50 mg/mL) in the disc diffusion method were prepared in a plastic test tube containing the appropriate broth medium to obtain various concentrations (50, 25, 12.5, 6.25, and 3.125 mg/mL). Fungal yeast inoculums were added to give 0.5 x10³–2.5 × 10³ CFU/mL. Each tube was covered with sterile cotton and incubated at 35 °C for 48 h. The test tube, which showed a clear appearance, indicated no fungal growth, and the concentration attributed to fungal growth inhibition was considered to be the MIC (mg/mL).

In vitro Antioxidant Activity Examination

Chloroform, methanol, and ethanol leaves extracts enriched with major secondary metabolites were assessed for their antioxidative potential using DPPH free radical and potassium ferric ion reduction antioxidant power (PFRAP) assays.

Anti-DPPH Activity

The antioxidative ability against DPPH oxidant of the aforementioned three extracts (chloroform, ethanol, and methanol), and the positive control (ascorbic acid) was examined following detailed procedures described elsewhere.^12,18 Six serially diluted solutions (5, 12.5, 25, 50, 75, and 100 μg/mL) of each extract were derived from the corresponding original solution (0.5 mg/mL). Next, 2 mL of fresh DPPH solution (0.004% g/mL in MeOH) was added to 2 mL of each dilution and incubated at 25 °C for half an hour. The absorbance of each tested analyte, including the DPPH solution, was measured three times at a wavelength of 517 nm. The antioxidative action of each solution against DPPH was determined by the percentage of scavenging activity, calculated using Equation (2):

(2)

where A₀ and A represent the absorbance of the DPPH solution and target analyte, respectively.

Potassium Ferric Ion Reducing Power (PFRAP) Analysis

The PFRAP of each extract was investigated using the K₃Fe(CN)₆-FeCl₃ procedure with six dilutions (5, 12.5, 25, 50, 75, and 100 μg/mL), and the absorbance values were measured at a wavelength of 700 nm.¹⁹ Ascorbic acid was employed as an antioxidant drug, and the overall antioxidative activity against ferric ions of each analyte was presented in terms of absorbance values.

Data Analysis

Multivariate analysis using SPSS software version 20 was used to calculate the mean ± standard deviation (SD) of individual values recorded from triplicate experimental analyses. One-way analysis of variance (ANOVA) was used to compare significant differences in the obtained results at a confidence level of 95%.

Results and Discussion

Percentage of Crude Extract Yield

In this study, the alcoholic solvents provided the highest percentage of extract yield with 17.56 ± 16% for methanol and 16.45 ± 19% for ethanol. The next lowest extract yield was recorded for chloroform (4.65 ± 11%), ethyl acetate (3.03 ± 0.6%), and n-hexane (1.73 ± 0.7%). This indicates that the plant may be enriched in polar phytochemical constituents. This was supported by qualitative phytochemical profiling, although the number of phytochemicals screened was limited. Therefore, methanol and ethanol are better solvents for the extraction of phytochemicals from the leaves powder of E. aethiopicum. In line with this, the same solvents were suggested to exploit polar compounds, such as flavonoids and phenolics (Amalraj et al, 2021).²⁰

Qualitative Phytochemical Profiles

The preliminary phytochemical profiles of various leaves extracts are presented in Table 1. Flavonoids, phenolics, and saponins were detected in all, except ethyl acetate, extracts. Phytosterols and terpenoids were positively screened for all extracts. However, alkaloids, free anthraquinones, and coumarins were absent in all extracts. The methanol and ethanol extracts demonstrated a strong presence of the tested class of compounds, both in number and quantity. The present phytochemical screening results indicate that the ethnomedicinal uses of E. aethiopicum leaves can be attributed to the presence of flavonoids, phenolics, terpenoids, saponins, and tannins (Table 1).

Table 1 Qualitative Chemical Profiles of Five Leaves Extracts

Total Contents of Phenolics, Flavonoids and Tannins

The concentrations of flavonoids, phenolics, and tannins in each extract are presented in Table 2. The total flavonoids content (TFC) of each extract was determined as milligrams of catechin equivalent per gram of dried crude extract (mg CE/g DCE) using the obtained equation (y = 0.003x + 0.2426; R² = 0.9938). Accordingly, the highest TFC content (5.38 ± 0.52 mgCE/1gDCE) was observed in the methanol extract followed by the ethanol (4.12 ± 0.10 mgCE/1gDCE) and chloroform (2.16 ± 0.13 mgCE/1gDCE) extracts (Table 2). The TFC was found to be significantly different (P ≤ 0.05). Based on the generated linear regression equation (y = 0.0018x + 0.0893, R² = 0.9972) of the gallic acid standard curve, the extracts showed a statistically significant difference (P< 0.05) in the total polyphenolics content (TPC) ranged from 13.93 ± 0.86–22.63 ± 0.69mgGAE/gDCE with the highest content recorded in the methanol extract (22.63 ± 0.69), followed by ethanol (21. 52 ± 0.56) (Table 2).

Table 2 Total Phenolic, Flavonoid and Tannin Contents (Mean ± SD, n = 3) of Three Different Leaves Extracts

Similarly, the total condensed tannin content (TCTC) in each extract was estimated using the equation: y = 0.0008x+ 0.0826 (R²= 0.995). The highest TCTC value was recorded for the methanolic extract (39.18 ± 38 mgCE/gDCE), followed by the ethanol extract (12.77 ± 0.14). In general, the obtained results (Table 2) were found to be in line with the intensity of the qualitative phytochemical profiles obtained (Table 1), which revealed that flavonoids, polyphenolics, and tannins were strongly detected in both methanol and ethanol extracts.

Antioxidant Activity Determination

Anti-DPPH Activity

The percentage scavenging activity and IC₅₀ values against DPPH free radicals of the chloroform, ethanol, and methanol extracts are presented in Figure 2 and Table 3. The results demonstrated that the methanolic extract displayed the best anti-DPPH activity (IC₅₀ value of 4.31μg/mL), which was slightly comparable to that of ascorbic acid (IC₅₀ value of 0.49 µg/mL). Whereas the ethanolic and chloroform extracts showed a weaker DPPH inhibitory effect with higher IC₅₀ values (6.73 µg/mL and 12.75µg/mL, respectively) compared to methanolic extract and ascorbic acid.

Table 3 DPPH Oxidant Scavenging Activity Percentage (Mean ±SD, n=3) and IC50 Values of Three Different Leaves Extracts

Figure 2 DPPH scavenging percentage (%) against six concentrations (µg/mL) of three leaves extracts (chloroform, ethanol and methanol) and ascorbic acid.

The DPPH solution (0.004%, (m/v), used for the estimation of antioxidant activity, contains a purple free radical that changes into a yellow color that is stable when reacting with plant extracts enriched with antioxidants.²¹ Our finding suggested that the alcoholic extracts demonstrated the greatest scavenging percentage against DPPH oxidant, which was in agreement with the highest total phenolic and flavonoid contents. This indicates that the plant is a potential source of natural antioxidants.

FRAP Assay

The ferric ion reduction antioxidative potentials recorded at various concentrations of the tested extracts are shown in Figure 3. The methanolic and ethanolic extracts presented the best reductive capacity with higher absorbance values (0.71 and 0.42, respectively), although it was found to be weaker than that of ascorbic acid (0.93) at a similar dose (200 μg/mL). Generally, a significant difference (P ≤ 0.05) in the FRAP mean value was observed between the tested extracts according to the obtained statistical data.

Figure 3 Absorbance of FRAP versus various dilutions (μg/mL) of three leaves extracts (chloroform, ethanol and methanol) and ascorbic acid.

The bright yellow color of ferric ions (Fe³⁺) in the ferricyanide complex ([K₃Fe(CN)₆]) was changed to dark blue ferrous ions (Fe²⁺) depending on the concentration of the plant extract.

Several studies have demonstrated that plants enriched with polyphenols, including flavonoids, are interesting classes of bioactive phytochemicals that exhibit antimastitis/breast related infections,^22,23 preventing morning sickness^24,25 and vomiting during pregnancy.^26,27 Hence, E. aethiopicum is a source of numerous phytochemicals and polyphenols that possess antioxidant activities and traditionally possess the aforementioned activities, supporting the present study.

In vitro Antimicrobial Activity Evaluation

The antimicrobial activities of the n-hexane, chloroform, and methanol extracts against the tested bacteria (E. coli and S. sciuris) and fungal pathogens (C. albidus and C. glaebosa) were presented in Table 4 and Figures S1–S4 (see Supplementary Materials for inhibition zone images). The result showed that all the extracts showed a positive inhibitory action on both C. albidus and C. glaebosa fungal species up to 50 mg/mL dose with respective diameter of cleared zone values of 7.25 ± 0.25–8.83 ± 0.38 mm and 7.25 ± 0.25–10.17 ± 0.38 mm. The n-hexane and methanol extracts revealed improved action on C. albidus (8.83 ± 0.38 to 12.17 ± 0.58 mm) and C. glaebosa (10.17 ± 0.38 to 14.83 ± 0.80 mm), respectively, at concentrations of 50–125 mg/mL. However, their activity was still weak compared to that of the fluconazole standard (22.92 ± 0.72 mm). Whereas not all the tested extracts (n-hexane, chloroform and methanol) showed any activity against the bacterial species at all concentrations, except the n-hexane extract which tried to block spreading of E. coli bacterium (7.00 ± 0.38 to 8.08 ± 0.24 mm) up to 50 mg/mL.

Table 4 In vitro Antimicrobial Activity Diameter of Cleared Zone Values (Mean ± SD, Millimeter) of Three Different Leaves Extracts Against Human Pathogenic Fungal and Bacterial Strains

MIC Test

All the tested extracts were subjected to antifungal activity evaluation to determine their MIC values. This was because they showed good activity against the two fungal strains at a minimum concentration (50 mg/mL) using the agar disc diffusion method discussed above. Accordingly, both the methanol and n-hexane extracts showed MIC of 12.5 mg/mL, while the chloroform extract exhibited 25 mg/mL against both C. albidus and C. glaebosa fungal species.

Overall, The current promising antifungal activity of n-hexane extract could be due to the presence of various important phytochemicals including terpenoids, phytosterols, flavonoids, and phenolic acids.^28–30 The activity observed in methanolic and chloroform extracts may also be attributed to the flavonoids and polyphenolic constituents.³¹ Congruently, previous studies also reported that fungal pathogens cause infection in or around the human breast,^32–34 and nausea and vomiting.^35–37 Therefore, this is the first report on the antifungal activities of the E. aethiopicum leaves extracts, which support the traditional claims or the folkloric usage of this plant and could be a source of novel antifungal drugs.

Conclusion

In the present study, methanol and ethanol extracts showed better extraction efficiency by providing a comparatively higher extract yield. Flavonoids, phenolics, phytosterols, tannins, and saponins were observed in almost all extracts, with a strong presence observed in the alcoholic extracts. The highest total flavonoid, phenolic, and tannin contents were recorded in the methanol and ethanol extracts. The methanol extract showed the highest antioxidant activity against DPPH, although it was not comparable to that of ascorbic acid. All tested extracts presented good antifungal action against both C. albidus and C. glaebosa species. However, against the bacterial species, no extracts showed any activity at all concentrations, except the n-hexane extract, which inhibited the growth of E. coli bacterium up to 50 mg/mL. In general, this study concluded that methanol and ethanol could be used as extraction solvents for target compounds, such as flavonoids and phenolics, from E. aethiopicum leaves. This plant could also be considered as a potential source of antifungal agents. Therefore, as this study is reported here for the first time, it can be used as preliminary information to isolate bioactive compounds and evaluate additional biological activities of E. aethiopicum leaves.