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Effects of solvent extraction on the phytoconstituents and in vitro antioxidant activity properties of leaf extracts of the two selected medicinal plants from Malawi

BMC Complementary Medicine and Therapies volume 24, Article number: 317 (2024) Cite this article

Abstract

This study evaluated and compared the phytochemical and antioxidant properties of the solvent extracts of Azadirachta indica A. Juss and Vernonia amygdalina Del leaves. Methanolic and aqueous extracts showed high (P ≤ 0.05) extract yields (in %), compared to chloroform and ethyl acetate extracts from both V. amygdalina and A. indica leaves. The study exhibited high phytochemical content in methanol and aqueous extracts compared to chloroform and ethyl acetate extracts, confirming the potential for medicinal use. V. amygdalina methanol and aqueous extracts had higher (P ≤ 0.05) total phenolic content (TPC), in mg GAE/gDW, (158.810±0.846 and 217.883±0.265, respectively) than chloroform (37.574±0.118) and ethyl acetate (104.758±0.236) but higher ethyl acetate content in A. indica extracts. Low polar solvents extracted high (P ≤ 0.05) total flavonoids, in mgQE/gDW, (367.051±0.858 and 149.808±0.009) compared to high polar solvents (14.863±0.071 and 54.226±0.014 ) in V. amygdalina while as in A. indica leaf extracts, low polar solvents showed high TFC ( 658.469±3.451 and 275.288±10.490) compared to high polar solvents (26.312±0.063 and 48.858±0.063) respectively. In vitro total antioxidant capacity, in mg/g, was higher in polar solvents than in low-polar solvents, ranging from 34.300±1.784 to 121.015±6.839 for A. indica ethyl acetate and methanolic extracts. A strong correlation between TPC and tannic acid content was observed, except in A. indica methanolic extracts of A. indica. Ferric reducing power was high, except for V. amygdalina chloroform and methanol leaf extracts, which were lower (P≤ 0.05) than that of the standard ascorbic acid. The study revealed that high polar solvents, such as methanol and water, are more efficient in the extraction of antioxidants from A. indica but lower in V. amygdalina extracts. High phytochemical content and antioxidative capacity could be significant in treating various diseases in humans.

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Introduction

Plants have been a source of herbal medicine and food since ancient times, and are a significant source of basic medicine in developing countries, even before the discovery of biomedicine [1,2,3]. The demand for herbal medicines and nutraceuticals as complements, supplements, and alternative sources of medicines and natural products for primary health has increased [4,5,6]. A World Health Organization (WHO) report indicated that a lack of healthcare services in developing countries led every four people out of five to depend on medicinal plants to cure basic ailments [7]. Globally, plants have been subjected to pharmaceutical experiments, with about one-fourth of medicines and drugs introduced on the market being produced from naturally synthesized plant bioactive molecules [8, 9]. Herbal and medicinal plants are used in developing countries to prevent and treat infectious diseases [1, 10]. In Africa, including Malawi, many rural poverty-stricken people, indigenous people, and traditional medicine practitioners use medicinal plants to treat many ailments and diseases in humans and livestock [1, 10].

Medicinal plants contain bioactive molecules, referred to as phytochemicals that define the antioxidant activities of plants and plant-based products [11, 12]. Phytochemicals are naturally synthesized primary and secondary biochemicals in ethnomedicinal plant leaves, fruits, and roots, with defensive and protective mechanisms against different types of diseases [12, 13]. Plant phytochemicals include plant primary and secondary bioactive compounds such as flavonoids, phenols, terpenoids, alkaloids, glycosides, and tannins [14, 15].

In Malawi, the potential of various ethnomedicinal plants for treating various diseases has been documented [16, 17]. Phytochemical screening of plants and herbs used to treat infectious diseases in Malawi and other African communities has been conducted [18,19,20]. These ethno-medicinal plants include neem (Azadirachta indica) and Vernonia amygdalina known as bitter leaf in English [2, 20].

Neem (Azadirachta indica) belongs to the family Meliaceae and was originally grown in India, but grows in tropical and subtropical regions of the world [21, 22]. Azadirachta indica contains various phytochemicals including alkaloids, triterpenoids, flavonoids, and phenolic compounds [23] with Azadirachtin being the most active biochemical compound [24, 25]. A. indica trees have significant pharmacological and industrial applications, and their leaves are potential drugs for diabetes, eczema, ringworms, anti-inflammatory, anti-hyperglycemic, and fever [21]. Barks, seeds, leaves, and roots have the potential to heal or cure diseases, such as diabetes, fever, skin infections, leprosy, intestinal helminthiasis, viral diseases, and respiratory diseases in children, and are antifungal, antibacterial, and antimalarial [26].

Bitterleaf (Vernonia amygdalina) is a perennial shrub that belongs to Astereceae family [27] and grows in tropical Africa as well as in West Africa where is used as a vegetable soup [28]. In some African countries, Vernonia amygdalina leaves are used to treat diabetes, malaria, helminth infections, intestinal disorders, and fever [29, 30]. Some authors have reported that plants contain various phytochemicals that have antioxidative effects and are important pharmaceuticals [31, 32]. The phytochemicals in V. amygdalina include phenols, alkaloids, flavonoids, tannins, and saponins [31, 33]. Therefore, the medicinal properties of V. amygdalina are defined by the presence of these phytochemicals in their tissues [6].

Research has been conducted on ethnomedicine and ethnobotany in Malawi. Plant and herbal medicines have been reported to cure many diseases and infections, such as diarrhea, asthma, pneumonia, general body pain, yellow fever, malaria and COVID-19 related diseases and symptoms [16, 17, 19]. Studies have shown that the phytochemical composition of medicinal plants is defined by geographical climate change, stage of maturity, and harvesting stage [34,35,36,37]. Furthermore, Altemimi et al. [38] reported that the quality and quantity of phytochemicals from medicinal plants and the efficacy of antioxidants depend on the nature of the solvent used in the extraction process. Although A. indica and V. amygdalina have been studied in many countries [21, 28, 39, 40], varied quantities have been reported, which have been attributed to many factors like climate, soils, and extraction methods among others [41,42,43], yet there are no such studies in Malawi, and this information and knowledge is useful for predicting the potential pharmacological and toxic effects of the selected medicinal plants as well as the dose which also determines the efficacy, and toxicity of phytochemicals. The authors gathered information on the names and usage of medicinal plant species in Malawi [16, 17, 44, 45]. Furtermore, there is limited information on the phytochemical and antioxidative effects of medicinal plants and herbs from different solvents in Malawi. Therefore, this study evaluated the effects of solvent extraction on the phytochemical composition and antioxidant capacity of two selected medicinal plants from Malawi, namely; Azadirachta indica and Vernonia amygdalina, to confirm their potential for medicinal use or purposes.

Materials and methods

Medicinal plant leaf sampling and identification

Azadiracta indica and Vernonia amygdalina are naturally (wild) growing plants in Malawi. Therefore, Azadirachta indica plant leaves were randomly sampled from Chikwawa districts at latitude 16° 11'S and longitude 34° 46'E, in the southern region, whereas Vernonia amygdalina leaves were sampled from Bunda area at latitude 14° 11'S and longitude 33° 46'E, in Lilongwe district, in central region of Malawi respectively. A. indica and V. amygdalina leaves were identified and authenticated at Lilongwe University of Agriculture and Natural Resources, Bunda Campus, by Mr. Ian Saini, a Senior Agronomy/ Botany Technician, in the Department of Crop and Soil Sciences. The sample identification was done by comparing them physically with the available specimens. The leaves were washed with distilled water several times and air-dried in the shade for 7-10 days. The leaves were ground through a 1 mm sieve using a Thomas-WILEY model 4 Laboratory Mill. The powdered samples were stored in airtight containers and refrigerated for chemical analysis [46].

Plant extract preparation

Samples (5 g) of each plant species were macerated in 50 ml of distilled water (aqueous extract), 80% methanol, chloroform, and ethyl acetate in 100 ml capped sampling bottles. The mixture was shaken on an automatic shaker at 160 rpm for 24 h and filtered using Whatman No. 1 filter paper. The plant leaf diagrammatical extraction process was done as presented in Fig. 1 below:

Fig. 1
figure 1

Medicinal plant solvent extraction process

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The residues were re-extracted once to exhaust the extraction process of bioactive compounds from the plant samples. The total extracts were poured into small glass vials of known weights and air-dried under a stream of air to concentrate the mixture. The extracts were stored at 4°C for further phytochemical and antioxidant activity analyses [47].

Medicinal plant extract yield

The extracts were placed in small glass vials of known weight and air-dried under a stream of air to evaporate the solvent. The dry sample extracts were then reweighed, and the total plant extract yield was calculated as the percentage of the original ground plant dry weight [47].

Qualitative phytochemical screening from the extracts

Qualitative phytochemical screening of the ethno-medicinal plant secondary metabolites involved the analysis of phenols, flavonoids, tannins, cardiac glycosides, and saponins using various methods described by Mechqoq et al. [48] with some modifications.

Test for total phenols

The crude plant extracts (0.5 g) were weighed in a 20 ml beaker and 5 ml of 5% FeCl3 was added. The mixture formed a dark green color, indicating the presence of total phenols.

Test for flavonoids

The crude sample extracts (0.5 g) was weighed in a 20 ml beaker, and 5 ml of distilled water, 2 ml of dilute ammonia solution, and 1 ml concentrated H2SO4 were mixed in a beaker. The mixture turned yellow, indicating the presence of the flavonoids.

Test for tannins

The crude plant extracts (0.5 g) was weighed in a 20 ml beaker, gently boiled in 5 ml of distilled water for 2 min, and cooled. FeCl3 solution (three drops) was added to the cold mixture, and the mixture changed to blue/green precipitates, which indicated the presence of tannins.

Test for cardiac glycosides

The plant extracts (0.5 g) was mixed with 2 ml of concentrated sulphuric acid in a test tube. Slowly, 2 ml of 10% ferric chloride solution was added down the side of the test tube so as to form two separate layers. A brown, violet, or greenish ring color was observed at the interface, indicating the presence of cardiac glycosides.

Tests for saponins

The plant extracts (0.5 g) were mixed with 5 ml of distilled water, shaken for 2 min, and boiled for 1 min. The mixture produced froths, indicating the presence of saponins.

Determination of phytochemical composition from the medicinal plant extracts

Determination of total phenolic content

The total phenolic content of the aqueous, 80% methanolic, chloroform, and ethyl acetate extracts of Azadirachta indica and Vernonia amygdalina was spectrophotometrically determined [49]. The extracts were diluted to a concentration of 1 mg/ml as a stock solution, and 1 ml of the extract stock solution was added to 2.5 ml of 10% Folin-Ciocalteu reagent. After 5 min, 2 ml of 7.5% Na2CO3 was added to a 20 ml test tube. Standard samples were prepared in the range of 0-6 mg from a stock solution of gallic acid (1 mg per ml) and subjected to the same treatment as the samples. The samples and standards were allowed to stand for 20 min at room temperature for color development and diluted to 10 ml. The absorbance of the samples and standards was measured at 765 nm using a UV spectrophotometer. The total phenolic content was calculated from the standard gallic acid curve using a linear equation and was expressed as mg gallic acid equivalent per gram of dry weight (mg GAE/g DW).

Determination of total tannins content

Total tannins content (TTC) of the leaf extracts was calorimetrically evaluated with minor modifications [50]. The extracts and required reagents were mixed as follows; 1 ml of the extracts was mixed in the test tube with 0.5 ml of 1:10 v/v Folin-Ciocalteau reagent, 0.5 ml of concentrated Na2CO3 was added and the solution was diluted to 10 ml with distilled water. A stock solution of tannic acid was prepared as a standard reagent (1 mg/ml tannic acid) and 0-6 mg of standard samples were prepared in different test tubes. Folin-Ciocalteu reagent (0.5 ml) and concentrated Na2CO3 were added to the test tubes. The samples were allowed to stand for 30 min for color development, and absorbance was measured at 760 nm using a UV spectrophotometer. The standard linear curve of tannic acid was used to calculate the total tannin content as mg of tannic acid equivalent (mg TAE/g DW) of the dry weight samples.

Determination of flavonoid content

The total flavonoid content (TFC) was determined by the aluminum chloride colorimetric method using quercetin as a standard [48]. The extracts were diluted to 1 mg/ml, and 1 ml of the diluted extracts was mixed with 1 ml of 5% sodium nitrite and 1 ml of 10% aluminum chloride (w/v ethanol) solution in separate test tubes. The test tubes were allowed to stand for 20 min at room temperature to allow color development. The standard solution of quercetin was prepared from a stock solution of 1 mg/ml, dissolved in 50% ethanol by pipetting 1-6 ml into various test tubes. The standards were treated similarly to the plant leaf sample extracts. After color development, the samples and standards were diluted to 10 ml using distilled water. The absorbance of the mixture was measured at 510 nm wavelength using a UV spectrophotometer. The calibration curve of quercetin was used to calculate the TFC expressed as mg quercetin equivalent/g of dry weight samples (mg QE /g DW).

Antioxidant capacity assay of the medicinal plants extracts

Quantification of total antioxidant capacity by phosphomolybdenum method

The method described by Prieto et al. [51], with minor modifications, was used to analyze total antioxidant capacity (TAC) of leaf extracts. This chemical reaction involves the reduction of Mo(VI) to Mo(V) by plant extracts, resulting in the formation of green/blue phosphate complexes under acidic sample conditions. The sample extracts (1 ml) was mixed with 1 ml of 0.6M sulphuric acid, 28mM sodium phosphate and 4mM ammonium molybdate in clean test tubes, which were capped and incubated in a water bath at 950C for 90 min. Standard samples were prepared from a stock solution of 1 mg/ml ascorbic acid in the concentration range of 1-6 mg and were treated as samples. The absorbance of both sample extracts and standards was measured after cooling at 695 nm using a UV spectrophotometer. The TAC was calculated from the standard ascorbic acid calibration curve and expressed as a fraction of mg ascorbic acid equivalent (AAE) per gram of dry weight (mg AAE/g DW).

Determination of Ferric Reducing Power (FRP) capacity of the medicinal plant extracts

Ferric reducing power of the medicinal plant extracts was conducted using the F3+ reduction to F2+ colorimetric method with minor modifications [52] (Oyaizu, 1986). In this assay, plant extracts with reducing ability reduces potassium ferricyanide (K3Fe3+(CN)6) to K4Fe2+(CN)6 which reacts with freshly prepared ferric chloride to ferric-ferrous complex as follows:

  • K3Fe3+(CN)6+ Plant extract/Iron chelator → K4Fe2+(CN)6 + Plant extract/Iron chelator. Then K4Fe2+(CN)6 + FeCl3 → KFe3+Fe2+(CN)6 + 3KCl.

The sample extracts (2 ml) was mixed with 1 ml of 0.2 M reagents (sodium phosphate buffer (pH 6.6) and 1% potassium ferricyanide (1%) in dry clean test tubes. The samples were then allowed to stand for 20 min in a water bath at 50°C and cooled after the addition of 10% (w/v) trichloroacetic acid. The samples were then centrifuged for 10 min at 3000 rpm to settle the precipitate. Then, 1 ml of the clear solution was transferred into the test tube and 1.0 ml of distilled water and freshly prepared ferric chloride solution (0.1 %) were added for color development. Standard ascorbic acid solutions in the range of 0-6 mg were prepared from a stock solution of 1 mg /ml and further treated as sample extracts. The absorbance of the samples and standards was read at 700 nm after 10 min of observation for color development using a UV spectrophotometer. A standard ascorbic acid and sample extracts calibration curve was constructed, and the high absorbance of the reaction mixtures showed high reducing power of the sample extracts against the standard ascorbic acid absorbance.

Statistical analysis

Chemical analysis was performed three times for each sample. Statistical package for social sciences (SPSS) version 20, was used for statistical data analysis. Means and standard errors of the chemical parameters and graphs were performed as one way of analyzing the data using one-way analysis of variance (ANOVA) on the effect of solvent extraction on the chemical parameters of medicinal plant extracts to test for significant differences at P≤0.05.

Results and discussion

Extraction yields

Extraction of the ground ethno-medicinal leaf samples was performed using four different solvents based on their increasing solvent polarity: ethyl acetate < chloroform < 80% methanol < distilled water. Figure 2 presents the extraction yields (in percent) of the four ethno-medicinal leaf extracts. The extracts yield ranged from 1.91±0.11 to 13.30±0.28 for V. amygdalina and A. indica leaves respectively. In V. amygdalina leaves, methanolic extraction resulted in high extract yield (13.30±0.28) content compared to aqueous (11.44±0.23), chloroform (4.50±0.14) and ethyl acetate (4.46±0.01) extractions. Similar results were observed in A. indica in which the methanolic extract had the highest extract yield (3.11±0.17) compared to aqueous (3.0±0.17), chloroform (2.77±0.28), and ethyl acetate (1.91±0.11) extracts. In this study, high extract yields were obtained from V. amygdalina leaves compared to A. indica irrespective of the type and polarity of the extraction solvents. This result further indicated that optimal phytochemical extraction from both samples was achieved using high polar solvents, such as 80% methanol and water, compared to low polar solvent systems. Plants contain low concentrations of bioactive molecules therefore, optimal extraction of biomolecules from plants depends on the choice of a suitable extraction solvent [53]. Previous studies employed various extraction solvents, resulting in different biological activities and yield content [40].

Fig. 2
figure 2

Plant solvent extracts yields in percent

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Phytochemical screening of the two ethno medicinal plants

The effect solvent extraction on qualitative phytochemical composition in the two ethno medicinal plants

The qualitative phytochemical compositions of the ethno-medicinal plant extracts obtained using the four solvents are presented in Table 1. The study revealed that V. amygdalina and A. indica leaf extracts contain high concentrations of plant secondary metabolites, such as phenols, flavonoids, tannins, steroids, cardiac glycosides, and saponins. However, the study showed that aqueous extracts had the highest phytochemical composition, followed by 80% methanol, chloroform, and ethyl acetate extracts, in both V. amygdalina and A. indica leaf extracts. Other studies reported that in Vernonia amygdalina, 80% methanolic extracts qualitatively contains more polyphenols, saponins, and flavonoids than tannins and glycosides [31] whereas in this study, high flavonoids contents were extracted in the methanolic extracts compared to the other phytochemicals. In a similar study, the aqueous extracts of V. amygdalina showed a higher concentration of saponins, tannins, and flavonoids than steroids, and phenolic compounds were absent [54]. Other studies have reported that methanolic, ethyl acetate, aqueous, and chloroform extracts of A. indica contain flavonoids, saponins, tannins, steroids, and alkaloids which was consistent with this study [55]. Aqueous leaf extracts of A. indica have been reported to contain saponins, flavonoids, steroids, polyphenols and tannins [22]. Therefore this study revealed that both high polar and low polar solvents can be used in phytochemical extraction from ethno-medicinal plants, but high polar solvents can optimize the extraction efficiency. The study further revealed that V. amygdalina and A. indica have high concentrations of phytochemicals and could therefore be useful in the herbal medicine, pharmaceutical, and nutraceutical industries.

Table 1 Phytochemical screening of the three solvent extracts
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Effect of solvent extraction on phytochemical content of the two ethno medicinal plants

The phytochemical contents (mg/g DW) of the two ethno-medicinal plants extracted using the four solvents are presented in Table 2. In the extraction of ethno-medicinal plant biomolecules, the polarity of both the extraction solvents and the bioactive molecule of interest as a solute should be considered before conducting the extraction process [38]. In this study, two high polar solvents (water and 80% methanol) and two low polar solvents (ethyl acetate and chloroform) were employed.

Table 2 Phytochemical composition and TAC of the two various medicinal plant solvent extracts
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The study showed that ethyl acetate extracts had high (P≤0.05) total phenolic compounds (TPC), in mg GAE/g DW, in both A, indica (374.923±0.354) and V. amygdalina (104.758±0.236) leaf extracts. However, methanol and aqueous extracts registered 158.810±0.846 and 217.883±0.265 in V. amydalina and 186.781±0.068 and 291.743±0.245 in A. indica compared to 37.574±0.118 and 27.840±0.068 for the chloroform extracts of both V. amydalina and A. indica leaf extracts, respectively. A. indica and V. amygdalina chloroform leaf extracts exhibited the lowest (P≤0.05) TPC values of 27.840±0.068 and 37.574±0.118, respectively, compared to the methanolic, aqueous, and ethyl acetate extracts. However, aqueous extracts had high (P≤0.05) TPC values of 291.743±0.245 and 217.883±0.265 compared with 186.781±0.068 and 158.810±0.846 for A. indica and V. amygdalina leaf extracts, respectively. This study revealed that chloroform, a low polar solvent, was less effective in extracting polyphenol compounds from the two medicinal plants (P ≤ 0.05). A similar observation was made when extracting polypneols from Tunisian Quercus coccifera L. and Juneperus phoenicea L. fruit [53]. In related studies, methanolic leaf extracts of V. amygdalina recorded a low TPC value of 3.81 [55] and 25.2±2.62 and 14.79±0.53 [56] for methanolic and ethyl acetate extracts compared to the values obtained from this study.

Other authors reported that A. indica 80% methanolic leaf extracts contained low TPC value of 69.55 [37] and in Oman, while Al-Hashemi & Hossain [55] reported similar results, in which ethyl acetate was more effective in extracting TPC (35.8), followed by methanol (12.7) chloroform (9.6), and aqueous leaf extract (4.2) respectively. The study in general revealed that less polar solvents are more effective for TPC extraction from V. amygdalina and A. indica leaves. This is because the low polarity of EA results in the formation of non-polar electrostatic interactions between phenolic compounds and the EA solvent, which increases solubility [57]. This study showed that A. indica and V. amygdalina from Malawi contained high concentrations of plant biomolecules in the form of TPC compared to those from previous studies, which are significant in both biomedicine and ethno-medicine.

The total flavonoid content (TFC) in mg QE/g DW ranged from 14.863±0.071 to 658.469±3.451 for V. amygdalina aqueous and A. indica ethyl acetate extracts, respectively. The study revealed that low polar solvents, ethyl acetate, and chloroform extracted high (P≤0.05) concentrations of TFC compared to the high polar solvents, methanol and water, from both A. indica and V. amygdalina leaves. Therefore, TFC was highest (P≤0.05) in A. indica ethyl acetate (658.469±3.451) and V. amygdalina (367.051±0.858) ethyl acetate leaf extracts and lowest (P≤0.05) in the aqueous leaf extracts of V. amygdalina (14.863±0.071) and A. indica (26.312±0.063), respectively. However, the study revealed that A. indica had a high (P≤0.05) concentration of TFC compared to V. amygdalina leaf extracts. Related research conducted in Nigeria reported a high TFC value of 1250 mg/g from V. amygdalina 70% ethanolic leaf extracts compared to the results from this study [4], and a high TFC mean value of 119 mg/g, was reported in India, in A. indica methanolic leaf extract compared to 48.858±0.063 in this study [37]. Other authors have reported a high TFC value of 138 in A. indica aqueous extracts compared to 26.094±0.064 in our study [22]. The study showed that low polar solvents were effective in extracting TFC, indicating that V. amygdalina and A. indica leaves have high concentrations of non-polar flavonoids such as isoflavones, flavones and flavonols [58].

Epidemiological and clinical studies have shown that flavonoids are significant in scavenging free oxygen radicals, culminating in the prevention and treatment of chronic degenerative diseases, such as cancer [56, 59]. Therefore, the high flavonoid content observed in the EA and chloroform extracts in this study indicates that A. indica and V. amygydalina leaf extracts could be a potential source of ethno-medicinal plants for the prevention and treatment of cancer.

Total tannin content (TTC), in mg TAE/g DW, ranged from 18.598±0.025 to 410.279±0.387 with V. amygdalina aqueous extracts and A. indica ethyl acetate extracts, respectively, which showed the lowest (P≤0.05) and highest (P≤0.05) values. The methanolic and aqueous extracts exhibited high (P≤0.05) TTC values of 204.395±0.075 and 319.255±0.269, respectively, in A. indica compared to 23.664±0.808 and 18.598±0.025, respectively, for V. amygdalina extracts. However, chloroform extracts had the lowest (P≤0.05) TTC values of 41.117±0.013 and 30.466±0.074 in V. amygdalina and A. indica compared to those of the other three solvents. In addition, other authors have reported high TTC values of 444 and 693.3 in V. amygdalina methanolic leaf extracts compared to 23.6640.808 in this study [48, 57]. This study revealed that ethyl acetate was more effective in extracting tannins (410.279±0.387) from A. indica than other solvents such as aqueous (319.255±0.269), methanol (204.395±0.075), and chloroform (30.466±0.074). Hydrolyzable tannins are beneficial to human health because of their antimutagenic, anticancer, and antioxidant [60]. However, high doses of tannins are reported to be harmful to human health [61]. Tannins form complexes with both minor and major elements, such as minerals (calcium), proteins, amino acids, and carbohydrates, resulting in the reduced bioavailability of these elements to humans [62]. However, the consumption of tannins less than 1.5-2.5g per day is safe with no side effects [63].

This study showed that no single organic or inorganic solvent is more efficient for extracting phytochemicals from medicinal plants. This is because each solvent extracted various amounts of phytochemicals from the leaves of the two medicinal plants used in this study. Therefore, a high degree of accuracy in various plant phytochemical extractions can be achieved by employing various or multiple solvents with different polarity indices in a single extraction process [38].

Furthermore, the study showed that A. indica from Malawi contained high concentrations of plant biomolecules, followed by V. amygdalina which is significant in both biomedicine and ethno-medicine. The variability in the phytochemical composition between the previous studies and this study could be defined by the environmental changes, climatic factors, geographical location, and stage of maturity of the medicinal plants [64].

The effects of solvent extraction on Total Antioxidant Capacity (TAC) of the two ethno medicinal plants

The total antioxidant capacity (TAC), in mg AAE/g, of the two ethno-medicinal plant leaf extracts is presented in Table 2. Plant antioxidant capacity is defined by phytochemical composition and is therefore responsible for the prevention and delay in the development of non-infectious diseases, such as diabetes, heart diseases, and cancer [65]. A. indica methanolic and aqueous extracts had high (P≤0.05) TAC values of 121.015±6.839 and 92.422±1.072, respectively, compared to 51.164±0.328 and 26.094±0.064 for V. amygdalina methanolic and aqueous extracts, respectively. The study showed that low polar solvent extracts (chloroform and ethyl acetate) had low (P≤0.05) TAC values of 66.184±1.511 and 64.644±0.080, and 39.734±3.700 and 34.300±1.784 from V. amygdalina and A. indica compared to those of methanol and aqueous extracts. Other authors reported higher absorbance of ethanolic leaf extracts of V. amygdalina fresh tissues compared to standard ascorbic acid absorbance, indicating that V. amygdalina leaf tissues had high total antioxidant capacity [4]. This study showed that high polar solvents, such as methanol and water, are more efficient in the extraction of antioxidants from A. indica and low polar solvents extracted more antioxidants from V. amygdalina leaves. This observation is in agreement with previous studies, which indicated that high polar solvents, such as methanol, have high efficiency in the extraction of antioxidants [38, 66].

The effects of solvent on the ferric reducing power of the two ethnomedicinal plants

The ferrous reducing power of the ethnomedicinal plants is presented in Fig. 3. The ferric reducing power assay is defined as the reduction of Fe3+/ferricyanide complex to Fe2+/ferrous form, indicating the antioxidant power of the plant leaf extract. The yellow color of the leaf extract changes to either Perls Prussian blue or green color with reference to the reduction capacity of the plant leaf extracts which is measured at 700 nm using a UV spectrophotometer [11]. The ferric reducing power assay is described as a practical and sensitive method for measuring the total antioxidant capacity of plant and plant-based pharmacological products [67, 68]. The results from this study showed that various extracts of A. indica and V. amygdalina leaves had high electron-donating power, which correlated with the sample concentration. The ferric reducing antioxidant power capacity of the extracts was higher (P≤0.05) than that of standard ascorbic acid, except for V. amygdalina chloroform leaf extract, whose absorbance was below (P≤0.05) that of standard ascorbic acid. The maximum ferric reducing antioxidant power of the extracts was obtained in A. indica ethyl acetate extracts (1.783), followed by ethyl acetate V. amygdalina (1.760), aqueous A. indica (1.615), and aqueous V. amygdalina (1.609). The ethno-medicinal plant extracts from this study had high ferric reducing antioxidant power compared to the absorbance of the standard ascorbic acid at 2 mg AAE/ml, denoting high ferric reducing power from the leaf extracts, except for V. amygdalina chloroform (0.477) and methanolic extracts (1.002). Some authors have reported a concentration-dependent increase in ferric reducing power absorbance from V. amygdalina in high polar absolute ethanol (50% and 70% ethanol leaf extracts), which was in agreement with the findings of this study [4]. The high ferric reducing antioxidant power of V. amygdalina methanol and aqueous extracts indicates that polar solvents are effective in extracting electron-donating biomolecules from plants, and the extracts could act as primary and secondary antioxidants. Similarly, in A. indica, aqueous and methanolic extracts proved to have high electron-donating biomolecules compared to low polar chloroform and ethyl acetate. This study further indicated that V. amygdalina and A. indica leaves have high concentrations of polar reductants.

Fig. 3
figure 3

Ferric Reducing Power of the two selected medicinal plant leaves. AA= ascorbic acid, AI=A. indica

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The effect of solvent extraction on correlation between TAC and TPC of the two medicinal plants

Various solvents were used to assess the correlation between TPC and TAC based on solvent polarity. Results in Tables 3 and 4 present the Pearson's correlation coefficients (r) colour matrix and the coefficient of determination (R2) for the TPC and TAC of the various medicinal plant leaf extracts respectively. The findings revealed a strong correlation between TPC and TAC from various plant extracts, regardless of the polarity of the solvent used, except for the 80% methanol extracts from A. indica as depicted in Tables 3 and 4. However, a significant (P≤0.05) correlation was observed in the chloroform extracts, followed by the aqueous, ethyl acetate, and 80% methanolic extracts in A. indica. However, in A. indica methanolic extracts, a weak correlation (r = 0.510) was observed, suggesting that the TPC extracted by methanol had a weak effect on the TAC properties of the plant leaves. In V. amygdalina, a significant (P≤0.05) correlation was observed in 80% methanol extracts, followed by EA, chloroform, and aqueous extracts. The study showed that the correlation between the TPC and TAC depends on the type of the solvent used in the extraction of the biomolecules. These findings are in agreement with reports that a positive correlation exists between TPC and total antioxidants capacity [69]. The higher correlation between TPC and TAC indicated that the TPC extracted by both high polar and low polar solvents in this study could define the TAC properties of the plant leaf extracts.

Table 3. Correlation colour matrix for V. amygdalina TPC and TAC
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Table 4 Correlation between TPC and TAC of the two medicinal plant leaf extracts
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Conclusion

The present study showed that aqueous, 80% methanolic, chloroform, and ethyl acetate leaf extracts of V. amygdalina and A. indica had high concentrations of phytochemicals, such as phenols, flavonoids, and tannins, and total antioxidant capacity. The study revealed that high polar solvents, such as methanol and water, are more efficient in extracting antioxidants from A. indica whereas low polar solvents were effective in antioxidant extraction from V. amygdalina leaves. The strong correlation was observed between TPC and TAC indicating that phenolic compounds from the solvent extracts defined the antioxidant properties of V. amygdalina and A. indica leaves. The high total antioxidant activity of the extracts could be attributed to their high phenolic, flavonoid, and tannin contents. The study showed that the chemistry of various plant bioactive compounds has different chemical properties, which have been defined by differences in extract solubility, yields, phytochemical composition, and antioxidant properties. The findings from this study have revealed that the two medicinal plants from Malawi had high concentrations of phytochemicals, which defined their high in vitro antioxidative capacity and could be significant in treating various diseases in humans. Although the study has presented very important results about antioxidant activity, the limitation of the study is that the samples were collected in a few areas, one season, and one stage of plant growth, of which, according to the literature, differences in these could generate different or similar results, although the study design is consistent with the literature. Therefore, future studies should also investigate the effects of these variations on the studied parameters. Furthermore, both in vitro and in vivo antimicrobial effects of the plants extracts need to be investigated in the future studies.

Availability of data and materials

The data for this study are included within the manuscript. Additional data are available from the corresponding author upon request.

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Acknowledgements

The authors would like to thank Mr. Ian Saini, a Senior Agronomy/Botany Technician, in the Department of Crop and Soil Sciences, for identifying the two medicinal plants used in this study.

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Authors and Affiliations

  1. Basic Science Department, Faculty of Agriculture, Lilongwe University of Agriculture and Natural Resources, Bunda Campus, P.O. Box 219, Lilongwe, Malawi

    Lesten Eliez Chisomo Chatepa & Bonface Mwamatope

  2. Pharmacy Department, Kamuzu University of Health Sciences (KUHeS), P.O. Box 360, Chichiri, Blantyre 3, Malawi

    Ibrahim Chikowe

  3. Food Science and Technology Department, Faculty of Food and Human Sciences, Lilongwe University of Agriculture and Natural Resources, Bunda Campus, P.O. Box 219, Lilongwe, Malawi

    Kingsley George Masamba

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  4. Kingsley George Masamba
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LECC Conceived and designing the study. He did sample chemical analysis and interpretation, performed statistical analysis, drafted the manuscript. BM performed statistical analysis, reviewed, revised and edited the manuscript. IC reviewed, revised and edited the manuscript. KGM Reviewed, revised and edited the manuscript.

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Chatepa, L.E.C., Mwamatope, B., Chikowe, I. et al. Effects of solvent extraction on the phytoconstituents and in vitro antioxidant activity properties of leaf extracts of the two selected medicinal plants from Malawi. BMC Complement Med Ther 24, 317 (2024). https://doi.org/10.1186/s12906-024-04619-7

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