In vitro Screening for Antioxidant, Antimicrobial, and Antidiabetic Properties of Some Korean Native Plants on Mt. Halla, Jeju Island.

In this study, Prunus padus, Lonicera caerulea, Berberis amurensis, and Ribes maximowiczianum, which are mainly distributed on Mt. Halla, Jeju Island, have been investigated for their antioxidant, antimicrobial, and antidiabetic activities. The methanol extracts of R. maximowiczianum leaves and P. padus branches exhibited significant and dose-dependent antioxidant activity including electron-donation ability and reducing power. To analyze the antimicrobial activity, each extract was tested by a serial two-fold dilution method against five selected gram-positive bacteria and four gram-negative bacteria, and this suggested that P. padus branches possessed the maximum antimicrobial activity against most of the gram-positive bacteria tested. In addition, the methanol extracts of P. padus branches exhibited the highest α-glucosidase inhibitory activity with an IC50 value of 1.0±0.1 μg/ml, indicating that P. padus is a promising source as a herbal medicine.

Natural products, mostly from plants, have begun to gain worldwide interest for promoting healthcare, and have been used as conventional or complementary medicines due to toxicity and side effects of synthetic drugs[1]. In addition, natural products are known not only as a rich source of structurally diverse substances with a wide range of biological activities, but also as a primary source for synthesized drugs[12]. Therefore, the investigation of the pharmaceutical properties of medicinal plants and the analyses of their natural products are an important aspect when developing alternative or adjunctive therapies. The biological and pharmaceutical activities in medicinal plants are mostly mediated by the presence of secondary metabolites including phenolic and flavonoid compounds. These compounds exhibit a wide range of pharmaceutical properties, such as antioxidant, antimicrobial, antiinflammatory and anticancer properties[3]. Although a variety of plants are known to be good sources of these compounds, their contents are dependent on a number of factors including the climatic conditions, ripeness of the material, their tissues, and genetic factors[4].

Jeju Island in South Korea is known as a crossroads for several migration routes[5]. In addition, due to its geographical position, elevation and topography, the vertical distribution of temperatures and pressures from a subtropical to a subarctic zone allow for the generation of a unique ecosystem on Jeju Island[67]. So far, over 1,990 species including 719 species of edible plants have been classified[89]. This indicates that Jeju Island has a rich diversity of plants. Prunus padus L., Berberis amurensis var. quelpaertensis Nakai, Lonicera caerulea L., and Ribes maximowiczianum Kom., which are mainly distributed on Mt. Halla, Jeju Island, have been widely used as a traditional medicine, with beneficial effects in numerous diseases such as stroke, neuralgia, hepatitis, diarrhea and ocular disorder[101112]. The phytochemical analysis revealed that P. padus, L. caerulea are a rich source of anthocyanins and flavonoids, which display potential health-promoting effects[1113]. In addition, berberine (isoquinoline alkaloid), which possesses various biological activities including antimalarial, anticancer and antiAlzheimer disease, has been isolated from the roots and stem bark of Berberis species[12]. These indicate that these plants might be potential resources as a crude drug and dietary health supplements, although there are only a few systematic studies regarding their pharmaceutical potentials. Therefore, in this study, leaves and branches of P. padus (fig. 1a), L. caerulea (fig. 1b), B. amurensis (fig. 1c), and R. maximowiczianum (fig. 1d) have been investigated for their antioxidant, antimicrobial, and antidiabetic activities. Methanol extracts from branches of P. padus exhibited a high level of radical scavenging activity, reducing power, and α-glucosidase-inhibition activity compared with other extracts. These findings suggest that P. padus is a new potential source for herbal medicine.

Fig. 1

Pictorial image of plants used in study.

General view of Prunus padus L. (a), Lonicera caerulea L. (b), Berberis amurensis var. quelpaertensis Nakai (c), and Ribes maximowiczianum Kom. (d).

MATERIALS AND METHODS

Branches and leaves of P. padus, L. caerulea, B. amurensis, and R. maximowiczianum were obtained from the Research Institute for Hallasan (Jeju). The ground materials were soaked with methanol for 24 h, and were sonicated at 55° in an ultrasonic bath (Power sonic 520, Hwashin co., Korea). After filtration, each extract was evaporated using a rotary vacuum evaporator.

Analysis of total phenolic and flavonoid content:

The total phenolic contents were determined with Folin-Ciocalteu reagent by using gallic acid as a standard phenolic compound. Each extract (0.1 ml) was mixed with 50 µl of 2 N Folin-Ciocalteu reagent. After 5 min, the mixture was mixed with 0.3 ml of 20% sodium carbonate, and incubated for 15 min at room temperature. Then, 1 ml of distilled water was added to the mixture. The absorbance was measured at 725 nm using a UV-spectrophotometer (UV-1800, Simadzu, Tokyo, Japan). The level of the total phenolic compounds in each extract was calculated as μg of gallic acid equivalents per gram of extract using the equation obtained from the standard gallic acid graph.

To analyze the total flavonoid content in each extract, 0.5 ml of each extract was added to test tubes containing 0.1 ml of 10% aluminum nitrate (w/v), 0.1 ml of 1 M potassium acetate, and 4.3 ml of 80% ethanol. After 40 min incubation at room temperature, the absorbance was determined at 415 nm. The total flavonoid content was determined in micrograms of quercetin equivalents (QE) per gram of extract.

Analysis of electron-donation ability:

Based on the 1,1-diphenyl-2-picryl-hydrazil (DPPH) radical scavenging activity, the EDA of each extract was determined. The different concentrations of each extract in 4 ml methanol were mixed with 1 ml DPPH (0.15 mM in MeOH). The mixture was incubated for 30 min at room temperature, and then the absorbance was measured at 517 nm using a UV/Vis spectrophotometer. The EDA was calculated as a reduction rate in the absorbance by using the following equation: EDA (%)=[1-(A1/A0)]×100, where A1 is the absorbance value of the sample, and A0 is the absorbance value of the control. The means and standard errors were calculated from three independent experiments.

Determination of the total reduction capability via Fe3+-Fe2+ transformation:

To analyze the total reducing power of all the extracts, different concentrations of extracts (100, 200 and 300 µg/ml) were mixed with 0.5 ml of 0.2 M sodium phosphate buffer (pH 6.6) and 0.5 ml of 1% potassium ferricyanide. After incubation at 50° for 20 min, 2.5 ml of 10% trichloroacetic acid was added to the reaction mixture, and then centrifuged at 650 rpm for 10 min. The upper layer (0.5 ml) was mixed with distilled water (0.5 ml) and ferric chloride (0.1 ml, 0.1%). The absorbance was measured at 700 nm using a UV/Vis spectrophotometer.

Determination of antimicrobial activity:

The test strains used for the analysis of antimicrobial activity included five gram-positive bacteria; Bacillus atrophaeus (KACC 14742), Kocuria rhizophila (KACC 14744), Micrococcus luteus (KACC 14819), Staphylococcus epidermidis (KACC 14822), and Bacillus subtilis subsp. Spizizenii (KACC 14741), and four gram-negative bacteria; Klebsiella pneumoniae (KACC 14816), Enterobacter cloacae (KACC11958), Salmonella enterica subsp. enterica (KACC 10769), Pseudomonas aeruginosa (KACC 10186). All strains were obtained from the Korean Agricultural Culture Collection (KACC) in South Korea. The degree of antimicrobial activity was assayed by a serial two-fold dilution method, to determine the minimum inhibitory concentration (MIC) of each extract, as described by Olajuyigbe and Afolayan[14].

α-Glucosidase inhibitory effect:

The α-glucosidase inhibitory effect of each methanol extract was assayed according to the procedure described previously by Kim et al.[15], with minor modifications. 50 μl of each extract was mixed with 50 μl α-glucosidase (0.5 U/ml) and 50 μl of 0.2 M potassium phosphate buffer (pH 6.8), and the mixture was incubated at 37° for 15 min. 3 mM of the substrate (p-nitrophenyl glucopyranoside; pNPG) added to the mixture to start the reaction. The reaction mixture was incubated at 37° for 10 min and was stopped by adding 750 μl of 0.1 M Na2 CO3. The α-glucosidase activity was analyzed by measuring the p-nitrophenol released from pNPG at 405 nm using a spectrophotometer. The α-glucosidase inhibitory effects of each extract were calculated as: Inhibition rate (%)=[1-(Abssample–Absblank)/Abscontrol]×100, where Abssample represents the absorbance of the experimental sample, Absblank represents the absorbance of the blank, and Abscontrol represents the absorbance of the control.

Statistical analysis:

Data were subjected to an analysis of variance (ANOVA), and the means were compared via Duncan's multiple range tests at P<0.05, which were used to determine the significance of the means.

RESULTS AND DISCUSSION

A number of plants are considered to be good sources of phenolic and flavonoid compounds, which have pharmaceutical properties, such as antioxidant and antimicrobial activities[16]. Therefore, plant-derived phenolic and flavonoid compounds have earned considerable interest. To investigate the pharmaceutical properties of P. padus, L. caerulea, B. amurensis and R. maximowiczianum, we firstly determined the total phenol (TPC) and flavonoid content (TFC) from methanol extracts of these plants. The highest level of total phenolic compounds (2956±152 μg GAE/g) was found in R. maximowiczianum leaf extract (RmL), whereas B. amurensis branch extract (BaB) contained the lowest level of TPC (986±6 μg GAE/g) (Table 1). In the case of TFC, the extract of L. coerulea leaves (LcL) and RmL exhibited a higher level of TFC compared with other extracts. In addition, the branch extracts of P. padus (PpB, 1735±43 μg GAE/g) and L. caerulea (LcB, 1310±14 μg GAE/g) contained higher levels of TPC compared with their leaf extracts, PpL (1180±49 μg GAE/g) and LcL (1074±49 μg GAE/g), respectively, whereas higher levels of TFC were found in PpL (210.8±2.6 μg QUE/g) and LcL (371.8±2.6 μg QUE/g) compared with PpB (32.3±1.4 μg QUE/g) and LcB (96.5±5.3 μg QUE/g) (Table 1). In addition, the extracts from the leaves of R. maximowiczianum (RmL) and B. amurensis (BaL) showed higher levels of TPC and TFC than their branch extracts. Although these differences might be due to genetic, seasonal, and agronomic factors, these findings indicate that the leaves of R. maximowiczianum are a potential rich source of phenolic and flavonoid compounds.

TABLE 1

TOTAL PHENOLIC CONTENT AND TOTAL FLAVONOID CONTENT OF METHANOL EXTRACTS FROM KOREAN NATIVE PLANTS ON MT. HALLA

Free radicals produced by several redox reactions in the human body are implicated in contributing to protein oxidation, DNA damage, cancer, immunosuppression, inflammation, diabetes and neurodegenerative disorders such as Alzheimer's and Parkinson's diseases[17181920]. The natural compounds from plants such as vitamins (ascorbate, tocopherol, and cartenoids), polyphenolics and flavonoids play an important role in the defense against free radicals. Consequently, they have been used as supplementary antioxidants[121]. The presence of high levels of TPC and TFC in RmL indicates that the leaves of R. maximowiczianum might be a good source for an antioxidant agent. To investigate this antioxidant activity, we analyzed the EDA of methanol extracts from the selected plants using DPPH method. As shown in Table 2, RmL displayed the highest antioxidant activity (RC50=33.8±0.2 µg/ml) compared with other extracts, supporting the idea that the polyphenolic constituents are responsible for the EDA of R. maximowiczianum. A similar phenomenon has been observed in the extract of P. padus leaves and branches. However, LcL exhibited a higher EDA compared to LcB (Table 2), although LcL contained a lower level of TPC than LcB (Table 1). This might be due to the presence of a higher level of TFC in LcL, and suggests that flavonoids are major players as free radical scavengers in L. coerulea.

TABLE 2

ELECTRON DONATION ABILITY OF METHANOL EXTRACTS FROM KOREAN NATIVE PLANTS ON MT. HALLA

To further characterize the antioxidant properties in the extracts, we investigated Fe3+-Fe2+ transformation in the presence of each extract. As we expected, RmL exhibited the highest reductive capacity compared to other extracts (Table 3). RmL (100 µg/ml) exhibited a 0.74±0.03 (OD700 value), while 300 µg/ml RmB (methanol extract of R. maximowiczianum branch) displayed 0.64±0.01 (OD700 value). This indicates that polyphenolic compounds are the major naturally occurring antioxidants in R. maximowiczianum. In the case of B. amurensis extract, BaB displayed higher reductive capacity (Table 3), as well as a higher EDA (Table 2) than BaL, although BaB contained a lower level of TPC and TFC compared to BaL (Table 1). One of the possibilities for this non-correlation is that some of the polyphenols, which exist in BaB, are extremely active owing to their structural characteristics even if they are present in smaller quantities[22]. Other possibilities are that non-polyphenol-type compounds such as polysaccharides, which possess strong antioxidant activities[23], might be the active compounds in BaB.

TABLE 3

REDUCING POWER OF METHANOL EXTRACTS FROM KOREAN NATIVE PLANTS ON MT. HALLA

Although a number of new antibiotics have been developed during the last few decades, multiple drug resistance in human pathogenic microorganisms has increased due to indiscriminate use of commercial antibiotics[24]. In addition to this problem, antibiotics are associated with side effects such as immunosuppression, allergic reactions and hypersensitivity[25]. Therefore, alternative antibiotics including plant extracts and phytochemicals are needed to develop treatments for infectious disease. To investigate antimicrobial activity, we analyzed MIC of methanol extracts from the selected plants using the serial two-fold dilution method. PpB and LcB showed antimicrobial activity against most of the gram-positive bacteria tested, whereas none of the extracts, except PpB, exhibited any activity against gram-negative bacteria (Table 4). This might be due to differences in the cell wall composition between gram-negative and gram-positive bacteria. PpB was most active against Kocuria rhizophila (MIC=125 µg/ml) in comparison to all the bacteria tested. In addition, LcB showed antimicrobial activity against Kocuria rhizophila (MIC=250 µg/ml) and Bacillus subtilis (MIC=250 µg/ml). However, leaf extracts of both plants (P. padus and L. coerulea) contained no or lower antimicrobial activity compared with their branch extracts (Table 4). This suggested that the antimicrobial activity of P. padus and L. coerulea extracts might be mediated by TPC rather than by the TFC.

TABLE 4

ANTIBACTERIAL ACTIVITY OF METHANOL EXTRACTS FROM KOREAN NATIVE PLANTS ON MT. HALLA

World ethnobotanical information indicates that a number of herbal medicines from plants (more than 800 plants) are used for controlling hyperglycemia[26]. In type 2 diabetes mellitus (DM), α-glucosidase inhibitors such as acarbose, 1-deoxynojirimycin and genistein, which are isolated from natural sources, are beneficial in delaying glucose intake with low hypoglycemic effect[2728]. These suggest the potential and opportunity of medicinal plants to be used for the prevention and management of DM. To identify the potential of P. padus, L. caerulea, B. amurensis and R. maximowiczianum as antidiabetic agents, the methanol extracts from each plant were tested for α-glucosidase inhibitory activity. Extracts from P. padus, and L. caerulea branches significantly inhibited α-glucosidase, whereas LcL showed the lowest inhibitory effect (Table 5). In addition, the leaf extracts and branch extracts of each plant showed different levels of inhibitory effect on α-glucosidase. Interestingly, PpB, LcB, and BaL contained higher levels of TPC and α-glucosidase inhibitory activity than PpL, LcL, and BaB, respectively (Tables 1 and 5). Plant phenolic compounds are known to modulate the enzymatic breakdown of carbohydrate due to their ability to bind with α-glucosidase[29]. Thus, the variation in α-glucosidase inhibitory activity between the organic extracts might be due to the level of phenolic compounds. Based on metabolite profiling of P. padus leaves, six phenolic compounds including astragalin and chlorogenic acid have been identified[30]. Astragalin is known as a glycation inhibitor[31] and antioxidant agent[32], and it has an inhibitory effect on α-glucosidase activity[33]. In addition, chlorogenic acid is a highly hydrophilic natural compound containing a catechol group like catechin, which effectively inhibits α-glucosidase and α-amylase activities[3435]. Although chlorogenic acid, known as an antioxidant agent[36], exhibited a low level of α-glucosidase inhibitory activity, alkyl chlorogenic acid derivatives have been suggested as potential α-glucosidase inhibitors[35]. Taken together, these findings indicate that astragalin and chlorogenic acid (or its derivatives) should be the major bioactive compounds of P. padus.

TABLE 5

ALPHA-GLUCOSIDASE INHIBITION ACTIVITIES OF METHANOL EXTRACTS FROM KOREAN NATIVE PLANTS ON MT. HALLA

In this study, we analyzed the antioxidant, antimicrobial, and antidiabetic activities of P. padus, L. caerulea, B. amurensis, and R. maximowiczianum. The overall results of the present study suggest that the methanol leaf extract of R. maximowiczianum could be useful as a source of natural antioxidant agents. In addition, the branch extract of P. padus was shown to possess notable pharmaceutical activities, indicating that P. padus should be considered as a useful source for herbal medicine. The variation in pharmaceutical activities between organic extracts indicates that the comparative analysis of the metabolome between branch extracts and leaf extracts will be required for the isolation and characterization of the active compounds in P. padus.

Financial support and sponsorship:

Nil.

Conflicts of interest:

There are no conflicts of interest.