The leaves of Premna serratifolia (syn. P.obtusifolia), locally known as arogo or orogo, are consumed as food ingredient in Tentena, Poso, Middle Sulawesi (Celebes) especially as ingredient for fish-soup. P. serratifolia is a tropical shrub or a small tree (family Lamiaceae) that can grow up to one and half meters. The leaves of this plant were reported to have a strong but not disagreeable odor and are consumed by the people in Tentena along with other ingredients.1-3 In several countries such as India and the Philippines, Premna species are used as Ayurvedic drug. There are several species of Premna, such as Premna integrifolia L., P. serratifolia L. (syn. P.obtusifolia R.Br.), and Premna mucronata Roxb. All Premna were reported to have medicinal properties, such as carminative, diuretic, spasmolytic, hypoglycaemic, expectorant and depurative for the treatment of stomach disorders, oedema, constipation, piles and cardiac diseases.4 Most of the studies used bark, fruit, root, and stem as raw material, whereas only a few report on the bioactivities of P. serratifolia leaves. Recently, bioactive components have been isolated from methanol extracts of the leaves and their bioactivities have been demonstrated. Diterpens and triterpenes were separated as bioactive compounds from the leaves of P. serratifolia.5-6 However, there has been very little information on bioactivities of aqueous leaf extracts, such as those prepared by infusion and decoction which are safe for human consumption and relevant for domestic environment. Therefore, it is necessary to perform a novel study to substantiate its local use as a nutraceutical leafy vegetable.7-8
The objectives of this study were to investigate the α-glucosidase inhibitory and antioxidant activities of infusion and decoction from leaves of P. serratifolia. The leaf extracts were analyzed for their total phenolic content and LC-MS profiles.
MATERIALS AND METHODS
Chemicals and plant material
All solvents and chemicals used in the experiments were of analytical grade and purchased from Sigma-Aldrich (St. Louis, USA) or Santa Cruz Biotechnology (Dallas, USA).
Premna serratifolia L. leaves were collected in September 2016 from Tentena, Poso, Middle Sulawesi, Indonesia. The plant materials have been identified and authenticated by one of the author, KHT, using two identification articles.1-2 A voucher herbarium is kept in the laboratory (herbarium voucher number: KWL017).
Preparation of extracts
Leaves were sorted, dried under shadow, and powdered using a mechanical grinder. The powder was stored in an airtight container at room temperature for further analysis. Both the infusion and decoction were of aqueous leaf extracts of P. serratifolia. The infusion was prepared by hot water extraction i.e. 2 g of leaf powder was immersed in 200 ml of boiling water and allowed to cool for 15 mins. The decoction was prepared by boiling water extraction i.e. 2 g of leaf powder was immersed in 200 ml of boiling water and boiled until 100 ml remained.
The UV–VIS spectra were recorded on a UV–Vis spectrophotometer BioChrom Libra S-22 in a spectral range between 210 and 700 nm. Quartz cells were used for all absorbance measurements.
LC-MS analysis was performed using a Mariner Biospectrometry, equiped with a binary pump (Hitachi L 6200). The HPLC was interfaced with a Q-tof mass spectrometer fitted with an ESI (Electrospray Ionisation) source with positive ion mode. Full-scan mode from m/z 100 to 1200 was performed with a source temperature of 140°C. An HPLC column (Shimp-pack C8, 150 × 6 mm i.d.,) was used. Methanol with 0.3% formic acid was used as solvent and delivered at a total flow rate of 1 ml/min. A 5 µl sample was injected and eluted isocratically.
Determination of total phenolic content
The total phenolic contents of aqueous extracts of P. serratifolia were determined by Folin-Ciocalteu method9 with slight modification. An aliquot (0.5 ml) of sample was mixed with 2.5 ml of Folin-Ciocalteu reagent (10% v/v). The solution was left to stand for 10 min at room temperature. The reaction was then neutralized using sodium carbonate. After incubation for 2 hours in darkness at room temperature, the absorbance was measured at 765 nm using spectrophotometer. The total phenolic content was estimated based on a linear regression equation from a standard curve of gallic acid in methanol (12.5 – 200 µg/ml, R2 = 0.997) and the results were expressed as µg gallic acid equivalent (µg GAE/ml).
α-Glucosidase inhibition assay
The α-glucosidase inhibitory activities of aqueous extracts (infusion and decoction) of P.serratifolia leaves were evaluated according to the method previously reported10 with some modifications. A 50 µl of sample and standard (acarbose) at various concentrations was mixed with 50 µl phosphate buffer (50 mM, pH 6.8) and 50 µl of α-glucosidase (0.5 unit/ml). After pre-incubating for 10 min at 37oC, 100 µl of substrate (1mM p-nitrophenyl-α-D glucopyranoside) was added to the reaction mixture and incubated for 20 min at 37oC. The reaction was terminated by the addition of 750 µl of Na2CO3 (100 mM). The absorbance was read at 405 nm. For the control solution, all procedures were followed except that the sample was replaced by buffer. The percentage of inhibition was calculated using the following equation:
Where A (control): absorbance of control, A (sample): absorbance of the sample. The concentration of the samples and positive control required to inhibit 50% of the enzyme activity was defined as IC50 and was determined from the graph plotted against the percentage inhibition. The IC50 values were expressed as µg GAE/ml and compared with standard.
α-Glucosidase inhibition mechanism
The inhibition mechanism on α-glucosidase activity was determined by conducting a kinetic study of the inhibition of α-glucosidase by aqueous extract of P. serratifolia leaves.11 The kinetic assay was performed using p-nitrophenyl-α-D-glucopyranoside as a substrate in increasing concentrations (0.15 – 1 mM), in the absence and presence of the extract at different concentrations (0.04, 0.13, and 0.17 μg GAE/ml). Double reciprocal plots were generated based on the Lineweaver-Burk method to analyse the mechanism of inhibition by the extract.
DPPH radical scavenging activity
The antioxidant activity of aqueous extracts from P. serratifolia L was determined based on procedure described in the literature12 with minor modification. DPPH solution (0.6 mM in ethanol) was prepared and 1 ml of this solution was added to 3 ml of extract and standard solutions (BHT, ascorbic acid, and α-tocopherol) at various concentration (10 – 100 µg/ml). The reactions were incubated for 30 min in darkness at room temperature and the absorbance was read at 517 nm with spectrophotometer. Ethanol (3 ml) in place of extract was used as control. The percentage inhibition activity was calculated according to the following equation:
Where A(control): absorbance of control, A(sample): absorbance of the sample. The percentage of inhibition was plotted against concentration to calculate the IC50 which is defined as the amount of antioxidant required to decrease the inhibition of DPPH radical by 50%.13 The IC50 values were expressed as µg GAE/ml and compared with the standards.
Ferrous ion chelating activity
Ferrous ion can be monitored spectrophotometrically by measuring the formation of red colour Fe(II)-Ferrozine complex.14 However, in the presence of other chelators, the complex formation is interfered, leading to a decrease in red colour. Measurement of colour reduction allows the estimation of binding ability of chelators present in test samples. The ability of aqueous extracts of P. serratifolia and standard (EDTA) to chelate iron (II) was estimated according to the method described previously.15 In this method, 0.4 ml extracts and EDTA at various concentrations were added with 0.5 ml FeSO4 (0.2 mM). To start the reaction, 0.2 ml ferrozine (0.5 mM) was added and the total volume was adjusted to 4 ml with water. The reaction was immediately vortexed and was left to stand in the dark at room temperature for 10 min. The absorbance was read at 562 nm using spectrophotometer. Iron chelating ability was calculated using the following equation:
Where A (control): absorbance of control, A(sample): absorbance of the sample. The concentration of extracts required to chelate 50% of the Fe (II) ion (IC50) was calculated from the graph plotted against the percentage of inhibition. The IC50 value was expressed as µg GAE/mL and compared with standard.
RESULTS AND DISCUSSION
Total phenolic content (TPC)
The total phenolic content (TPC) of the leaf decoction was higher than the infusion sample, i.e. 539.26 ± 7.44 μg GAE/ml compared to 347.81 ± 0.21, respectively. The difference in phenolics may be due to the more concentrated sample obtained from the decoction procedure. Furthermore, the TPC of aqueous extracts was found to be dependent on the infusion time, the amount of leaves, and the aquadest temperature.
Inhibition of α-glucosidase
Both infusion and decoction extracts exhibited strong α-glucosidase activity, with IC50 lower than the positive standard acarbose (Table 1). The inhibitory activities of infusion and decoction samples were 200 and 18x103 times better than acarbose. These results suggest that both extracts are more potent than acarbose in inhibiting α-glucosidase. It was observed that when compared with infusion, the capacity for α-glucosidase inhibition of leaf decoction was 90 times stronger.
*µg Gallic Acid Equivalent/ml.
**Values are mean ± SD (n=3).
Previously, many plants have been identified of having more potent inhibitors of α-glucosidase than acarbose.16-17 Our findings provide further evidence that plant aqueous extract as demonstrated by P. serratifolia leaves exhibited stronger inhibition against α-glucosidase than acarbose.
The type of inhibition of the aqueous extract was analysed using Lineweaver-Burk plots. The results reveal that the inhibition was of competive mode, similar to that of acarbose (Figure 1).
In this study, the antioxidant activities of infusion and decoction of P. serratifolia leaves were investigated using two different in-vitro assays, namely DPPH radical scavenging assay and ferrous ion chelating activity assay. DPPH radical scavenging activities of both aqueous extracts and the standard compounds are shown in Table 2. Both infusion and decoction samples showed strong radical scavenging activity. Additions of extracts induced a rapid increase in inhibition at 517 nm. The activities of leaf infusion and decoction as expressed by IC50 were stronger than the standards ascorbic acid and BHT, but weaker than α-tocopherol. The activities may be attributed to the hydrogen donating ability of the phenolic components in the extracts.
*µg Gallic Acid Equivalent/ml.
**Values are mean ± SD (n=3).
Furthermore, it was found that both infusion and decoction samples demonstrated strong chelating activity for ferrous ions when compared with EDTA. As can be seen in Table 3, the percentage inhibition of the complex Fe (II)-ferrozine formation is linearly increased with increasing concentration of the aqueous extracts, indicating that both extracts readily bound ferrozine in a concentration-dependent manner. When the chelating activity was expressed in terms of its GAE content, the IC50 values of both extracts were lower than that of the standard EDTA, indicating stronger activities than the standard. These results suggest that the extracts may afford for protection from oxidative damage in cell.18 It has been observed that the leaf decoction has stronger activity when compared with infusion sample. The results suggest that decocting the leaf may increase the capacity for ferrous ion chelation.
*µg Gallic Acid Equivalent/ml.
**Values are mean ± SD (n=3).
Compared with the result of antioxidant study of P. serratifolia leaves by Uppin et al (2017), the present study reported stronger antioxidant activities for both infusion and decoction. The difference could be due to the difference in solvent systems used, which were MeOH, EtOH and chloroform.
Chemical characteristics of the extracts
The UV spectra of both infusion and decoction extracts showed two major absorption bands. The first one with the absorption maximum in the spectral range between 250 and 285 nm (λmax 261.8 nm) and the second one with the maximum in the range between 320 and 380 nm (λmax 340.9 nm). The absorption peaks in these spectral ranges indicated the presence of phenolic compounds. The LC-MS profile yielded one dominant peak of 132 (100%) m/z fragment and 170 (2.3%), and 201 (2.55%) m/z fragments (Figure 2).
Based on the MS characteristics at LC-MS analysis, the tentative compound is caffeic acid.19 In addition, the aforementioned UV spectra are similar with that of caffeic acid spectra, with absorption maximum at 321 and a shoulder at 278 nm, which is characteristic of phenolpropenyl compounds. It has been reported by several literatures that caffeic acid has antidiabetic effect and antioxidant activity.20-22 Moreover, the present study confirmed significant and positive correlations beween the levels of TPC and inhibition of α-glucosidase or antioxidant activities (Figure 3), which are in agreement with previous studies.23
In this present study, it is evidenced that aqueous extract of P. serratifolia L. possess antioxidant activity in-vitro. The antioxidant activity is evident