Polyphenols are one of secondary metabolite in plants that have beneficial role on protective effect of cardiovascular system including improve endothelial function.1 Endothelial dysfunction is primarily due to reduction in Nitric Oxide (NO) bioavailabilty, impairment of endothelium- dependent vasorelaxation caused by a loss of nitric oxide bioactivity in the vessel wall.2 Endothelial dysfunction can result from and/or contribute to several disease development, as occured in hypertension, diabetes mellitus and hypercholesterolemia.1-2 Arginase is manganese metalloenzyme.3 Upregulation of Arginase can reduce the bioavailability of NO through competition between arginase with Nitric Oxide Syhthase (NOS) in using L-arginine as substrate, which can cause endothelial dysfunction.1-2 Potent inhibitor arginase such as S-(2- boronoethyl)-L-cysteine (BEC), 2-(S)-amino-6-boronohexanoic acid (ABH)) and (omega)-hydroxy-nor-L-arginine (nor-NOHA) have some problem to be more developed. BEC and ABH might confer a potential toxicity and pharmacokinetic problem due to their boronic acid functionality.4 On the other hand, nor-NOHA have very short half-life.5 According to this fact, it is necessary to find arginase inhibitor from natural sources. Until now, study on arginase inhibitor from plants were still limited.
Previous study showed that phenolic compounds such as quercetin, kaempferol, epicatechin has inhibitory effect on bovine arginase with IC50 31.2, 179.1, and 19.9 µg/ml.1 Melastoma malabathricum containing those three phenolic compounds6 and other phenolic compounds so we hypothesized that extracts from different plant of this plant may have arginase inhibitory effects. The plant were also chosen based on their pharmacological effect on patogenesis some diseases related to upregulation of arginase such as hypertension,7-8 hyperlipidemia9 and wound healing.10 To test this hypotesis, we investigated the inhibitory effect on arginase of different parts of this plant using in vitro methods. According to literature search, inhibitory effect on arginase from these different parts of this plants have never been studied. In order to see the relationship between arginase inhibitory effect and polyphenolic content, total phenolic content of each extract was also determined.
MATERIALS AND METHODS
nor-NOHA acetate (Cayman, US), gallic acid (Sigma Aldrich, India), aqua bidestilata, bovine liver arginase (Sigma, Singapore), foline-ciocalteu (Merck, Germany), L-arginine (Sigma, Singapore), maleic acid (Sigma, Singapore), mangan sulfate (Sigma, Singapore), urea assay kit (Abnova, Taiwan), dimethyl sulfoxide (Merck, Germany).
Parts of M. malabathricum were collected in July 2017 from central of Kalimantan. Plants were identified by Indonesia Science Institution, Center for Plant Conservation-Bogor Botanical Garden, Indonesia.
Preparation of samples
The part of the plant were collected and cleaned, dried at room temperature, and then powdered and stored in an air tight glass container. 50 g of each plant part was extracted by maceration with 500 mL of 70% ethanol as the solvent in first day at room temperature, and continued macerated with 250 mL of 70% ethanol in second until fourth day. The extract then evaporated using a rotatory vacuum evaporator, and then dried in water bath on 50°C.
Determination of percent yield (%)
The extraction yield was calculated for each extract using the formula below :
Percent yield (%) = a/b × 100
(a) is dry weight of extract and (b) soaked samples material
Arginase inhibition assay
The inhibition of arginase activity was performed using the enzyme protocol (Sigma Aldrich)11 and urea assay kit obtained from Abnova Corporation, Taiwan (KA 1652)12 with slight modification. N-hidroksi-nor-L-arginin (nor-NOHA) acetate was used as the standard inhibitor. Briefly, 10 µL of the 100 µg/ml extract solution, 15 µL of arginase solution (1 U/mL) and 20 µL L-arginine 570 mM as the substrate of arginase, was added into the well. The mixture were shaked and incubated for 30 min at 37oC to have the complete reaction. In control wells the extract was replaced by dimethyl sulfoxide. The reaction was stopped by addition of 100 µL of urea assay kit (kit A : kit B = 1 : 1) into each well. The absorbance of urea was measured at 430 nm after incubation in room temperature 25oC for 60 min, by a microplate reader (Versamax microplate reader, USA).
Percent of inhibition was calculated using the formula:
Abs1 is the absorbance of the control, Abs2 is the absorbance of the control blank, Abs3 is the absorbance of sample, and Abs4 is the absorbance of sample blank. IC50 value represents the concentration of inhibitor required to achieve 50% of enzyme inhibition.
The qualitative phytochemistry test were conducted according to Indonesian Materia Medica13 and Harborne.14 Alkaloid test with Mayer, Dragendorff, and Bouchardat reagents; flavonoid test with Shinoda and Wilson Tauböck reaction; tannin test with gelatin test, gelatin-salt test, and test with ferrous (III) chloride; saponin test with foaming test; terpenoid test with Liebermann-Burchard reagent.
Determination of total phenolic content
Total phenolic content was determined using the 96-well microplate with Folin–Ciocalteu reagent, adapted from Ahmad et al.15 with slight modification. 20 μL of the diluted extract or standard solution were added to 100 μL of reagent 25% Foline–Ciocalteu and homogenized by shaken for 60 s in 96-well microplate, and then allowed to stand for 4 min. After that, 75 μL of sodium carbonate solution (100 g/L) were added into the well and the mixture was shaken for 1 minute. After incubated for 2 h at room temperature, the absorbance was measured at 750 nm using the microplate reader (Versamax Microplate Reader). Extract or standard was substituted by ethanol as a blank. The standard curve for quantifying the phenolic contents was prepared by using gallic acid dilutions (6.25–200 mg/L). All samples were performed in triplicate.
The results showed that 70% ethanol extract of Melastoma malabathricum leaves gave the highest activity with percent inhibition of 81.26% ± 5.27 (Table 1) and IC50 62.43 µg/mL with equation y = 0.8163x + 0.9606, R2 = 0.9876.
|No.||Part of the plant||Percent yield (%)||Inhibition of Arginase (%)|
|1||Stem||8.44||61.61 ± 4.56|
|67.63 ± 7.61|
73.39 ± 9.39
81.26 ± 5.27
Final concentration of the extract solution in the Arginase inhibition assay was 100 µg/ml.
Data are mean ± SEM for triplicate measurements.
The leaves of Melastoma malabathricum as appears in Figure 1.
The extract was further investigated to determine the source of activity. The common phytochemistry content from plant such as flavonoid, alkaloid, terpenoid, steroid, tannin, and saponin have identified (Table 2).
Note : - = absent, + = present
Total phenolic content
The result of total phenolic content determination from 70% ethanolic extracts of Melastoma malabathricum leaves, flowers, stem and fruits extract are shown in Table 3. Calibration curve from gallic acid showed maximum absorbances at 750 nm wavelength (equation y = 0.0655x + 0.045, R2 = 0.9931). Total phenolic content of extracts could be ranked in the following order: M. malabathricum flower > leaves > fruits > stem.
Investigated samples were successively extracted by maceration method using 70% ethanol. Maceration method is cold extraction method by soaking raw materials for certain time in appropriate solvent. This method was chosen to avoid any degradation of any thermolabil active compounds during extraction which can caused by heat process.16-17 We used 70% ethanol as solvent due to the polarity of polyphenol as target compound.18 as studied before that some phenolic compounds has arginase inhibitory activity.1 Some polar and semi polar compounds could be extracted by 70% ethanol. The inhibitory effect of three 70% ethanol extracts on arginase activity was performed with in vitro method using L-arginine as substrate. Arginase will hydrolized L-arginine to L-ornithine and urea. Those condition will be accomplished by cleaving the guanidino group from arginine. It converts one positively charged amino acid into another to yield urea.19 It is a colorimetric reaction of urea, o-phthalaldehyde, and N-(1-naphthyl)ethylenediamme (NED).20 N-(1-naphthyl)ethylenediamme (NED) could be replaced with primaquin21 in kit urea assay.
Prior to the arginase inhibition test of Nor-NOHA as positive control and extracts as samples, optimization of substrate concentration was done with concentration of enzyme 1 U/mL, and some concentration of L-Arginine as substrat. The result showed that the optimum concentration of the substrate was at concentration of 570 mM. Based on this data, we chose L-arginin 570 mM for further experiments. Extracts assay prepared by dilluted with dimethyl sulfoxide (DMSO) and aquabidestilata. Samples need to be mixed first with DMSO to assist the dissolution of the samples.
In this study, nor-NOHA acetat was used as positive control because nor-NOHA is one of the potent arginase inhibitor with Ki 500 mM.22 Arginase inhibition by nor-NOHA was done by displacing the metal bridging hydroxide ion of arginase with N-hydroxy group of nor-NOHA22 In current study, determination of IC50 of nor-NOHA acetate was done at concentration 1,2,3,4,5 µg/mL. Ranging of concentration based on product information of nor-NOHA acetate that this compound exhibiting an IC50 value of 10-12 µM or 2,963-3,556 µg/mL. In some other research, studies of arginase inhibition of nor-NOHA not only tested using in vitro methods, but also developed to in vivo. Pre clinical trial in adult spontaneously hypertensive rats showed that Nor-NOHA decreases blood pressure and improves the reactivity of resistance vessels.23 Small scale clinical trial showed that nor-NOHA infusion increased endothelium-dependent vasodilatation in patients with coronary artery disease and diabetes mellitus type 2.24
Arginase inhibition test on four samples showed that highest inhibitory effect was on Melastoma malabathricum leaf extract. Melastoma malabathricum in Indonesia known as senduduk, senggani, harendong or karamunting.6 Leaves of Melastoma malabathricum possessed antihypertensive,25 wound healing activity.6 It contains some phenolic compound such as quercetin,6 epicathecin, epicathecin galat, kaempferol6,26 which is suspected as responsible compound for inhibitor of mamalia arginase1 also cinnamic acid derivate27 which can be also responsible for the inhibitory effect on arginase.28 These compound worked synergistically in the extract and showed higher arginase inhibitory effect on arginase than other Part extract of the plant. In other study, flavonol and epicatechin from Cocoa showed inhibition of arginase in HUVEC,29 Flavanone from Scutellaria indica showed inhibition of arginase in mice kidney lisate.29 The result of total phenolic content of M. malabathricum flower extract showed highest phenolic content of this plant compared to other parts of plant extract. It can be considered that arginase inhibitor activity of this plant was not related with the high phenolic content. According to Thernier and Pudlo30 the catechol group suggested interacts with Asp 129 which is involved in metal bridge formation for the two Mn2+ cofactors in the site of arginase.
Total phenolic content determination from samples was measured using colorimetric method. Foline-Ciocalteu reagent use in this study was reacted with phenolic compound in the samples and created blue complex colour that could be read by the microplate reader.The relationship between the arginase inhibitory activity of extracts from different plant of M. malabathricum with their phenolic content was determined using pearson test correlation. The result indicated no correlation (p>0.05) between phenolic content of the samples with arginase inhibitory activity.
The current study indicate that 70% ethanolic extracts of Melastoma malabathricum (L). leaves exhibited highest arginase inhibitory activity among the three plant extracts,. This preliminary study could be used as early stage to develop arginase inhibitor from natural sources. Further study need to be carried out to identify the bioactive constituents.