The Effect of Antioxidant activity, Total Phenols and Total Flavonoids on Arginase Inhibitory Activity on Plants of Genus Sterculia

. The Effect of Antioxidant activity, Total Phenols and Total Flavonoids on Arginase Inhibitory Activity on Plants of Genus Sterculia


INTRODUCTION
The genus of Sterculia was included in the subfamily of Sterculioideae, the family of Malvaceae and right now becomes the family of Sterculiaceae, 1 Sterculia consists of 200 species. The stem, wood, leaves, fruit, and roots of the Sterculia have been used traditional medicine in many countries to treat various diseases, including digestive diseases, diabetes, respiratory diseases, and skin diseases. In addition, the genus Sterculia has been studied and has activities as antimicrobial, anti-inflammatory, antioxidant and anticancer 2 cytotoxic and immunomodulatory activities, 3 anti-nociceptive and anti-inflammatory, 4 sedative 5 antibacterial 6 and anti-TB. 7 The genus Sterculia contains of compounds flavonoids and their derivatives, terpenoids mostly as triterpenoids, coumarins, alkaloids and other groups such as phenolic acid, phenyl propanoid, fatty acids, sugar and some steroids. 8 The literature study confirms that the main content of the genus Sterculia was flavonoids which include flavones, C-glycoside flavones, flavonols, flavan, isoflavones, isoflavan and anthocyanins. Other phenolic compounds such as phenolic acid, propanoid phenyl, coumarin, lignans and lignin. 2 Indonesia has plants of genus Sterculia: Sterculia macrophylla Vent. was found in Sumatra, Maluku and Papua. Sterculia rubiginosa Zoll. ex Miq. was found in Sumatra. Sterculia parkinsonii F. Muell was found in Papua. Sterculia stipulata Korth and also sterculia comosa. Arginase was an enzyme responsible for converting L-arginine to L-ornithine and urea. The substrat was L-arginine. This substrat used for Arginase and nitric oxide synthase (NOS), they use same substrat, so arginase competes with NOS for arginine. 9,10 Nitric oxide (NO) production has been correlated to arginase activity in vessels, such as in physiological and pathological conditions on hypertension, 11 diabetes, 12,13 erectile dysfunction, 14 atherosclerosis, 15,16 and endothelial disfunction. 16 Some secondary metabolites have arginase inhibitory activity such as phenol and flavonoids. 18 It was interesting to find the relationship between total phenols, total flavonoids and antioxidant activity with the inhibitory enzyme arginase on Sterculia.

Extraction
The powder from woods and leaves of Sterculia plants (20 g) were extracted by using a solvent continously with n-hexane, ethyl acetate and methanol. The ratio between powder and solvent was 1:10. Each extract was concentrated with a rotary evaporator at 50 °C, then continued using a waterbath at 50 °C. Ethyl acetate and methanol extracts were tested for their activity as an arginase inhibitor.

Arginase inhibitor activity
The method for determined the arginase inhibitor activity used the procedure from the Kit and has been slightly modified. This procedure has also been carried out in previous studies. In the preliminary research, the concentration of the extracts in the well was made 100 μg/ml. The extract (50 mg) was added with 400 μl DMSO to dissolve, added with aquabidestillata to 5 ml (Stock 1). This solution was taken 1 ml and diluted with aquabidestillata to 2 ml (stock 2). From the stock 2, 90 μl was taken and diluted with aquabidestilata to 1 ml (stock 3). This solution (stock 3) would be tested for arginase inhibitory activity. Ten (10) µl extracts solution (stock 3) were added to the well, added 15 μl enzyme (1 U/ml), added 25 μl of L-arginine (570 mM) solution and shake for 5 sec. Incubated at 37 °C for 30 min. After incubation added with 100 μl urea kits A and B (1: 1), shake for 5 s. Incubate for 1 h at room temperature. The absorbance was read at 430 nm. The concentration extract for this activity was 100 µg/ml in well. The nor-NOHA as a positive control was performed under the same conditions and determined the IC 50 .

Antioxidant activity
The Antioxidant activity used the DPPH method from Bobo garcia (2015) with a slight modification. 19 For the antioxidant activity, the concentration of extracts were used 100 µg / ml. Twenty (20) µl extract in methanol added 180 µl DPPH 150 µmol /l solution in methanol, put into the well. The mixture shake for 60 s, incubate for 40 min in a dark place. The absorbance results was read at 517 nm. Methanol was used to replace the extract as a control. Quercetin was used as a positive control. The antioxidant activity was calculated as follow:

Total phenols
The determination of total phenols content used the method from Farasat (2014) with microplate as an instrument. 20 The concentration of the extract in the well was 100 µg/ml. Twenty (20) µl extracts in methanol (1000 µg/ml ) were added in the well, added 100 µl of Folin reagent (Folin-Ciocalteu: destilate water = 1:10). After 4 min, the mixture added 80 µl of Na 2 CO 3 7.5%. The incubation was carried out for 2 h in a dark place at room temperature. The absorbance was read at 600 nm. The standard curve was used gallic acid (3.125; 6.25; 25; 50; 100 µg/ml). Total phenols content was calculated as gallic acid equivalent (mg)/gram dry extract (mg GA /g extract).

Total flavonoids
The determination of total flavonoids content used the method from Farasat (2014) with microplate as an instrument. 20 The concentration of the extract in the well was 100 µg/ml. Twenty (20) µl extracts in methanol (1200 µg/ml) were added in the well, 20 µl aluminium chloride 10%, Added 20 µl potassium acetate 1 M and 180 µl distilled water. The mixture was incubated for 30 min at room temperature. The absorbance was read at 415 nm. The standard curve was used quercetine (3; 6; 9; 12; 18; 24 µg/ml). Total flavonoids was calculated as quercetine equivalent (mg)/gram dry extract (mg QE/g extract).

Phytochemical screening and TLC chromatogram
The chemical compounds in the active extracts were determined by the method of Harbone and Indonesian pharmacopoeia. 21,22 The content of the chemical compounds: tannins, alkaloids, flavonoids, phenols, saponins and anthraquinones. The active extracts were determined the profile of TLC chromatogram.

Statistical analysis
The multiple linear regression was used as a statistical analysis to find the relationship between antioxidant activity, total phenols and total flavonoids on arginase inhibitory activity. The total phenols, total flavonoids and antioxidant activity as independent variable, and dependent variable was arginase inhibitory activity.

Arginase activity
Methanol extract was an active extract from plants in the genus Sterculia. This active extract in wood and leaves. The results showed in table 1 and table 2. The IC 50 for nor-NOHA as a positive control was 3.773 µg / ml. The result showed on table 2 and figure 1.

Antioxidant activity
The DPPH was method to determined the antioxidant activity. Quercetine was used as a positive control, the IC 50 of quercetine was 5.63 µg/ml. The result of antioxidant activity showed on table 3 and figure 2.

Determination of total flavonoids and total phenols
The determination of total flavonoid was used quercetine as a standard.

Phytochemical screening
The active extract as an arginase inhibitor was determined the chemical constituents.
The results showed on

Statistical analysis
The multiple linear regression used for statistical analysis to find the effect of antioxidant activity, total phenols and total flavonoids to arginase inhibitory activity. The Significance value 0.000 (*P<0,05) it was meant that Ho was rejected. It can be said that total phenol, total flavonoids and antioxidant activity have an affect to the arginase inhibitory activity. The value of beta coefficient for total phenol was 0.891; the value of total flavonoid was -0.224 and the value of antioxidant activity was -0.053, it can be concluded that the total phenols more contributed         for the arginase activity than total flavonoids and antioxidant activity. The VIF (Variance Inflance Factor) values showed for total phenols 2.338, total flavonoids 1.444 and for antioxidant 2.430, from the three independent variables showed that there was no multicolinerity. The multiple linear regression with 3 independent variables as follow: Y = 28.289 + 0.128 X 1 -0.497 X 2 -0.069 X 3 . X 1 = independent variable (total phenols), X 2 = independent variable (total flavonoids), X 3 = independent variable (antioxidant activity).
Endothel dysfunction was related to arginase activity, one of the disease was hypertension. L-arginine was a substrate that used by NOS and arginase. Under physiological conditions NOS maintains the health of blood vessels by producing NO. Arginase produces ornithine, which would be metabolized to polyamine for tissue growth and proline for collagen. Under pathological stimulation with the presence of RhoA/ ROCK, arginase activity would be increase so that it would deplete the substrate NOS, L-arginine. When NOS does not have enough substrate, it will become unbound and produce more superoxide (O 2-) than NO. Increased production of polyamines and proline can also cause pathological and vascular stiffness. 23,24 Availability of NO will affect the regulation of vascular tone and maintenance of vascular integrity, 2 The inhibition of arginase activities by phenol, flavonoids, among of them were chlorogenic acid, quercetine, epicatechin, wogonin, ( 25 Flavonoids such as luteolin, fisetin can inhibit the arginase enzyme. 26 Our previous study showed that Sterculia macrophylla which has arginase activity also have high of antioxidant activity and total flavonoids. 27 The woods of Sterculia more active than leaves. And the methanol extract more active than ethyl acetate extract for inhibit arginase. The recent study the stem bark of Caesalpini turtuosa have the arginase activity with the IC 50 11.58 µg/ml for methanol extract and 33.81 µg/ml for ethyl acetate extract. 28 This result was same with our study. Sterculia contain phenol compounds and flavonoids as the abundant compound. 1 Interesting to examine whether the arginase inhibitory activity of from the genus Sterculia was influenced by antioxidant activity, total phenol levels and total flavonoids. The analytical results by multilinear regression analysis showed that total phenol was more contributed for this activity than total flavonoids and antioxidant activity. Sterculia comosa and Sterculia macrophylla have a high inhibitory activity on arginase. It is need more research to know the chemical compound which was responsible for this activity.

CONCLUSION
The total phenols of the plant of Sterculia genus responsible for the arginase inhibitory activity. The most active plants was Sterculia comosa woods. Based on this study Sterculia comosa woods may be used for many diseases causes by endothelial dysfunction.