In vitro inhibition of α-amylase enzyme, Phytochemical study and Antioxidant capacity for Cupressus sempervirens extracts growing in arid climate

 

Zineb Rahmani¹*, Ali Douadi², Zehour Rahmani²

¹Laboratory of Valorisation et Promotion des Ressources Sahariennes (LVPRS), Univ Kasdi Merbah BP 511 Route Ghardaia, Ouargla, Algeria - 30000

²Univ Kasdi Merbah Ouargla, Department of Chemistry, Faculty of Mathematics and Matter Sciences, BP 511 Route Ghardaia, Ouargla, Algeria - 30000

 

ABSTRACT:

Cupressus sempervirens (Cupressaceae) “cypress” is growing in the Mediterranean basin; it is a medicinal and an aromatic plant it used in treatment of many diseases in traditional medicine. This paper aims to determining antioxidant potential of C. sempervirens extracts and evaluating their inhibitory effects on α-amylase enzyme. The antioxidant properties of the crude extract and its factions were assessed by phosphomolybdenum, DPPH assay and α-amylase inhibitory activity was evaluating by spectrophotometric methods. Results showed that all extracts have high reductive activity better than butylated hydroxyanisole (BHA) with Ascorbic Acid Equivalent Anti-oxidant Capacity (AEAC) values ranged between 789.04 ± 32.47 and 28.18 ± 3.09 mM, while the greatest radical scavenging activity was found at ethyl acetate fraction with IC₅₀ value 10 µg/ml, whereas in α-amylase enzyme inhibitory activity, ethyl acetate fraction of fruits showed the highest α-amylase inhibitory activity (43.35 %). While the inhibitory patterns of ethyl acetate fraction of fruits (EFF) and ethyl acetate portion of tannin extract for leaves (EFT) were competitive type.

 

 

 

INTRODUCTION:

In the last years medicinal and aromatic plants have been known as an inexhaustible source of bioactive compounds. A scientific study has concentrated on natural antioxidants as a treatment for many diseases rather than synthetic drugs which have side effects.¹ Oxidative stress  are the disequilibrium between the production of interactive species resulting from the process of respiration and many other factors on the one hand and the defenses of the organism on the other², which  result from most diseases such as diabetes through the production of  Reactive oxygen species (ROS) during hyperglycemia.

 

For this, many studies is focused on medicinal plants as naturally sources for treatment or Prevention of the complications of the diabetes^3,4.

 

Climatic conditions, ecological and soil factors can influence the quantity and quality of secondary metabolites of plants^5,6, among these plants a cypress that affiliate to Cupressaceae family, which is widely used in traditional medicine. Cupressaceae is a family of gymnosperm plants. It contains about 19 genera and 130 species of trees and shrubs⁷. The genus includes up to 25 species⁸. The genus Cupressus (Cupressaceae) is composed of 12 species spread in North America, in the Mediterranean region and in subtropical Asia at high altitude⁹. Cupressus sempervirens (Cupressaceae) “cypress” are endemic to the Mediterranean area¹⁰. Popularly, it is known as “sarwel” that is considered to be a medicinal tree as its dryish leaves are used for stomach pain as well as to treat diabetes¹¹. Its dried cones are used and applied as anti-inflammation, toothache, laryngitis and astringent¹². The cypress resin is used orally to treat cough and rheumatic affections. In external use, it is used against cracks, crevices and foot ulcers; the oil is applied on wounds to treat scars. The Boiled of fruits is used internally as anti-diarrhea and antihemorrhagic¹³. In previous studies, plant extracts of this family showed inhibitory properties of the enzyme Acetyl cholinesterase¹⁴. The main target of this study is examining antioxidant properties for C. sempervirens leaf extracts and their inhibitory effects on α-amylase enzyme.

 

MATERIAL AND METHODS:

Chemicals and reagents:

Folin–Ciocalteu reagent, Gallic acid, Quercetine, Catechin, Ascorbic acid, 2,2-diphenyl-1-picrylhydrazyl (DPPH), Vanillin, Butylated hydroxyanisole (BHA), α-amylase of Aspergillus oryzae were provide by  Sigma-Aldrich, Ammonium molybdate, Sodium carbonate Na₂CO₃, Aluminium chloride AlCl₃, Hydrochloric acid, Sodium dihydrogen phosphate NaH₂PO₄ were provide by Biochem chemopharma. All other chemicals and solvents used in this study were of analytical grade.

 

Instrumentation:

The proposed work was carried out on a UV/VIS Spectrophotometer (SPECTROSCAN 80 DV), All weighing was done on electronic balance, Plant extracts preparation was carried out by using  Rotary evaporators (ISOLAB GmbH) for degassing solvent.

 

Preparation of plant extracts /fractions:

The used parts of the plant (leaves) were dehydrated in a well ventilated venue at room temperature. Plant-dried powder (100 g) was extracted three times with methanol-water (70:30 v/v) at room temperature after obtaining hydromethanolic extract were evaporated to dryness, recovered with distilled water and partitioned successively using dichloromethane, ethyl acetate and n-butanol. The extracts and the remaining aqueous fraction were concentrated under reduced pressure and then re-dissolved with minimum of methanol or water then were conserved at 4°C.

 

Determination of total Phenolic Contents:

Total phenolic content in the crude extract of leaves and its factions of C. sempervirens was determined using Folin-Ciocalteu reagent¹⁵. Briefly; 0.1ml of the sample was mixed with 0.5 ml freshly prepared dilute (1:10) Folin–Ciocalteu reagent. After 5min, 2ml of (20%) Na₂CO₃ were added, the blend was shaken and reacted for 30min at room temperature in the dark. The absorbance was read at 760nm and the results were expressed as mg Gallic acid rewards per gram of plant dry weight (mg GAE/g).

Determination of total flavonoid content:

The concentration of total flavonoid was estimated by aluminum chloride colorimetric method¹⁶ with slight adjustments. To sum up, 0.5ml of 2% AlCl₃ ethanol solution was added to 0.5ml of extract. The reaction mixture incubate for 30min at room temperature then the absorbance is read at 430nm and the results were expressed as mg Quercetine rewards per gram of plant dry weight (mg QE/g).

 

Determination of total tannin content:

The concentration of total tannin in the crude extract of leaves and its factions of C. sempervirens was estimated by using acidified vanillin method¹⁷. In short, 400μL of extract sample was added to 3ml of vanillin solution (4% in ethanol) and 1.5ml of concentrated hydrochloric acid. After 15min of incubation the absorbance is read at 500 nm. The results were expressed as mg catechin equivalent per gram of plant dry weight (mg CE/g).

 

Phosphomolybdenum assay:

The test is based on the reduction of Mo (VI) to Mo (V) by antioxidants samples; this reduction is materialized by the forming of a greenish complex (phosphate/Mo (V)) at an acid pH; the assay was applied as characterized by P. Kasangana et al. with slight modifications¹⁸. An aliquot of 0.1ml different concentrations of the extract was mixed with 1ml reagent solution (H₂SO₄ (0.6 M), NaH₂PO₄ (28 mM) and Ammonium molybdate (4mM). The samples are incubated at 95°C for 90minutes. Once cooling the tubes to room temperature, the absorbance of each is read at 695 nm against a blank. A standard blank solution included 0.1ml of H₂O and 1ml of the reagent solution mentioned above. It was incubated in the same conditions as above; the ascorbic acid was used as standard of positive reference, and the results were expressed as mM equivalent ascorbic acid.

 

Determination of antiradical activity:

Free radical scavenging activity against 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical was assessed by using the method characterized by Kim et al.¹⁹ and with slight modifications²⁰, 1ml of different concentrations of the extract was added to 1 ml of a 250μM DPPH^· ethanol solution, at room temperature and in the dark, the mixture is incubated for 30min, the absorbance of the samples were read at 517nm, the negative control (methanol with DPPH solution), inhibition of free radical DPPH in percent (I %) was calculated in following way:

 

 

Where (Abs₀; Abs₁) are the absorbance at 30min of the negative control and the sample, respectively.

Assay for α-amylase inhibition:

The activity of α-amylase is carried out according to Ademiluyi protocol with slight modification²¹. The principle of this method is based on quantifying of the free aldehyde and ketone groups of the reducing sugars (maltose equivalent) the result of the hydrolysis of starch by α-amylase. Briefly; an aliquot of 0.5ml of 1% (w/v) starch solution in 0.02M sodium phosphate buffer (pH 6.9) were mixed with 0.5ml of 1.3UI/ml α-amylase of Aspergillus oryzae (Enz-no:3.2.1.1) prepared in the same buffer solution (pH 6.9) and 50μl of the plant extract; then the mixture is incubated at 37°C for 30minutes. The reaction is stopped by adding 1ml of 3,5-dinitrosalicylic acid reagent (DNSA) (1 g of DNSA is dissolved in 40 ml of distilled water. At this solution 30g of Potassium sodium tartrate tetrahydrate are added with stirring then addition of 20ml of a 2N NaOH solution makes the reagent clear with an orange color, the volume obtained is adjusted to 100ml with distilled water); then the samples is incubated for 10 min at 100°C. After cooling, it is diluted with10ml of distilled water. A blank was set without plant extract and another without enzyme, substitute it by similar quantities of buffer solution (pH 6.9); next the absorbance is read at 540 nm against a blank. The α-amylase inhibition was expressed as a percentage of inhibition and is calculated by the following equation:

 

Where I% percentage of inhibition (Abs _C; Abs _S) are the absorbance at 540 nm in absence and in the presence of inhibitor (extract) for the sample, respectively.

 

Mode of α-Amylase Inhibition:

determination of inhibition pattern and the kinetic study of this reaction according to protocol described by Moein with slight modification²², a dilution series (2.5 to 20 mg/ml) of a starch solution as a substrate was examined  in the presence and without of inhibitor (extract 0.5 mg/ml) with the same previous steps. The reducing sugar released from starch was estimated by using maltose standard curve and converted to reaction velocities. The type of inhibition for extracts on 𝛼-amylase was analyzed by a Lineweaver-Burk plot and using Michaelis-Menten kinetics²³.

Statistical analysis:

Statistical analysis was performed using Origin Pro 8 software.  Data are presented as mean ± standard deviation (SD)

 

RESULTS AND DISCUSSION:

Extraction yield, total phenolic, flavonoid and tannin contents:

Crude extract of leaves and its factions of C. sempervirens were prepared to examine the total phenolic and tannins contents, flavonoid concentration and antioxidant activity. The yield of the methanolic extract of C. sempervirens leaves was estimated at 27.71 %. For the fraction, the highest yield was recorded at WF (10.43 %). from another side, the lowest yield of extraction was in dichloromethane fraction (0.098 %) (Table 1).

 

The total phenolic content of the crude extract and the fractions are reported as Gallic acid equivalent per gram dry weight (mg GAE/g DW). The highest content was recorded in crude extract and water fraction (34.34 ± 2.03 and 23.81 ± 0.865 mg GAE/g DW respectively) (Table 1). The total flavonoid content of the plant extracts studied expressed as equivalent of quercetin per gram of dry weight (mg EQ / g), The highest amount of flavonoids were present in the crude extract and butanol fraction (0.546 ± 0.04 and 0.1775 ± 0.0023 mg QE/g DW respectively) (Table 1). The concentration of total tannins was expressed as catechin equivalent per gram of dry weight (mg EC /g DW), the higher content of tannins was recorded in crude extract and water fraction (11.02 ± 0.455 and 3.47 ± 0.089 mg CE/g DW respectively) (Table 1)

 

Analysis uncovered the existence of flavonoids, terpenoids, resins, tannins, steroids and phenols in the raw extract and the fractions of C. sempervirens. These secondary metabolites are accountable for various biological activities. Therefore, lot of attention has been given to natural products derived mainly from plants^24-26.

 

Antioxidant activity of leaf extracts from C. sempervirens was evaluated using the DPPH, Phosphomolybdenum (PPM)

 

Table 1: Total phenolic, flavonoid and tannin contents

*Results are expressed as mean of 3 values ± standard deviation

 

 

Phosphomolybdenum assay (PPM):

This test is based on the reduction of molybdenum from the oxidation stage (VI) to the oxidation stage (V) by the plant extract which contains antioxidant agents. This reduction is materialized by fashioning of a bluish green colored phosphate/Mo (V) complex in the acidic medium²⁷  the antioxidant activity is measured according to a term called (AEAC) Ascorbic Acid Equivalent Anti-oxidant Capacity. The AEAC is defined as the molar concentration of the ascorbic acid solution, which has a reducing power equivalent to a solution of 1M concentration of test compound. Results revealed that all extracts have high reductive activity better than BHA with AEAC values ranged between 789.04±32.47 and 28.18±3.09 mM (Fig 1); The highest AEAC values were recorded in water fraction and crude extract (789.046 ± 32.473 mM and 515.25±27.88 mM respectively), while the lowest effects were shown by dichloromethane fraction (28.186±3.09 mM) (Table 2). During this test; the hydrogen and the electron are transferred from the reducing compound (antioxidant, extracts) to the oxidizing complex (PPM). This transfer depends on the redox potential, the pH of the medium and the structure of the antioxidant compound²⁸  the results revealed that the crude extracts, butanol and water fractions of C. sempervirens leaves recorded the high amounts of flavonoids and phenols as that this fractions exhibited  high values  AEAC of antioxidant activities. On the other hand, we found a good correlation between AEAC and phenols, tannins and flavonoids contents with a coefficient of determination R² = 0.98, 0.974, 0.463 respectively.

 

Fig. 1: Total antioxidant activity of C. Sempervirens extracts dichloromethane (D F), ethyl acetate (EF), butanol (BF) and water (WF) and crude extract (CE)

 

Antiradical activity:

DPPH^• test is a method largely used for free radical scavenging activity assessment. The antioxidant activity of the various tested samples of C. sempervirens against stable DPPH^• (2-diphenyl-2-picrylhydrazyl hydrate) was determined by spectrophotometric method. The values of IC₅₀ ranged from 10.97±0.947 to 560.7±41.7µg/ml (Table 2). The greatest scavenging activity was found at ethyl acetate fraction and n-butanol fraction (55.4±3.627 µg/ml), while the lowest scavenging activity was recorded in the dichloromethane fraction (560.7±41.7 µg/ml). The fractions and the crude extract showed better scavenging activity than the dichloromethane fraction. IC₅₀ values for DPPH^• scavenging activities of C. sempervirens extracts and ascorbic acid were compared and shown at Figure 2

 

 

Fig. 2:  Antiradical activity of C. Sempervirens extracts, BHA and ascorbic acid

 

The DPPH^• of violet color is reduced to a yellow compound in the presence of antioxidant compound, which can grant hydrogen atom and it has a maximum absorption at 517 nm²⁹.  The good scavenging activity of DPPH^• radical recorded at ethyl acetate, n-butanol fractions and crude extract, which have the greatest contents of phenols, tannin and flavoniodes. Polyphenols possess many antioxidant properties because of the mobility of phenolic hydrogen, so they are able to trapping oxygen free radicals and in particular peroxide radicals³⁰. While the total amount of phenols, tannins and flavonoids showed low correlation with DPPH antiradical activity with values of correlation coefficient R² = 0.17, 0.08, 0.118 respectively.

 

The interaction of flavonoids with many radicals has been used in several studies to define the major elements of antioxidant activity. Flavonoids (Flav-OH) are thermodynamically capable of reducing oxidative free radicals (R^•) such as superoxide, peroxyl radical, alkoxyl radical and OH^• by hydrogen transfer. The resulting aryl radical (Flav-O^•) can react with another free radical to form a stable quinone structure. In addition, the aroxyl radical can interact with oxygen to give a quinone and a superoxide anion. Therefore, the ability of flavonoids to act as antioxidants depends not only on the redox potential of the Flav-O^•/Flav-OH pair, but also on the reactivity of the aroxyl radical^31-33.

 

 

Table 2:  DPPH scavenging, total antioxidant activities

Extract	DPPH  (IC50 µg/ml) *	total antioxidant activities (AEAC mM) *
Ethyl acetate fraction	10.97 ± 0.947	233.616 ±  4.67
BHA	11.50 ± 0.315	1.277 ±  0.145
Ascorbic acid	13.42 ± 1.583	-
Butanol fraction	55.4 ± 3.627	508.215 ±  41.48
Crude extract	116.7 ± 11.629	515.25 ±  27.88
Water fraction	139.9 ± 2.867	789.046 ±  32.47
Dichloromethane fraction	560.7 ± 41.7	28.186 ±  3.09

*Results are expressed as mean of 3 values ± standard deviation

 

α-amylase inhibition:

To assess the influence of the seven extracts (leaves and fruits) of the species Cupressus sempervirens on the activity of α-amylase in vitro; a starch concentration of 1% (w/v) was used as substrate in the presence and absence of inhibitor at different concentration of each extract. The highest inhibition at 0.538 and 0.1894 mg/ml was observed in ethyl acetate fraction and crude extract of fruits with 43.35 and 35.94 %, respectively. While the highest inhibition in leaves was recorded at ethyl acetate tannin fraction with 35.43% at 0.616 mg/ml (Fig 3)

 

 

Fig. 3: Inhibition rate of α-amylase activity for C. Sempervirens extracts

 

Study of inhibition type in the enzymatic activity of α-amylase:

To define the pattern of extracts inhibition against α-amylase activity, we chose the ethyl acetate fraction of fruits (EFF) and ethyl acetate fraction of tannins extract for leaves (EFT) (which has high inhibitory power).

 

Figure 04 shows that the α-amylase enzyme follows a Michaelis kinetics V = f [S]. According to this Figure, the relation between the concentration and the enzymatic activity is proportional in a linearity domain which stops at a starch concentration of 12 mg/ml where the curve has constant value are represented by plateau, which means that the active site of the α-amylase is saturated.

 

Fig. 04:  Pattern of inhibition of α-amylase in the presence and absence of inhibitor

 

To study the behavior of the inhibition of extracts (EFT, EFF) on α-amylase ; the kinetics of the enzyme was  analysed by the Lineweaver-Burk curve (1/V=1/[S]) while  the latter allowed to determine the different kinetic parameters of α-amylase: the maximum velocity V_max and the Michaelis-Menten constant K_M; The values of V_max and K_M are thus obtained by curve fitting (Figure 05) where the resulted plot has a straight line with slope of K_M/V_max, y-intercept of 1/V_max, and X-intercept of -1/K_M

 

Lineweaver-Burk plot allowed us not only the determination of the type of inhibition, but also the other kinetic parameters namely the maximum velocity (V_max) and the inhibition constant (K_i) of the two extracts (EFT, EFF) which are calculated from Figure 05 by the projection of the point of intersection of the lines on the X-axis according to equation (1), kinetic parameters values shown at Table 3.

 

 

Where

[I]: Concentraction of the inhibitor
K_i: Inhibition constant

: Apparent Michaelis constant
K_M:  Michaelis–Menten constant

 

 

 

Table 3:  Kinetic parameters of extracts  

 

Figure 05 shows that the inhibition of Aspergillus oryzae α-amylase by ethyl acetate fraction of fruits (EFF) and ethyl acetate fraction of tannins extract for leaves (EFT) was a competitive type of inhibition with K_i values of 0.760, 1.852 µg/µl respectively. (EFF) was the best inhibitor for α-amylase than (EFT).

 

Fig. 05: Lineweaver-Burk plot of a-amylase inhibitory by ethyl acetate fraction (EFT, EFF)

 

The assay showed that the extracts (EFT, EFF) contains α-amylase inhibitory compounds when compared this results in case to the absence of inhibitor and through to the enzymatic activity for each sample (without inhibitor, EFT and EFF). This activity is perhaps due to the high quantity of polyphenols, flavonoids, and tannins and also to the chemical nature of these compounds, while is found that some naturally flavonoids act as inhibitors of human α-amylase³⁴. Also some studies indicate that there is a high correlation between the levels of enzyme inhibition and tannins content. The competitive type of inhibition can be explained by that the two extracts studied have compounds bearing hydroxyl groups close to those of the substrate, which moved it from the active site of the enzyme. These inhibitors are able to occupy the active site of α-amylase ^35,36. This study revealed that extracts of C .sempervirens have high contents of flavonoids and phenols where Tadera et al. tested several flavonoid compounds for their inhibitory activity against α-amylase³⁷. On furthermore Lo Piparo et al. they found that the inhibition of α-amylase enzyme by flavonoids is correlated with the number of hydroxyl groups in their B-ring. These compounds inhibit α-amylase by the formation of hydrogen bonds between its hydroxyl groups and the residues of the active site of this enzyme and formation of a conjugated π-system that stabilizes the interaction with the active site³⁴. A previous study assessed the anti-diabetic activities of flavonoids in vivo and in vitro in which it was found that it has a strong inhibitory effect³⁸. Lili Kandra et al. showed in their study that tannins is as an effective inhibitor of α-amylase as Acarbose and indicate a higher stability for the enzyme-inhibitor complex than the enzyme-substrate-inhibitor complex enzyme-substrate-inhibitor (ESI); the tannins bind to α-amylases; interconnection can take place on secondary site or the active site of the enzyme, these bonds generating complexes of the enzyme-inhibitor type, since the tannins act as competitive inhibitors of α-amylases³⁹. This explains the effectiveness of cypress extracts in inhibition of α-amylase because they contain the large amount of tannins.

 

ACKNOWLEDGEMENT:

The experiments of this study was done in Laboratory of Valorisation et Promotion des Ressources Sahariennes (LVPRS) of Kasdi Merbah University in Ouargla, Algeria and the authors express their sincere gratitude to Pr Mohamed HADJADJ head of the laboratory. We also thank Prs Mokhtar SAIDI and Hocine DENDOUGUI to their help to accomplish this work.

 

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

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Received on 06.09.2019         Modified on 28.09.2019

Accepted on 14.10.2019         © AJRC All right reserved