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Phytochemical Screening, Determination of Antioxidant Activity and Alpha-Amylase Inhibitory Activity of Nypa fruticans Sugar
Available online at www.sciencedirect.com
ScienceDirect Materials Today: Proceedings 19 (2019) 1745–1751
www.materialstoday.com/proceedings
ICCSE 2018
Phytochemical Screening, Determination of Antioxidant Activity and Alpha-Amylase Inhibitory Activity of Nypa fruticans Sugar Wan Mazliena Aliana Wan Sabri, Syed Ahmad Tarmizi Wan Yusop, Ahmad Hafizi Sukairi, Mohd Razip Asaruddin* Department of Chemistry, Faculty of Resource Science and Technology, Universiti Malaysia Sarawak, 94300 Kota Samarahan, Sarawak, Malaysia
Abstract Nypa fruticans Wurmb belongs to the family of Arecaceae and one of the most widely distributed palms in the mangrove forests in the Southeast Asia. Nypa palm sugar or known as “Gula Apong” are expected to benefit wider community as a substitute sweetener. In this study, phytochemical screening, antioxidant activity and alpha-amylase inhibitory activity of water (H2O) extract, methanol (MeOH) extract, ethanol (EtOH) extract and ethyl acetate (EtOAc) extract were conducted. Phytochemical screening of H2O extract, MeOH extract and EtOH extract indicated the presence of saponin, terpenoids, phenol, flavonoids and tannins while EtOAc extract only shown the presence of terpenoid, phenol and tannin. The antioxidant activity of extracts was determined based on 2, 2-diphenyl-1-picryhydrazyl (DPPH). The EC50 value for H2O extract, MeOH extract and EtOH extract, EtOAc extract and ascorbic acid were 221.3 mg/mL, 576.0 mg/mL, 183.7 mg/mL, 1331.0 mg/mL and 28.6 mg/mL respectively. The antidiabetic activity of nypa palm sugar extracts was determined by the inhibition of alpha-amylase. Based on the results, IC50 value for extracts were 545.1 mg/mL (H2O extract), 124.2 mg/mL (MeOH extract), 109.4 mg/mL (EtOH extract) and 44.37 mg/mL (acarbose). The results also revealed that EtOH extract exhibited highest inhibition compared to other extracts. The highest inhibition of the EtOH extract was 44.35% which observed at a concentration of 100 mg/mL. © 2019 Elsevier Ltd. All rights reserved. Selection and peer-review under responsibility of the scientific committee of the International Conference on Chemical Sciences and Engineering: Advance and New Materials, ICCSE 2018. Keywords: Nypa fruticans Wurmb; nypa palm sugar; phytochemical screening; DPPH; alpha-amylase
* Corresponding author. Tel.: +6017-9325604. E-mail address: [email protected]; [email protected] 2214-7853 © 2019 Elsevier Ltd. All rights reserved. Selection and peer-review under responsibility of the scientific committee of the International Conference on Chemical Sciences and Engineering: Advance and New Materials, ICCSE 2018.
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1. Introduction Nypa fruticans Wurmb belongs to the Arecaceae family, grow in brackish water. It widely distributed India, Thailand, Myanmar, Malaysia, Philippines and Northern Australia [1]. N. fruticans sap used as raw materials and cooked for several hours to make N. fruticans sugar. Phytochemical is defined as a non-nutrient that produced by plants. The plants can produce several bioactive constituents which can protect against the free radical damage [2]. The primary constituents consist of carbohydrates, chlorophylls and simple sugar, while secondary constituents consist of alkaloids, saponins, flavonoids, steroids, tannins and phenolic compounds [3]. Antioxidants are defined as a compound that can inhibit or delay the inhibition of oxidizable products by scavenging free radicals [4]. 2, 2diphenyl-1-picrylhydrazyl (DPPH) is a stable free radical which comprised an unpaired valence at one atom of nitrogen bridge. Thus, DPPH will accept an electron or hydrogen radical to become a stable diamagnetic molecule which widely used in radical scavenging activity. Diabetes mellitus (DM) can be divided into two different types which are Type 1 diabetes (insulin-dependent diabetes) or also known as juvenile-onset diabetes and Type 2 diabetes (non-insulin dependent diabetes). Type 2 diabetes is the most common form of diabetes in the world. One of the treatments of DM is by inhibiting the converting enzymes which are α-amylase [5]. α-amylase is a protein enzyme which hydrolyzes linked polysaccharides such as starch and glycogen to yield simple sugars such as maltose and glucose. This study aims to carry out phytochemical screening of some extracts of N. fruticans sugar (H2O, MeOH, EtOH and EtOAc extract), to determine the DPPH radical scavenging activity of extracts and lastly to find out the inhibitory activity of alpha-amylase on extracts. 2. Materials and Methods 2.1 Chemicals Reagents and chemicals were purchased from Sigma Aldrich (methanol and alpha-amylase enzyme), R&M Chemicals (ethanol, hydrochloric acid, chloroform and 3,5-dinitrosalicylic acid), HmbG Chemicals (ethyl acetate and sulphuric acid), Alfa Caesar (DPPH), Abacus 50 (acarbose) and ChemAR (ascorbic acid). 2.2 Instruments The solvents were concentrated to dryness using a rotatory evaporator (BUCHI model B-490) and UltravioletVisible (UV-Vis) Spectrophotometer (Jasco V-630) was used to determine the absorbance of antioxidant and antidiabetic activity. 2.3 Sample Collection and Sample Preparation Nypa fruticans sugar was supplied by local producer from Beladin, Sarawak. About 2 kg of sample was extracted by using methanol, ethanol, ethyl acetate and water for three days at room temperature. The extracts were filtered using Advantec filter paper (150 mm) into a conical flask. The filtrates were evaporated to dryness using rotatory evaporator. All the extracts which MeOH, EtOH, EtOAc and H20 extracts were stored in refrigerator. 2.4 Phytochemical screening 2.4.1 Test for saponin About 4 mL of distilled water was added to 1 mL of crude extracts (MeOH, EtOH, EtOAc and H20 extracts). The mixture of extract and distilled water was shaken vigorously and observed for 10 minutes. A stable foam persists for 10 minutes indicate the positive result for saponin [6].
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2.4.2 Test for Tannins Exactly 1.0 g of extracts were taken in 20 mL of warm distilled water and filtered using filter paper. 1 mL of 1% FeCl3 was added into 5 mL of filtrate. The formation of green or black colour indicates the presence of tannins [7]. 2.4.3 Test for Phlobatannins About 2 mL of extracts was added with 2 mL of 1% HCl and boiled with the help of hot plate stirrer. Red-coloured precipitate indicates the presence of phlobatannins [8]. 2.4.4 Test for Terpenoids 0.5 g of extract was dissolved in 10 mL of distilled water and filtered using filter paper. Exactly 2 mL of chloroform and 3 mL of concentrated H2SO4 were added into 5 mL of aqueous plant extract. The formation of reddish-brown color indicates the presence of terpenoids [9]. 2.4.5 Test for Flavonoid About 10 mL of distilled water was added into 0.5 g of each extract. Then, 5 mL of dilute ammonia and 1 mL of concentrated H2SO4 were added into the aqueous filtrate of extracts. The presence of flavonoid was determined by the formation of a yellow colour [10]. 2.4.6 Test for Phenolic Compound Exactly 0.5 g of extract was added to 5 mL of distilled water. A few drops of 5% FeCl3 were added to the solution. Dark green or black colour indicates the presence of the phenolic compound [11]. 2.5 Biological assay 2.5.1 DPPH radical scavenging assay The DPPH was used to determine the antioxidant activity of the extracts with some adjustments [12]. 6 mg of extract was dissolved in 6 mL of methanol and 20 mg of DPPH was dissolved in 100 mL of methanol. The stock solution was prepared with different concentrations by transferring 10, 30, 50, 100, 300 and 500 μL of sample into vials. Exactly 0.8 mL of methanol was added into diluted solutions of stock solutions for each vial and mixed with 0.5 mL of DPPH solution and incubated in the dark room for 60 minutes. The control solution was prepared by mixing 1.8 mL of methanol and 0.5 mL of DPPH solution. Ascorbic acid was used in this study as a positive control. The absorbance of the mixture was recorded at 517 nm. The DPPH radical scavenging activity is expressed as a percentage of inhibition (%) and it can be calculated as the equation below:
𝑃𝑒𝑟𝑐𝑒𝑛𝑡𝑎𝑔𝑒 𝑜𝑓 𝑖𝑛𝑖ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 % =
𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝐶𝑜𝑛𝑡𝑟𝑜𝑙 − 𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝑆𝑎𝑚𝑝𝑙𝑒 𝑥 100% 𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝐶𝑜𝑛𝑡𝑟𝑜𝑙
2.5.2 Alpha amylase inhibitory activity Alpha-amylase enzyme was used to determine the inhibitory activity of N. fruticans sugar. This inhibition assay was done by using the method proposed with some modification [13]. The sample was prepared by mixing 10 mg of extracts and 10 mL of distilled water with different concentrations which 20, 40, 60, 80 and 100 mg/mL. Acarbose as a positive control was prepared by dissolving 10 mg of acarbose tablet in 10 mL of distilled water. Exactly 200 μL of α-amylase enzyme and 400 μL of 1% starch solution were added into samples and incubated at 36.5 °C for 20 minutes.
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After 20 minutes, the reaction was stopped using 800 μL of 3,5-dinitrosalicylic acid (DNS) reagent and placed into boiling water for 5 minutes and cooled for 3 minutes. The samples were diluted with distilled water and the absorbance of the mixture was measured at 530 nm by using UV-Vis Spectrophotometer. The blank control was prepared by mixing of 1 mL distilled water, 400 μL of 1% of starch solution and 200 μL α-amylase enzyme. The percentage of inhibition was calculated based on the equation below:
𝑃𝑒𝑟𝑐𝑒𝑛𝑡𝑎𝑔𝑒 𝑜𝑓 𝑖𝑛𝑖ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 % =
𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝐶𝑜𝑛𝑡𝑟𝑜𝑙 − 𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝑆𝑎𝑚𝑝𝑙𝑒 𝑥 100% 𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝐶𝑜𝑛𝑡𝑟𝑜𝑙
3. Results 3.1 Phytochemical screening Based on the phytochemical screening analysis conducted, the results of the chemical constituents were summarized in Table 1 below: Table 1: Results for phytochemical screening of Nypa fruticans extracts Test
MeOH
EtOH
extract
extract
EtOAc
H20 extract
extract
Saponin
+
+
-
+
Tannins
+
+
+
+
Phlobatannins
-
-
-
-
Terpenoids
+
+
+
+
Flavonoid
+
+
-
+
Phenolic Compound
+
+
+
+
(+): Presence of constituents, (-): Absence of constituents
3.2 Biological Assay 3.2.1 DPPH Radical Scavenging Assay Table 2 shows the result for the percentage inhibition and EC50 value for the DPPH radical scavenging assay. Figure 1 shows the percentage of restraint against the log concentration of N. fruticans sugar extracts. Table 2: Percentage inhibition and EC50 of Nypa fruticans sugar extracts Compound concentration (ppm)
Percentage Inhibition (%) H2O MeOH EtOH
EtOAc
Ascorbic acid
10 30 50 100 300 500
2.46 5.65 16.95 26.04 55.77 77.40
0.12 3.03 3.34 3.45 24.52 24.64
95.90 96.36 96.81 96.13 95.44 95.22
8.10 15.72 18.43 22.60 38.08 50.12
13.51 13.76 24.82 35.14 63.63 69.04
EC50 (mg/mL) H2O MeOH
EtOH
EtOAc
Ascorbic acid
221.3
183.7
1331.0
28.6
576.0
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Figure 1: The percentage of inhibition against log concentration of extracts.
3.2.2
Alpha-amylase inhibitory activity
The percentage inhibition of α-amylase of the extracts against log concentration of extracts was plotted in comparison with the acarbose as shown in Figure 2. Table 3 shows the result for the percentage of inhibition and IC50 value for each of the extract. Table 3: The percentage inhibition and IC50 of N. fruticans sugar extracts Compound concentration (ppm)
H2O
Percentage Inhibition (%) MeOH EtOH Acarbose
20
1.02
5.34
3.08
25.40
40
2.05
11.09
9.03
37.47
60
3.08
17.45
19.30
67.45
80
3.90
31.62
34.29
72.69
100
7.19
40.04
44.35
80.90
H2O
MeOH
545.1
124.2
IC50 (mg/mL) EtOH
109.4
Figure 2: The percentage inhibition of α-amylase against log concentration of extracts and acarbose.
Acarbose
44.37
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4. Discussion Phytochemical screening analysis for different extracts of N. fruticans sugar showed the presence of active constituents as saponins, tannins, terpenoids, flavonoids and phenolic compound as shown in Table 1. All the extracts including MeOH, EtOH, EtOAc and H2O extract gave a negative result for the phlobatannins. Flavonoids and phenols as bioactive constituents are considered as good antioxidant and anticancer [14]. The dietary intake of flavonoids and phenols also can lead to protect human being from non-communicable (NCD) diseases such as cardiovascular diseases and cancer [15]. DPPH radical scavenging assay was performed for H2O, MeOH, EtOH and EtOAc extract. The percentage of extracts were calculated to determine the scavenging activity. It can be observed by reaction of DPPH with the antioxidant compound in extracts. Table 2 shows the percentage of inhibition and EC50 value for extracts of N. fruticans sugar. Based on the result, EtOH extracts showed the most potent antioxidant properties compare to other extracts. Also, flavonoids constituents which exist in EtOH extract possessed great antioxidant properties through scavenging free radical [16]. Figure 1 shows the ability of extracts to scavenge DPPH radical compared to ascorbic acid. The higher the concentration of extracts, the higher the radical scavenging activity. DPPH radical, which forms a purple solution, turn to yellow
coloured is caused by the reaction with the antioxidant compound that can donate a hydrogen atom [17]. The degree of discolouration of the solution indicates the potential for radical scavenging activity [18]. The inhibitory activity of H2O, MeOH, EtOH extracts on alpha-amylase was conducted in this study. The result of percentage inhibition of extracts and the IC50 value shown in Table 3. Based on the result, at the maximum concentration (100 ppm), H2O, MeOH, EtOH extract were shown 7.19%, 40.04% and 44.35% of enzyme inhibition activity while IC50 valued 545.1 mg/mL, 124.2 mg/mL and 109.4 mg/mL. The extracts were compared with the positive control, acarbose, which inhibit 80.90% enzyme activity at maximum concentration (100 ppm) with the IC50 valued 44.37 mg/mL. Acarbose was used as a positive control as its potent alpha-amylase inhibitor. 5. Conclusion The phytochemical screening showed that N. fruticans sugar extracts contain active constituents as saponins, tannins, terpenoids, flavonoids and a phenolic compound. MeOH extract, EtOH extract and H2O extract showed the presence of saponins, tannins, terpenoids, flavonoids and a phenolic compound. EtOAc extract only revealed the presence of tannins, terpenoids and a phenolic compound. In DPPH radical scavenging activity and alpha-amylase inhibitory activity, EtOH extract showed the highest inhibition compare to other extracts which are 77.40% and 44.35% respectively. In conclusion, EtOH extract was the most potent in antioxidant and antidiabetic properties. Hence, further studies should be conducted to determine the ability of active constituents of N. fruticans sugar to other biological activity such as antimicrobial and anticancer activity. Acknowledgements My sincere gratitude goes to my family. I am also grateful to Dr. Mohd Razip Asaruddin, my supervisor who guided and advised me throughout my research and studies. Thank you for being a committed supervisor who helps me to understand my research. Other than that, I am also grateful to Universiti Malaysia Sarawak and Faculty of Resource Science and Technology (FRST) for making it possible for me to research the N. fruticans sugar. Lastly, I also would like to acknowledge Kampung Beladin for their supply of N. fruticans’s sugar. References [1] Joshi, L., Kanagaratnam, U. and Adhuri, D., 2006. Nypa fruticans: Useful but Forgotten in Mangrove. Reforestation Programs. Resilience, Rights and Resources: Two Years of Recovery from the tsunami in Coastal Zone Aceh (Indonesia). [2] Suffredini, I.B., Sader, H.S., Gonçalves, A.G., Reis, A.O., Gales, A.C., Varella, A.D. and Younes, R.N., 2004. Screening of antibacterial extracts from plants native to the Brazilian Amazon Rain Forest and Atlantic Forest. Brazilian journal of medical and biological research, 37(3), pp.379-384. R. T. Wang, “Title of Chapter,” in Classic Physiques, edited by R. B. Hamil (Publisher Name, Publisher City, 1999), pp. 212–213.
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[3] Kumar, A., Ilavarasan, R., Jayachandran T., Decaraman, M., Aravindhan, P., Padmanaban, N. and Krishnan, M.R.V. (2009). Phytochemical investigation on a tropical plant. Journal Nutritional., 8(1): 83-85. [4] Sulaiman, M., Tijani, H.I., Abubakar, B.M., Haruna, S., Hindatu, Y., Mohammed, J.N. and Idris, A., 2013. An overview of natural plant antioxidants: analysis and evaluation. Advances in Biochemistry, 1(4), pp.64-72. D. L. Davids, “Recovery effects in binary aluminum alloys,” Ph.D. thesis, Harvard University, 1998. [5] Ali, H., Houghton, P.J. and Soumyanath, A., 2006. α-Amylase inhibitory activity of some Malaysian plants used to treat diabetes; with reference to Phyllanthus amarus. Journal of ethnopharmacology, 107(3), pp.449-455. [6] Zohra, S.F., Meriem, B., Samira, S. and Muneer, M.A., 2012. Phytochemical screening and identification of some compounds from mallow. Journal Natural Product Plant Resources, 2(4), pp.512-6. [7] Edeoga, H.O., Okwu, D.E. and Mbaebie, B.O., 2005. Phytochemical constituents of some Nigerian medicinal plants. African journal of biotechnology, 4(7), pp.685-688. [8] Yadav, M., Chatterji, S., Gupta, S.K. and Watal, G., 2014. Preliminary phytochemical screening of six medicinal plants used in traditional medicine. International Journal Pharmacy and Pharmaceutical Sciences, 6(5), pp.539-542. [9] Wadood, A., Ghufran, M., Jamal, S.B., Naeem, M., Khan, A. and Ghaffar, R., 2013. Phytochemical analysis of medicinal plants occurring in local area of Mardan. Biochem Anal Biochem, 2(4), pp.1-4. [10] Ayoola, G.A., Coker, H.A., Adesegun, S.A., Adepoju-Bello, A.A., Obaweya, K., Ezennia, E.C. and Atangbayila, T.O., 2008. Phytochemical screening and antioxidant activities of some selected medicinal plants used for malaria therapy in Southwestern Nigeria. Tropical Journal of Pharmaceutical Research, 7(3), pp.1019-1024. [11] Mir, M.A., Sawhney, S.S. and Jassal, M.M.S., 2013. Qualitative and quantitative analysis of phytochemicals of Taraxacum officinale. Wudpecker Journal of Pharmacy and Pharmocology, 2(1), pp.001-005. [12] Mensor, L.L., Menezes, F.S., Leitão, G.G., Reis, A.S., Santos, T.C.D., Coube, C.S. and Leitão, S.G., 2001. Screening of Brazilian plant extracts for antioxidant activity by the use of DPPH free radical method. Phytotherapy research, 15(2), pp.127-130. [13] Nair, S. S., Kavrekar, V., & Mishra, A. (2013). In vitro studies on alpha amylase and alpha glucosidase inhibitory activities of selected plant extracts. European Journal of Experimental Biology, 3(1), 128-132. [14] Al Aboody, M.S., 2015. In vitro screening of Phytochemical, Antibacterial and Antioxidant activities of Rumex vesicarius L. International Journal Current Microbiology. Applied Sciences 4(9), pp.884-893. [15] Eman, A.A., 2011. Phytochemical screening on different plant parts of some succulent plants of Egypt. New York Sci J., 4, pp.15-18. [16] Harborne, J.B. and Williams, C.A., 2000. Advances in flavonoid research since 1992. Phytochemistry, 55(6), pp.481-504. [17] Kedare, S.B. and Singh, R.P., 2011. Genesis and development of DPPH method of antioxidant assay. Journal of food science and technology, 48(4), pp.412-422. [18] Prasad, N., Yang, B., Kong, K.W., Khoo, H.E., Sun, J., Azlan, A., Ismail, A. and Romli, Z.B., 2013. Phytochemicals and antioxidant capacity from Nypa fruticans Wurmb. fruit. Evidence-Based Complementary and Alternative Medicine, 2013.
ScienceDirect Materials Today: Proceedings 19 (2019) 1745–1751
www.materialstoday.com/proceedings
ICCSE 2018
Phytochemical Screening, Determination of Antioxidant Activity and Alpha-Amylase Inhibitory Activity of Nypa fruticans Sugar Wan Mazliena Aliana Wan Sabri, Syed Ahmad Tarmizi Wan Yusop, Ahmad Hafizi Sukairi, Mohd Razip Asaruddin* Department of Chemistry, Faculty of Resource Science and Technology, Universiti Malaysia Sarawak, 94300 Kota Samarahan, Sarawak, Malaysia
Abstract Nypa fruticans Wurmb belongs to the family of Arecaceae and one of the most widely distributed palms in the mangrove forests in the Southeast Asia. Nypa palm sugar or known as “Gula Apong” are expected to benefit wider community as a substitute sweetener. In this study, phytochemical screening, antioxidant activity and alpha-amylase inhibitory activity of water (H2O) extract, methanol (MeOH) extract, ethanol (EtOH) extract and ethyl acetate (EtOAc) extract were conducted. Phytochemical screening of H2O extract, MeOH extract and EtOH extract indicated the presence of saponin, terpenoids, phenol, flavonoids and tannins while EtOAc extract only shown the presence of terpenoid, phenol and tannin. The antioxidant activity of extracts was determined based on 2, 2-diphenyl-1-picryhydrazyl (DPPH). The EC50 value for H2O extract, MeOH extract and EtOH extract, EtOAc extract and ascorbic acid were 221.3 mg/mL, 576.0 mg/mL, 183.7 mg/mL, 1331.0 mg/mL and 28.6 mg/mL respectively. The antidiabetic activity of nypa palm sugar extracts was determined by the inhibition of alpha-amylase. Based on the results, IC50 value for extracts were 545.1 mg/mL (H2O extract), 124.2 mg/mL (MeOH extract), 109.4 mg/mL (EtOH extract) and 44.37 mg/mL (acarbose). The results also revealed that EtOH extract exhibited highest inhibition compared to other extracts. The highest inhibition of the EtOH extract was 44.35% which observed at a concentration of 100 mg/mL. © 2019 Elsevier Ltd. All rights reserved. Selection and peer-review under responsibility of the scientific committee of the International Conference on Chemical Sciences and Engineering: Advance and New Materials, ICCSE 2018. Keywords: Nypa fruticans Wurmb; nypa palm sugar; phytochemical screening; DPPH; alpha-amylase
* Corresponding author. Tel.: +6017-9325604. E-mail address: [email protected]; [email protected] 2214-7853 © 2019 Elsevier Ltd. All rights reserved. Selection and peer-review under responsibility of the scientific committee of the International Conference on Chemical Sciences and Engineering: Advance and New Materials, ICCSE 2018.
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1. Introduction Nypa fruticans Wurmb belongs to the Arecaceae family, grow in brackish water. It widely distributed India, Thailand, Myanmar, Malaysia, Philippines and Northern Australia [1]. N. fruticans sap used as raw materials and cooked for several hours to make N. fruticans sugar. Phytochemical is defined as a non-nutrient that produced by plants. The plants can produce several bioactive constituents which can protect against the free radical damage [2]. The primary constituents consist of carbohydrates, chlorophylls and simple sugar, while secondary constituents consist of alkaloids, saponins, flavonoids, steroids, tannins and phenolic compounds [3]. Antioxidants are defined as a compound that can inhibit or delay the inhibition of oxidizable products by scavenging free radicals [4]. 2, 2diphenyl-1-picrylhydrazyl (DPPH) is a stable free radical which comprised an unpaired valence at one atom of nitrogen bridge. Thus, DPPH will accept an electron or hydrogen radical to become a stable diamagnetic molecule which widely used in radical scavenging activity. Diabetes mellitus (DM) can be divided into two different types which are Type 1 diabetes (insulin-dependent diabetes) or also known as juvenile-onset diabetes and Type 2 diabetes (non-insulin dependent diabetes). Type 2 diabetes is the most common form of diabetes in the world. One of the treatments of DM is by inhibiting the converting enzymes which are α-amylase [5]. α-amylase is a protein enzyme which hydrolyzes linked polysaccharides such as starch and glycogen to yield simple sugars such as maltose and glucose. This study aims to carry out phytochemical screening of some extracts of N. fruticans sugar (H2O, MeOH, EtOH and EtOAc extract), to determine the DPPH radical scavenging activity of extracts and lastly to find out the inhibitory activity of alpha-amylase on extracts. 2. Materials and Methods 2.1 Chemicals Reagents and chemicals were purchased from Sigma Aldrich (methanol and alpha-amylase enzyme), R&M Chemicals (ethanol, hydrochloric acid, chloroform and 3,5-dinitrosalicylic acid), HmbG Chemicals (ethyl acetate and sulphuric acid), Alfa Caesar (DPPH), Abacus 50 (acarbose) and ChemAR (ascorbic acid). 2.2 Instruments The solvents were concentrated to dryness using a rotatory evaporator (BUCHI model B-490) and UltravioletVisible (UV-Vis) Spectrophotometer (Jasco V-630) was used to determine the absorbance of antioxidant and antidiabetic activity. 2.3 Sample Collection and Sample Preparation Nypa fruticans sugar was supplied by local producer from Beladin, Sarawak. About 2 kg of sample was extracted by using methanol, ethanol, ethyl acetate and water for three days at room temperature. The extracts were filtered using Advantec filter paper (150 mm) into a conical flask. The filtrates were evaporated to dryness using rotatory evaporator. All the extracts which MeOH, EtOH, EtOAc and H20 extracts were stored in refrigerator. 2.4 Phytochemical screening 2.4.1 Test for saponin About 4 mL of distilled water was added to 1 mL of crude extracts (MeOH, EtOH, EtOAc and H20 extracts). The mixture of extract and distilled water was shaken vigorously and observed for 10 minutes. A stable foam persists for 10 minutes indicate the positive result for saponin [6].
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2.4.2 Test for Tannins Exactly 1.0 g of extracts were taken in 20 mL of warm distilled water and filtered using filter paper. 1 mL of 1% FeCl3 was added into 5 mL of filtrate. The formation of green or black colour indicates the presence of tannins [7]. 2.4.3 Test for Phlobatannins About 2 mL of extracts was added with 2 mL of 1% HCl and boiled with the help of hot plate stirrer. Red-coloured precipitate indicates the presence of phlobatannins [8]. 2.4.4 Test for Terpenoids 0.5 g of extract was dissolved in 10 mL of distilled water and filtered using filter paper. Exactly 2 mL of chloroform and 3 mL of concentrated H2SO4 were added into 5 mL of aqueous plant extract. The formation of reddish-brown color indicates the presence of terpenoids [9]. 2.4.5 Test for Flavonoid About 10 mL of distilled water was added into 0.5 g of each extract. Then, 5 mL of dilute ammonia and 1 mL of concentrated H2SO4 were added into the aqueous filtrate of extracts. The presence of flavonoid was determined by the formation of a yellow colour [10]. 2.4.6 Test for Phenolic Compound Exactly 0.5 g of extract was added to 5 mL of distilled water. A few drops of 5% FeCl3 were added to the solution. Dark green or black colour indicates the presence of the phenolic compound [11]. 2.5 Biological assay 2.5.1 DPPH radical scavenging assay The DPPH was used to determine the antioxidant activity of the extracts with some adjustments [12]. 6 mg of extract was dissolved in 6 mL of methanol and 20 mg of DPPH was dissolved in 100 mL of methanol. The stock solution was prepared with different concentrations by transferring 10, 30, 50, 100, 300 and 500 μL of sample into vials. Exactly 0.8 mL of methanol was added into diluted solutions of stock solutions for each vial and mixed with 0.5 mL of DPPH solution and incubated in the dark room for 60 minutes. The control solution was prepared by mixing 1.8 mL of methanol and 0.5 mL of DPPH solution. Ascorbic acid was used in this study as a positive control. The absorbance of the mixture was recorded at 517 nm. The DPPH radical scavenging activity is expressed as a percentage of inhibition (%) and it can be calculated as the equation below:
𝑃𝑒𝑟𝑐𝑒𝑛𝑡𝑎𝑔𝑒 𝑜𝑓 𝑖𝑛𝑖ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 % =
𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝐶𝑜𝑛𝑡𝑟𝑜𝑙 − 𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝑆𝑎𝑚𝑝𝑙𝑒 𝑥 100% 𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝐶𝑜𝑛𝑡𝑟𝑜𝑙
2.5.2 Alpha amylase inhibitory activity Alpha-amylase enzyme was used to determine the inhibitory activity of N. fruticans sugar. This inhibition assay was done by using the method proposed with some modification [13]. The sample was prepared by mixing 10 mg of extracts and 10 mL of distilled water with different concentrations which 20, 40, 60, 80 and 100 mg/mL. Acarbose as a positive control was prepared by dissolving 10 mg of acarbose tablet in 10 mL of distilled water. Exactly 200 μL of α-amylase enzyme and 400 μL of 1% starch solution were added into samples and incubated at 36.5 °C for 20 minutes.
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After 20 minutes, the reaction was stopped using 800 μL of 3,5-dinitrosalicylic acid (DNS) reagent and placed into boiling water for 5 minutes and cooled for 3 minutes. The samples were diluted with distilled water and the absorbance of the mixture was measured at 530 nm by using UV-Vis Spectrophotometer. The blank control was prepared by mixing of 1 mL distilled water, 400 μL of 1% of starch solution and 200 μL α-amylase enzyme. The percentage of inhibition was calculated based on the equation below:
𝑃𝑒𝑟𝑐𝑒𝑛𝑡𝑎𝑔𝑒 𝑜𝑓 𝑖𝑛𝑖ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 % =
𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝐶𝑜𝑛𝑡𝑟𝑜𝑙 − 𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝑆𝑎𝑚𝑝𝑙𝑒 𝑥 100% 𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝐶𝑜𝑛𝑡𝑟𝑜𝑙
3. Results 3.1 Phytochemical screening Based on the phytochemical screening analysis conducted, the results of the chemical constituents were summarized in Table 1 below: Table 1: Results for phytochemical screening of Nypa fruticans extracts Test
MeOH
EtOH
extract
extract
EtOAc
H20 extract
extract
Saponin
+
+
-
+
Tannins
+
+
+
+
Phlobatannins
-
-
-
-
Terpenoids
+
+
+
+
Flavonoid
+
+
-
+
Phenolic Compound
+
+
+
+
(+): Presence of constituents, (-): Absence of constituents
3.2 Biological Assay 3.2.1 DPPH Radical Scavenging Assay Table 2 shows the result for the percentage inhibition and EC50 value for the DPPH radical scavenging assay. Figure 1 shows the percentage of restraint against the log concentration of N. fruticans sugar extracts. Table 2: Percentage inhibition and EC50 of Nypa fruticans sugar extracts Compound concentration (ppm)
Percentage Inhibition (%) H2O MeOH EtOH
EtOAc
Ascorbic acid
10 30 50 100 300 500
2.46 5.65 16.95 26.04 55.77 77.40
0.12 3.03 3.34 3.45 24.52 24.64
95.90 96.36 96.81 96.13 95.44 95.22
8.10 15.72 18.43 22.60 38.08 50.12
13.51 13.76 24.82 35.14 63.63 69.04
EC50 (mg/mL) H2O MeOH
EtOH
EtOAc
Ascorbic acid
221.3
183.7
1331.0
28.6
576.0
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Figure 1: The percentage of inhibition against log concentration of extracts.
3.2.2
Alpha-amylase inhibitory activity
The percentage inhibition of α-amylase of the extracts against log concentration of extracts was plotted in comparison with the acarbose as shown in Figure 2. Table 3 shows the result for the percentage of inhibition and IC50 value for each of the extract. Table 3: The percentage inhibition and IC50 of N. fruticans sugar extracts Compound concentration (ppm)
H2O
Percentage Inhibition (%) MeOH EtOH Acarbose
20
1.02
5.34
3.08
25.40
40
2.05
11.09
9.03
37.47
60
3.08
17.45
19.30
67.45
80
3.90
31.62
34.29
72.69
100
7.19
40.04
44.35
80.90
H2O
MeOH
545.1
124.2
IC50 (mg/mL) EtOH
109.4
Figure 2: The percentage inhibition of α-amylase against log concentration of extracts and acarbose.
Acarbose
44.37
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4. Discussion Phytochemical screening analysis for different extracts of N. fruticans sugar showed the presence of active constituents as saponins, tannins, terpenoids, flavonoids and phenolic compound as shown in Table 1. All the extracts including MeOH, EtOH, EtOAc and H2O extract gave a negative result for the phlobatannins. Flavonoids and phenols as bioactive constituents are considered as good antioxidant and anticancer [14]. The dietary intake of flavonoids and phenols also can lead to protect human being from non-communicable (NCD) diseases such as cardiovascular diseases and cancer [15]. DPPH radical scavenging assay was performed for H2O, MeOH, EtOH and EtOAc extract. The percentage of extracts were calculated to determine the scavenging activity. It can be observed by reaction of DPPH with the antioxidant compound in extracts. Table 2 shows the percentage of inhibition and EC50 value for extracts of N. fruticans sugar. Based on the result, EtOH extracts showed the most potent antioxidant properties compare to other extracts. Also, flavonoids constituents which exist in EtOH extract possessed great antioxidant properties through scavenging free radical [16]. Figure 1 shows the ability of extracts to scavenge DPPH radical compared to ascorbic acid. The higher the concentration of extracts, the higher the radical scavenging activity. DPPH radical, which forms a purple solution, turn to yellow
coloured is caused by the reaction with the antioxidant compound that can donate a hydrogen atom [17]. The degree of discolouration of the solution indicates the potential for radical scavenging activity [18]. The inhibitory activity of H2O, MeOH, EtOH extracts on alpha-amylase was conducted in this study. The result of percentage inhibition of extracts and the IC50 value shown in Table 3. Based on the result, at the maximum concentration (100 ppm), H2O, MeOH, EtOH extract were shown 7.19%, 40.04% and 44.35% of enzyme inhibition activity while IC50 valued 545.1 mg/mL, 124.2 mg/mL and 109.4 mg/mL. The extracts were compared with the positive control, acarbose, which inhibit 80.90% enzyme activity at maximum concentration (100 ppm) with the IC50 valued 44.37 mg/mL. Acarbose was used as a positive control as its potent alpha-amylase inhibitor. 5. Conclusion The phytochemical screening showed that N. fruticans sugar extracts contain active constituents as saponins, tannins, terpenoids, flavonoids and a phenolic compound. MeOH extract, EtOH extract and H2O extract showed the presence of saponins, tannins, terpenoids, flavonoids and a phenolic compound. EtOAc extract only revealed the presence of tannins, terpenoids and a phenolic compound. In DPPH radical scavenging activity and alpha-amylase inhibitory activity, EtOH extract showed the highest inhibition compare to other extracts which are 77.40% and 44.35% respectively. In conclusion, EtOH extract was the most potent in antioxidant and antidiabetic properties. Hence, further studies should be conducted to determine the ability of active constituents of N. fruticans sugar to other biological activity such as antimicrobial and anticancer activity. Acknowledgements My sincere gratitude goes to my family. I am also grateful to Dr. Mohd Razip Asaruddin, my supervisor who guided and advised me throughout my research and studies. Thank you for being a committed supervisor who helps me to understand my research. Other than that, I am also grateful to Universiti Malaysia Sarawak and Faculty of Resource Science and Technology (FRST) for making it possible for me to research the N. fruticans sugar. Lastly, I also would like to acknowledge Kampung Beladin for their supply of N. fruticans’s sugar. References [1] Joshi, L., Kanagaratnam, U. and Adhuri, D., 2006. Nypa fruticans: Useful but Forgotten in Mangrove. Reforestation Programs. Resilience, Rights and Resources: Two Years of Recovery from the tsunami in Coastal Zone Aceh (Indonesia). [2] Suffredini, I.B., Sader, H.S., Gonçalves, A.G., Reis, A.O., Gales, A.C., Varella, A.D. and Younes, R.N., 2004. Screening of antibacterial extracts from plants native to the Brazilian Amazon Rain Forest and Atlantic Forest. Brazilian journal of medical and biological research, 37(3), pp.379-384. R. T. Wang, “Title of Chapter,” in Classic Physiques, edited by R. B. Hamil (Publisher Name, Publisher City, 1999), pp. 212–213.
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