August 30, 2020
Archives • 2020 • vol.2 • 163-174
PHYTOCHEMICAL SCREENING AND ANTIOXIDANT PROFILING OF TWO MANGROVE SPECIES OF SUNDARBANS: Heritiera fomes and Sonneratia apetala Sarkar Kumar, Bidduth1*; Sarkar Kumar, Barno2; Das, Joydeep3; Modak, Prema4; Das, Ananya4; Halder, Satyajit4; Islam, Farhana5; Chowdhury Rani, Anita6; Kundu Kumar, Sukalyan4
1Department of Pharmacy, Ranada Prasad Shaha University, Narayanganj, Bangladesh 2Faridpur Medical College Hospital, Faridpur, Bangladesh 3Department of Zoology, Charuchandra College, University of Calcutta, Kolkata, India 4Department of Pharmacy, Jahangirnagar University, Savar, Dhaka- 1342, Bangladesh 5Department of Pharmacy, Jashore University of Science and Technology, Jashore, Bangladesh 6Department of Pharmacy, Jagannath University, Dhaka, Bangladesh
Abstract In search of prevention and cure of diseases, human have relied on plant kingdom from the very beginning of civilization. Mangrove forest has become an endless source of numerous medicinal plants from the very beginning. This present study was aimed to investigate phytochemicals present in methanolic leaf extracts of two mangrove species: Heritiera fomes and Sonneratia apetala. In this study both leaf extracts showed presence of important phytoconstituents namely: carbohydrate, alkaloid, tannin, steroid and many more. Besides assessment of DPPH free radical scavenging acitivity, total phenolic content, total flavonoid content and total antioxidant activity were done to evaluate the antioxidant potential of the selected leaf extracts. This investigation proved better antioxidant potential of Heritiera fomes than Sonneratia apetala in all experiments. Both of this plants can be great sources of novel phytochemicals with medicinal value demanding more extensive research works in future. Keywords: Medicinal plant, mangrove forest, phytochemicals, methanolic leaf extract, antioxidant
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Introduction is used for diabetes and goiter in rural Plants and natural products are used by areas. This plant is also used to cure pain people for century as food and medicines and fever in local areas [5]. Mahmud et al., for cure and prevention of diseases [1]. At (2014) [5] claimed about its significant present, more than 80% of the world antioxidant and antidiabetic property. Due population depends on traditional and to the presence of high amount of plant derived medicine. In the last century, procyanidins, Heritiera fomes can act both roughly 121 pharmaceutical products were as radical scavenger and 15-lipoxygenase formulated from traditional herbal sources inhibitor. [2]. Pharmacologists, microbiologists, Sonneratia apetala Buch.-Ham., abundantly biochemist, botanists, and natural- grown in the coastal areas in India, products chemists all over the world are Bangladesh, Malaysia, Australia, etc, is a currently investigating medicinal plants for fast growing mangrove of 7.2 m high with phytochemicals and lead compounds that quadrangular branches. According to could be developed for treatment of Bandaranayake (1998) [6], fruits and barks various diseases [3]. of the plants belonging to genus Mangroves are assemblages of halophytic Sonneratia have remedial activity against woody covering about 75% of the world’s asthma, febrifuge, ulcers, swellings, tropical coastline [4]. Their distinctive sprains, bleeding, and hemorrhages. There ecological behavior, morphology, and are few reports on the antibacterial and traditional uses made researchers to work antioxidant activities of fruit of S. apetala on them. Usually mangroves are rich in [7] and other Sonneratia species [8]. polyphenols and tannins. Mangrove leaves Hossain et al., (2013) [7] reported that S. contain phenols and flavonoids that serve apetala fruit extracts have antioxidant, as ultraviolet (UV) screen compounds. antidiabetic and antibacterial activities. Substances in mangroves have long been Jaimini et al. (2011) [9] determined the used in folk medicines to treat diseases as antibacterial potential of S. apetala leave they significant activity against animal, extracts. Also, Patra et al. (2015) [10] human, and plant viruses including human focused on both S. apetala leaf and bark immunodeficiency virus [5] extracts and they concluded that the extracts have potent antibacterial, The mangrove forest Sundarbans is antioxidant, antidiabetic and anticancer named after the tree Heritiera fomes L. properties. Hossain et al., (2013) [7] found (local name Sundri). It is an evergreen antihyperglycemic activity of seeds and medium sized tree, growing up to 25m in pericarps of S. apetala fruits in STZ induced height. This species is a well-known diabetic mice. So this study investigated mangrove plant for its significant for major phytochemicals of the crude traditional use(s) by the local traditional extract and ended up with assessment of health practitioners against various antioxidative acitivity of them. diseases in the southern areas of Bangladesh. H. fomes leaves, roots, and stems are used by rural people for the treatment of gastrointestinal disorders, skin diseases, and hepatic disorders. Bark
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Method apetala (Kaora) were collected from Place of study: This study was carried out Mangrove forest Sundarban (Karamjol at Natural Product Research Laboratory of area) which grow prominently throughout Department of Pharmacy, Jahangirnagar that area. They were referenced by skilled University, Savar, Dhaka- 1342. botanist (DACB- 54665 for H. fomes and DACB- 54904 for S. apetala. Collection of Plant Leaves:Fresh leaves of Heretiera fomes (Sundri) and Sonneratia Preparation of leaf extract: The fresh leaves were washed separately methanol for 48 hours using Soxhlet and carefully with distilled water to apparatus. Then the efficient and gentle remove any extraneous materials. Then removal of solvents from samples was leaves were air dried under shade for 7 done by Rotary Evaporator and then the days then dried in the oven at 65 °C. And extract left behind was stored at 4 °C in a then the dried leaves were pulverized into refrigerator. coarse powder. About 1 kg of the each A. Preliminary photochemical screening: powder was extracted with 2.5 L of It involves testing of leaf extracts of get general idea about their presence in Heretiera fomes and Sonneratia apetala for crude drug. The qualitative chemical tests their contents of different classes of for various phytoconstituents were compounds [11]. Phytochemicals were carried out for all the extracts of Heretiera detected by qualitative chemical test to fomes and Sonneratia apetala separately. 1. Tests for Carbohydrate: Procedure: Two drops of molisch’s a) Molisch’s test (General test for reagent (10% alcoholic solution of α- Carbohydrates): A red or reddish violet naphthol) need to be added to 2ml of ring will be formed at the junction of the aqueous extract. 2ml of conc. sulfuric acid two layers if a carbohydrate is present. On is allowed to flow down the side of the standing or shaking a dark purple solution inclined test tube so that the acid forms a will form. layer beneath the aqueous solution. b) Barfoed’s test (General test for cuprous oxide will be formed within 2 Monosaccharides): Red precipitate of minutes if a monosaccharide is present. Procedure: 1 ml of an aqueous extract of of Fehling’s solutions A and B then the plant material is added to 1 ml of boiled for a few minutes. Test for Barfoed’s reagent in a test tube and heat Glycosides: A yellow color develops in in a beaker for boiling water. the presence of glycoside. c) Fehling’s test: A red or brick-red Procedure: A small amount of an alcoholic precipitate will be formed if a extract of the fresh or dried plant material reducing sugar is present. is Procedure: 2 ml of an aqueous dissolved in 1 ml of water and few drops of extract of the plant material is added aqueous sodium hydroxide solution are to 1ml of a mixture of equal volumes added in it.
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2. Test for glucosides: 3. Test for Tannins: A small amount of an alcoholic extract of A small quantity of the extract is boiled the plant material is dissolved in water with 5 ml of 45% solution of ethanol for 5 and alcohol, solution is divided into two minutes. Each of the mixture is cooled and portions and need to be treated in the filtered. The different filtrates are used for following ways: the following test: a) One of them is boiled with a mixture a) Lead acetate test: A yellow or red of equal volume of Fehling’s solution precipitate is formed A and B is boiled. Note any brick-red Procedure: 5 ml of aqueous extract of the precipitate. plant material is taken in a test tube and a
b) Other portion is boiled with a few few drops of a 1% solution of lead acetate drops of dilute Sulphuric acid for about are added. 5 minutes, neutralized the mixture with sodium hydroxide solution, an b) Ferric chloride test: Test solution gives equal volume of mixture of Fehling’s blue green color with ferric chloride solution A and B is added and then Procedure: Test solution is treated with boiled. ferric chloride solution. c) Alkaline reagent test: yellow to red Procedure: Test solution is treated with precipitate within short time sodium hydroxide solution . 4. Test for Alkaloids: Procedure: Iodine and Potassium iodide A small volume of each extract is are dissolved in water and the volume is neutralized by adding 1 or 2 drops of dilute made 100ml with distilled water. H2SO4. This neutralized solution is treated c) Dragendroff’s Reagent: Alkaloids give with a very small amount of the following reddish brown precipitate with reagents and the respective color and Dragendorff’s reagent. precipitate formation is observed: Procedure: Basic bismuth nitrate and a) Mayer’s reagent: Alkaloids give cream Tartaric acid are dissolved in water. This color precipitate with addition of Mayer's solution is mixed with a solution reagent. containing Potassium iodide and water. Procedure: Solution (a) is prepared by d) Hager’s reagent: Alkaloids give yellow dissolving mercuric chloride in distilled color precipitate with Hager's reagent water. Procedure: Neutralized solution of extract Solution (b) is prepared by dissolving is mixed with 1% solution of picric acid. potassium iodide in distilled water. e) Tannic acid test: Alkaloids give buff color Solution (a) and (b) are mixed and the precipitate with volume was adjusted to 100 ml with Procedure: Neutralized solution of extract distilled water. is mixed with 10% Tannic acid solution. b) Wagner’s reagent: Alkaloids give a reddish 5. Test for Flavonoids: brown precipitate with Wagner's reagent A small quantity of the extract is heated with 10 ml of ethyl acetate in boiling water
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for 3 minutes. The mixture is filtered and Procedure: The extract is mixed with 2 ml the filtrates are used for the following of chloroform and concentrated H2SO4 test. (3ml) carefully to form a layer. a) Ammonium Test: A yellow coloration in 8. Test for Steroids: ammonia layer indicates the presence of Liebermann- Burchard’s Test: A greenish the flavonoid. color is produced which turns blue on Procedure: The filtrate is shaken with 1 ml standing if a steroid is present of dilute ammonia solution (1%). The layers were allowed to separate. Procedure: A small amount of a petroleum b) Aluminum Chloride Test: The light yellow ether extract of the plant material is colour indicates the presence of flavonoid dissolved in 1 ml of chloroform then 2 ml and when dilute NaOH and HCl is added the of acetic anhydride is added with 1 ml of yellow solution turns colorless. conc. sulphuric acid. Procedure: The filtrates are shaken with 1 9. Fats & Fixed Oils ml of 1% aluminum chloride solution and a) Stain test: The stain on 1 filter paper observed for light yellow color. indicates the presence of fixed oils. 6. Test for Saponin: a) Frothing test: Production of a persistent Procedure: The small quantity of extract frothing (which remains stable in heating) is pressed between two filter papers. Procedure: About 0.5 ml of extract is b) Saponification test: The formation of shaken vigorously with water in a test soap or partial neutralization of alkali tube indicates the presence of Fixed oils and b) Haemolysis Test: Appearance of Fats. Hemolytic zone. Procedure: A few drops of 0.5 N of Procedure: Add leaves extract to one drop alcoholic potassium hydroxide is added to of blood placed on a glass slide. small quantities of various extracts along 7. Test for Triterpenoids: with a drop of Phenolphthalein separately Salkowski test: A reddish brown and heated on a water bath for 1-2 hrs. coloration in the interface. B. Determination of antioxidant activities scavenging of DPPH free radical 1. DPPH Free Radical Scavenging Assay [12] (neutralization) is indicated by the deep violet color being turned into pale yellow DPPH is a reactive free radical that acts as or colorless. an electron acceptor (oxidant/ oxidizing agent) and causes oxidation other The potential antioxidant activity of plant substances. On the other hand, extracts was determined on the basis of antioxidants act as electron donors the scavenging activity of the stable 1,1- (reductant/ reducing agent). Antioxidants diphenyl-2-picrylhydrazyl (DPPH) free neutralize DPPH by being oxidized radical. Aliquots of 30 µL of a methanolic themselves. DPPH is found as dark- solution containing leaf extract were colored crystalline powder composed of added to 3 mL of a 0.004% MeOH solution stable free-radical molecules and forms of DPPH. Absorbance at 517 nm was determined after 30 min, and the percent deep violet color in solution. The
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inhibition activity was calculated. IC50 with methanol, resulting in the blank values denote the concentration of solution. A second aliquot of the stock sample required to scavenge 50% DPPH solution was transferred to another 10.0 free radicals. mL volumetric flask, a volume of the 2% 2. Determination of Total Phenolics: [13] AlCl3 was added and made to volume 13 Folin- Ciocalteu reagent was used for with methanol, which was named test this determination. 2 mL of 80% methanol solution. After 25 min the absorbance of containing 1% hydrochloric acid was used the test solution was measured at 430 for extraction of 200 mg of sample at nm against blank solution. The results room temperature on an orbital shaker were expressed as the amount of set at 200 rpm for about 2 h. After flavonoid (mg)/g of plant extract.
centrifugation for 15 min at 1000g, the 4. Determination of Total Antioxidant supernatant was decanted into 4 mL Capacity: [15] An aliquot of 0.1 ml of sample solution vials. The pellets were also extracted at same conditions. Total phenolics assay containing a reducing species (in water, was done after combination of methanol, ethanol, dimethyl sulfoxide or supernatant. One hundred microliters of hexane) was combined in an Eppendroff extract was mixed with 0.75 mL of Folin- tube with 1 ml of reagent solution (0.6 M Ciocalteu reagent which was previously sulfuric acid, 28 mM sodium phosphate diluted 10-fold with distilled water. This and 4 mM ammonium molybdate). The mixture was allowed to stand at 22 °C for tubes were incubated in a thermal block at 95°C for 90 min after capping tightly. 5 min. After that, 0.75 mL of sodium bicarbonate (60 g/L) solution was added After the samples had cooled to room to the mixture. Absorbance was temperature, the absorbance of the measured at 725 nm after keeping the aqueous solution of each was measured mixture for 90 min at 22 °C. Results are at 695 nm against a blank. A blank expressed as gallic acid acid equivalents. solution was prepared using 1 ml of 3. Determination of Total Flavonoid reagent solution and same solvent as the Content: [14] sample and it was incubated under the An aliquot of the stock solution of the same conditions as the the samples. extract was transferred to a 10.0 mL Antioxidant capacities was expressed as volumetric flask and made to volume equivalents of ascorbic acid. Results scavenging assay extract has been shown A. Phytochemical Screening of Plant in Figure 02. Extracts: 2. Total Phenol Content of Extracts Findings of phytochemical screenings are Total phenolic content of the presented in Table 01 Heritiera fomes (HF) and Sonneratia apetala (SA) leaf extract and was B. Antioxidant Profiling of Plant Extracts: determined by using the Folin- 1. DPPH Free Radical Scavenging Ciocalteu reagent and were Capacity of Extracts expressed as Gallic acid equivalents
Comparison of IC50 values between HF and (GAE) per gram of plant extract. SA leaf extract in DPPH free radical The total phenolic contents of the
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test fractions were calculated using contain the greater amount of the standard curve of Gallic acid (y= phenols than methanolic leaf 0.009x + 0.058; R2 = 0.999 for HF extract of Sonneratia apetala and y= -0.0016x+ 0.4482; R2= (Figure 03). 0.6773). Methanol extract of 3. Total Flavonoid Content of Extracts Heritiera fomes leaf was found to Aluminium chloride colorimetric method for HF and y = 0.0048x + 0.1055 was used to determine the total flavonoid R² = 0.9977 for SA) and was expressed as contents of methanolic leaf extracts of quercetin equivalents (QE) per gram of Heritiera fomes (HF) and Sonneratia the plant extract. Methanol extract of apetala (SA). The total flavonoid content leaves of Heritiera fomes was found to was calculated using the standard curve of contain the highest amount of flavonoid quercetin (y = 0.005x - 0.005; R2 = 0.996 (Figure 04) 4.Total antioxidant capacity of extracts alkaloids, steroids, fats & fixed oil, Result: Total antioxidant capacity of flavonoids, tannins and carbohydrates. methanolic leaf extracts of Heritiera fomes These phytoconstituents have their own (HF) and Sonneratia apetala (SA) were medicinal values. Antioxidant profile of evaluated by the phosphomolybdenum this two plants is considerable and can be method and were expressed as ascorbic good choice to reduce oxidative stress in acid equivalents (AAE) per gram of plant different disease states of our body. More extract. Total antioxidant capacity of the detailed study must be done for farther isolation leading to the pure compounds test samples was calculated using the standard curve of ascorbic acid (y = 0.006x and quantitation of phytoconstituents + 0.101; R2= 0.991 for HF and y = -0.001x + leading to different intensive in vivo 1.574; studies. R² = 0.8852 for SA ) (Figure 4.28 and 4.29). Acknowledgements Methanolic leaf extract of Heritiera fomes We are grateful to Professor Pijus Saha, was found to possess the higher total Chairman of Department of Pharmacy, antioxidant capacity than Sonneratia Jahangirnagar University for his kind help apetala. Total antioxidant capacity of the and inspiration for doing this experiment. extracts was found to decrease in the We would also like to thank Vice- following order: HF> SA (Figure 05). Chancellor of Ranada Prasad Shaha Discussion University for his kind inspiration to The leaves of Heritiera fomes, Sonneratia publish this research articles. apetala contain phytoconstituets like International Journal of Pharmaceutical Science, 1(1), 1-20. References 2. Verma, S. and Singh, S.P. (2008). 1. Raghavendra, H.L., Yogesh, H.S., Current and future status of herbal Gopalakrishna, B., et al. (2009). An medicines, Veterinary World, 1(11): 347- overview of herbal medicine. 350.
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10. Patra, J.K., Das, S.K., Thatoi, H. (2015). 3. Acharya, D. and Shrivastava, K. (2008). Phytochemical profiling and bioactivity Indigenous Herbal Medicines: Tribal of a mangrove plant, Sonneratia Formulations and Traditional Herbal apetala, from Odisha Coast of India. Practices. Aavishkar Publishers Chinese Journal of Integrative Medicine, Distributor, Jaipur- India, pp 440. 21(4): 274-285. 4. Bandaranayake, W.M. (2002). 11. De, S., Dey, Y.N., Ghosh, A.K. (2010). Bioactivities, bioactive compounds Phytochemical investigation and and chemical constituents of chromatographic evaluation of the mangrove plants. Wetland different extracts of tuber of Ecology and Management, 10: 421- Amorphaphallus paeoniifolius 452. (araceae). International Journal on 5. Mahmud, I., Islam, M.K., Saha, S. Pharmaceutical and Biomedical (2014). Pharmacological and Research, 1(5), 150-157. Ethnomedicinal Overview of Heritiera fomes: Future Prospects. Int. Sch. Res. 12. Braca, A., Tommasi, N.D., Bari, L.D. et Not, 1-13. al. (2001). Antioxidant Principles 6. Bandaranayake, W.M. (1998). from Bauhinia tarapotensis. Journal of Traditional and medicinal uses of Natural Products, 64(7), 892-895. mangroves. Mangroves Salt Marshes, 13. Singleton, V.L., Rossi, J.A. (1965). 2:133-148. Colorimetry of total phenolics with 7. Hossain, S.J., Basar, M.H., Rokeya, B. et phosphomolybdic- phosphotungstic al. (2013). Evaluation of antioxidant, acid reagents. Am J Enol Vitic, 16: 144- antidiabetic and antibacterial activities 158 of the fruit of Sonneratia apetala 14. Silva, L., Pezzini, B.R., Soares, L. (2015). (Buch.-Ham.). Oriental Pharmacy and Spectrophotometric determination of Experimental Medicine, 13(2): 95-102. the total flavonoid content in Ocimum 8. Saad, S., Taher, M., Susanti, D. et al. basilicum L. (Lamiaceae) leaves. Phcog (2012). In vitro antimicrobial activity of Mag, 11: 96-101. mangrove plant Sonneratia alba. Asian 15. Pac J Trop Biomed, 2:427-429. Prieto, P., Pineda, M., Aguilar, M. 9. Jaimini, D., Sarkar, C., Shabnam, A.A., (1999). Spectrophotometric Jadhav, B.L. et al. (2011). Evaluation of Quantitation of Antioxidant Capacity antibacterial properties of mangrove through the Formation of a plant Sonneratia apetala Buch. Ham Phosphomolybdenum Complex: leaf. World Applied Sciences Journal, Specific Application to the 14(11): 1683-1686. Determination of Vitamin E. Analytical Biochemistry, 269, 337- 341.
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Table 01: Phytochemical test results for Heritiera fomes and Sonneratia apetala Sl. Chemical Test Result of H. Result of S. no. Constituents fomes apetala
Fehling’s Test + + Carbohydrates 01 Molisch’s Test ++ + Barfoed Test ++ +
02 Glycoside Sodium hydroxide Test - - Fehling’s Test + + Glucoside 03 Dilute H2SO4 Test + +
Lead acetate test + +
Ferric chloride test + 04 + Tannin Alkaline reagent test + + Mayer’s Test ++ +
Wagner’s Test ++ +
05 Alkaloids Hager’s Test + + Dragendroff’s Test + + Tannic Acid Test ++ +
Ammonium Test ++ - Flavonoids 06 Aluminium Chloride Test ++ -
Foam Test + - Saponin 07 Haemolysis Test - -
08 Triterpenoids Salkowski Test + ++
09 Steroids Liebermann- Burchard’s ++ + Test Stain Test ++ + 10 Fats and Fixed Oils Saponification Test + + [++ = strongly present, + = present, - =absent]
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Figure 01: Leaves of Heritiera fomes (left) and Sonneratia apetala (right)
Figure 02: Comparison of IC50 values between HF and SA leaf extract in DPPH free radical scavenging assay (Values are the mean of experiments)
800 716,602 700
600
500 400
300 IC50 (µg/ml) IC50 200 100 61 17,75 0 HF SA Ascorbic Acid
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Figure 03: Comparison of total phenol content (Gallic acid equivalents, mg/gm) between HF and SA leaf extracts (Values are the mean of experiments and represented as mean ± SD)
1600 1489,16
1400
1200
1000
800
600
400
200 13,977 0 HF SA Figure 04: Comparison of flavonoid content between HF and SA leaf extracts (Values are the mean of experiments and represented as mean ± SD) 800
700 664,4
600
500
400
300
200
100 14,88904 0 HF SA
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Figure 05: Comparison of total antioxidant capacity (mg/gm, Ascorbic Acid Equivalent) between HF and SA leaf extracts (Values are the mean of experiments and represented as mean ± SD) 1000 880 900
800
700
600
500
400
300
200
100 15,3225 0 HF SA
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