Phytochemical Screening of Putat Air [Barringtonia racemosa (L.)] Spreng Herbal Plants Found in Bireun, Aceh, Indonesia

 

Nur Asnah Sitohang1, Effendy De lux Putra2, Hajjul Kamil3, Musri Musman4*

1Graduate School of Mathematics and Applied Science, Universitas Syiah Kuala,
Banda Aceh, 23111, Banda Aceh, Indonesia.

1Department of Maternity and Child Nursing, Faculty of Nursing, Universitas Sumatera Utara,

Medan, 20155, Sumatera Utara, Indonesia.

2Department of Pharmacy, Faculty of Pharmacy, Universitas Sumatera Utara,

Medan, 20155, Suamtera Utara, Indonesia.

3Department of Nursing Management, Faculty of Nursing, Universitas Syiah kuala,

Banda Aceh, 23111, Banda Aceh, Indonesia.

4Department of Chemistry Education, Faculty of Education and Teacher, Universitas Syiah Kuala,

Banda Aceh, 23111, Banda Aceh, Indonesia.

*Corresponding Author E-mail: musrimusman@unsyiah.ac.id

 

ABSTRACT:

Indonesia has a variety of medicinal plants. This medicinal plant has been widely used by the public in an effort to overcome health problems such as wound healing amid advances in science and technology. The discovery of various new medicinal compounds from natural materials further clarifies the important role of secondary metabolites of plants as medicinal raw materials. A natural ingredient that can be used as an alternative biological therapy for wound management is the putat air [Barringtonia racemosa (L.) Spreng.]. This plant is found in Gampong Pulo village, District of Peudada Bireun Regency, Aceh Province, Indonesia. B. racemosa is a type of local mangrove plant known as putat air. It grows wild along the banks of rivers, lakes and rice fields. Phytochemical screening results with Shinoda test of B. racemosa seeds in ethanol extract, ethyl acetate fraction and water containing flavonoids. Molisch test on ethanol extract, fraction of ethyl acetate and water containing glycosides. Foam test on ethanol extract, hexan fraction, ethyl acetate fraction and water fraction detected saponins. Using iron reaxation (III) chloride on ethanol extract and water fraction contains tannins Liebermann-Burchard test on fraction n – hexants containing steroids and triterpenoids. As the conclusion, B. racemosa seeds contain many secondary metabolites that can be used by the public as an alternative treatment of the disease.

 

KEYWORDS: Barringtonia racemosa (L.) Spreng, Flavonoida, Glikosida, Saponin, Tannin, Triterpenoid.

 

 


INTRODUCTION:

The plant has been used as an important source of medicine because of its bioactive components that are pharmaceutically significant since time immemorial. Medicinal plants can cure various diseases and certain disorders of the body due to the presence of plant bioactive compounds isolated from the secondary metabolites of plants.

 

 

The discovery of natural medicines that have been developed intensively in recent decades has proven the effectiveness of treatment. The effectiveness of folk remedies used for the treatment of certain diseases in humans has been widely verified through research1. Ethnic natural products have become the substitute a way to switch the synthetic infections and delaying the ageing progression2. Plant have almost variety of uses for human being. They are the richest source of various drugs containing pharmacological active constituents have been widely used in variety of field for the prevention and cure of disease3.

 

 

Indonesia has a variety of medicinal plants. This medicinal plant has been widely used by the public as an effort to overcome health problems such as wound healing amid advances in science and technology. The discovery of various new medicinal compounds from natural materials further clarifies the important role of secondary metabolites of plants as medicinal raw materials4. A natural ingredient that can be used as an alternative biological therapy for wound management is the putat air [Barringtonia racemosa (L.) Spreng.]. This plant is found in Aceh area, Gampong Pulo village. District of Peudada Bireun Regency. The geographical location of Bireuen Regency is between 04° 54' 00" - 05° 21' 00" LU and 96° 20' 00" - 97° 21' 00" BT. The area is 1,796.32 Km˛ (179,632 Ha), with an altitude of 0 - 2,637 meters above sea level.  The distance from the city of Banda Aceh is 233.9km.

 

B. racemosa is a type of local mangrove plant known as putat air. Barringtonia racemosa is a tropical plant that belongs to the family Lecythidaceae. It grows wild along the banks of rivers, lakes and rice fields5. It is about 4–8 m but can grow-up to 15m. The leaves are large and wide in the form of squares and oval. The size of leaves is approximately 8–35cm 4–13cm6. Barringtonia racemosa’s flower whitish pink attached to the staminal tube. The flowers bloom at night and fall the next morning. The fruit is the size of a small chicken egg. The shape of the fruit is cone to ovoinal with a size of approximately 1.5 x 2.5 inches. The fruit is coated with fibrous tissue that causes it to float when it falls into the water1.

 

Research proves that this plant is already prescribed in the ayurvedic literature of traditional Indian medicines for the treatment of dog bite wounds7. This fruit is also used to treat snakebite wounds, arthritis remathoids, diarrhea, asthma and painkillers8. The young shoots of B. racemosa are eaten raw as vegetables because they are believed to be effective in the treatment of hypertension, gastritis and cancer in Malaysia1. The leaves, roots and skin are pounded for itchy medicine and chicken pox9 .  Zulu people in South Africa use their fruit as a malaria drug10.

 

Pharmacological activity of this plant contains antimicrobial, antibacterial to good strains of gram-positive and gram-negative bacteria namely Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Shigella dysentriae, Vibrio cholerae and Proteus sp. This plant also contains antinociceptive (analgesic), antioxidant, anti-inflammatory and antifungal11. The ethyl acetate fraction of fruit extract Barringtonia racemosa has an anti-inflammatory effect on acute and chronic inflammation12. This study aims to identify secondary metabolites contained in  Barringtonia racemosa seeds through phytochemical screening and the results will be utilized as an alternative to wound treatment development. Wound is perhaps an inescapable event in the life on organism. Healing process begins during the early phase of inflammation, but reaches completion usually after the injurious influence has been neutralaized13. The rapid healing process can reduce the costs of the therapy and hospitalization but ideal availability of the drugs for wound healing therapy is limited due to the complex nature in the skin tissue. The novel medicine for wound healing and skin regeneration, herbal based therapeutics agents can be alternative medications14. Natural remedies are more acceptable in the belief that they are safer with fewer side effects than the synthetic ones. Herbal formulation have growing demand in the world15.

 

METHOD:

The criteria of B.racemosa fruit used as a research sample is an old fruit and falls to the ground. Physical condition is good, no damage caused by animals / insects and or diseases. The water putat fruit is immediately put in a sealed plastic bag. Before sealing first remove the air from the plastic bag. Next the plastic bag is placed in a cool place temperature of 4°C16.

 

Tools used are mixer, beaths, test tubes, vacuums, rotary evaporators, analytical balance sheets, spatula, mixing bats, measuring gourds, reagent bottles, drop pipettes, volume pipettes, petri dishes, gauze wire, three legs, spiritus, glass funnels, jars, test tube clamps, test tube racks, chambers, scales.

 

Materials used are seeds B. racemosa, ethanol 96%, hexane, ethyl acetate, methanol, dichloromethane, filter paper, HCl 2%, Mg band, ammonia 10%, chloroform, HCl 2 N, Dragendroff realaxation, Lieberman rearcew – Burchard, HCl 0.1%, Molish perekasi, H2SO4, FeCl3, hot water, distilled water, chloroform. The initial stage of extraction begins from the homogenization of simplisia followed by maceration of separation and phytochemical screening to detect flavonoids, saponins, glycosides, triterpenoids and steroids, alkaloids16.

 

RESULT:

Sample Preparation and Fractionation:

Ethanol Extract Preparation:

The ethanol extract is made by maceration, namely placing the powder in a container, then the solvent is added in the container. Then the mixture is stirred - within a certain time16. The maceration method in this study was carried out by means of B. racemosa simplicia powder and the liquid filter was 1:10, namely 500g of simplicia powder was put into a dark glass container, poured 75 parts of the filter liquid, namely 96% ethanol. The container is closed and stirred, left for 2 x 24 hours.

 


Table 1: The results of phytochemical screening of putat water seed gel (B. rasemosa )

S. No

Secondary Metabolites 

Reactor

Ethanol Extract

Fraction

n-hexane

Ethyl Acetate Fraction

Water Fraction

1.

Alkaloids

Dragendorff

Mayer

-

-

-

-

-

-

-

-

2.

Flavonoids

Mg+Amil Alcohol + HCl p    Powder

+

-

+

+

3.

Glycosides

Molisch+H2SO4

+

-

+

+

4.

Saponins

Hot Water/Shaken

+

+

+

+

5.

Tannins

FeCl3

+

-

-

+

6.

Triterpenes/Steroids

Liebermann-Burchard

-

+

-

-

 


Every 1 x 24 hours it is replaced by the saturated solvent with new solvent so that the chemical compounds contained in B. racemosa seeds can be drawn optimally. The maserate was evaporated using a rotating evaporator at 40°C until a thick extract was obtained, then dried16.

 

Preparation of Fractions:

Fractionation was carried out by the liquid-liquid extraction (ECC) method. A total of 10grams of thick extract of B. racemosa seeds were   dissolved in 10ml of ethanol, added 50ml of distilled water, then added with 50ml of n-hexane using a separating funnel, shaken and allowed to separate, then separated, then fractionated again using solvent hexane to obtain a clear fraction (do not give positive results with reagent Liebermann - Buchard). Then 50ml of ethyl acetate was added to the water fraction and allowed to separate.

 

Ethylacetate layer was separated and fractionation of the fraction was continued until clear ethylacetate (not give positive results with the addition of reagents FeC1 3 1%). The resulting collection of n-hexane, ethyl acetate fraction and water fraction are evaporated with a rotating evaporator at a temperature of 40°C until a thick extract is obtained, then dried and stored in a suitable place17.

 

The Extraction and Fractionation Result:

The extraction results obtained from the simplicia of B. racemosa seeds as much as 500g macerated with 96% ethanol solvent obtained 69g of B.racemosa seed ethanol extract with 13.8% yield. The fractionation of 10g of ethanol extract used n-hexane solvent, 2.5g of ethylacetate with a yield of 0.5% and a water fraction of 2.7g with a yield of 0.54%.

 

The results of Phytochemical Screening:

The results of phytochemical screening carried out on ethanol extract, n-hexane fraction, ethyl acetate fraction and water fraction of B. racemosa seeds can be seen in the following table 1.

 

Alkaloid detection by Mayer test did not form a yellow precipitate. Testing with the test Dragendorff also not found sediment in red, it means that the ethanol extract, fractions etil acetate, fractions of hexane a and fraction of water there are alkaloids16,18. Flavonoids test with the Shinoda test on ethanol extract, ethyl acetate fraction and water fraction formed red to dark red, indicating the presence of flavonoids. But the hexane fraction a does not appear red, meaning that there are no flavonoids16.

 

Detection of glycosides was carried out by using the Molisch test, in the ethanol extract, ethyl acetate fraction and water fraction a purple ring formed at the meeting of two layers indicating the presence of glycosides. But in the hexane fraction a was not found meaning that there were no glycosides16. Saponin testing was carried out by froth testing on ethanol extract, hexane fraction a, ethyl acetate fraction, water fraction formed a centimeter  foam layer indicating the presence of saponins16.

 

Detection of tannins using iron (III) chloride reagent. The results obtained in the fraction of ethyl acetate and water fractions ad a was positive for the tannins to give a blackish green. A blackish green color was formed in the extract after adding 1% FeC1 3 because tannins would react with Fe 3 +ions to form complex compounds. In the hexane fraction a and ethyl acetate fraction, there was no blackish green color, meaning that tannins were not detected19.

 

Triterpene detection done by testing Liebermann - Burchard on a fraction of hexane a formed a ring of chocolate between two layers showed that there are triterpenoids and steroids16,18. In the ethanol extract, ethyl acetate fraction and water fraction, triterpenoids and steroids were not found.

 

DISCUSSION:

The results of phytochemical screening by Shinoda test of Barringtonia racemosa seeds on ethanol extract, ethyl acetate fraction and water containing flavonoids. This study is in line with the research of 5 which states that the secondary metabolites found in Barringtonia racemosa are terpenes, triterpenoids, flavonoids, steroids and saponins20 stated that the extract of  fruit and seeds of B. racemosa showed flavonoids.21 states that B. racemosa seeds contain flavonoids.22 also stated that the flavonoids and carotenoids were very high in the ethyl acetate extract of the leaves of the shoots of B. racemosa. The shoot of this species was found to have three flavonoid compounds, namely quercetin, routine and kaempferol8.

 

Flavonoids are secondary metabolites which are found in many plants. Flavonoids are phenolic substances isolated from various vascular plants. Generally, flavonoids will be detected in all extracts. Environmental conditions, such as water pollution and ultraviolet radiation to trigger the production of flavonoids23. Flavonoids are the largest group of phenolic compounds in nature18,24 stated that flavonoid is one of the most common subcategories of polyphenols, which are found naturally in foods. Flavonoids are one of the bioactive compounds that can eliminate free radicals. Flavonoids have a chemical structure C6-C3-C6, two aromatic rings are bonded through a three-carbon chain link25.

 

Flavonoid has an important role in the mechanism of the plant defense system26. Flavonoids function as antioxidants, antimicrobials, photoreceptors, visual attractants and for filtering light. Flavonoids also act as phytoalexins, which are antimicrobial compounds in response to the presence of microorganisms27. The flovonoid content in the ethyl acetate extract of the dry leaves of B. racemosa = 38.6meRG/G28,22 extracts of leaves and stems of B. racemosa from Kedah contained 19.9meRg/G.29 stated that the flavonoid content in methanol extract was 239.35±2.02mg CE/100g and 170.96±1.28mg CE/100g for water extract (p <0.05).

 

The results of research with Molisch test on ethanol extract, ethyl acetate fraction and B. racemosa seed water contained glycosides. It is uncomplex sugars found in some plants. It is secondary metabolite compounds that bind to sugar compounds through glycoside bonds which consist of a combination of two parts of a compound, namely sugar (glycons) and non-sugar (aglycones). Glycosides play an important role in the living system of an organism. The function of glycosides in plants is regulation, protection, self-defense and health30.

 

The results of the study with u ji froth on ethanol extract, hexane fraction, the fraction of etyl acetate and water fraction seeds B.racemosa contains saponins. This study is in line with research conducted31states that k onstituen f itokimia are contained in the B. racemosa diantaran yes saponins, sterols and phenolic.20 also stated that e kstrak fruits and seeds B. racemosa showed saponin. The phytochemical content of the fruit and seeds of this plant is in the form of triterpenoid saponins20,21 states that B. racemosa seeds contain saponins.10 states that the seeds, skins and stems of B. racemosa contain saponins.32  stated that secondary metabolites such as diterpenes, triterpenoids and flavonoids (including polyphenols), steroids and saponins had previously been isolated from B. racemosa. Fruit seeds contain two sapogenins, namely, barringtogenol and barringtogenic acid.

 

Saponins are a group of glycoside compounds, can form colloidal solutions in water and foam when shaken. Saponins are steroids and triterpene glycosides, so named because of their soap-like properties. Element fat soluble (steroid or triterpene) and water soluble (sugar) present in one molecule causes saponins have properties such as detergent33. The emergence of foam in the saponin test indicates the presence of glycosides which have the ability to form foam in hydrated water into glycose and other compounds. These saponin compounds will tend to be attracted by semipolar solvents34. Saponin are plant glycosides that acquire their name from their soap – like action. Saponin accour naturally in fruit, roots, rhizome, leaf, bark etc and have historically been used in traditional system of medicine. Saponins are the utmost essential secondary metabolites from pharmaceutical and industrial point of view. The review is based on the recent phytopharmacological studies of saponin from year 2012 to 2013 even highlight to developments on saponins with their pharmacological activity35.

 

Saponins are surface active compounds and can cause foam when shaken in water36. Saponins provide a bitter taste. Saponins are secondary antioxidants, capable of inhibiting lipid peroxidation by forming hydroperoxides. Based on the research of 37saponins have antioxidant and antibacterial effects. Saponins function as antioxidants through the mechanism of increasing the formation of SOD and catalase38.

 

The results of the study using iron (III) chloride reagent on the ethanol extract of B.racemosa seeds and the water fraction containing tannin. The word 'tannins' refers to a heterogeneous group of polymer phenolic compounds, and was originally used to describe plant extracts used for animal skin tannin. Tannins are polyphenols that occur in vascular plants and are of two types; thick and hydrolyzed tannins39. Viscous tannins, which are also called proanthocyanidins, are the most important group of polyphenols studied in the plant kingdom40.  Tannins are the complex secondary metabolites has been used since earlier period as tanning agents and reported for astringent, anti-inflammatory, antidierrhoeal and antimicrobial activities41. Tannins are phenolic compounds that give a bitter and chelish taste, can react and agglomerate proteins or other organic compounds that contain amino acids and alkaloids. This compound plays an important role in protecting plants from predation by herbivores and pests, and as a regulatory agent in plant metabolism18. Tannins exhibited antibacterial activities against all the tested microorganisms42. Tannins are polyphenolic compounds, they are identified as better antiseptic due to the presence of phenolic group43,5 said that the skin of B.racemosa also contained high tannins29 stated that the total condensed tannin content in methanol extract was 33.11±0.61mg CE/100g and 13.38 ±0.87mg CE/100g in water extract (p <0.05). According to44, the abundance of hydroxyl groups and the presence of benzene rings give hydrophobic characters to tannins. This finding could be the reason why methanol extract gave higher tannin yields compared to water extract.

 

The results using Liebermann - Burchard test on a fraction of n-hexane containing steroids and triterpenoids. This study is in line with 11stated that the ethanol extract of the root of B. racemosa contained terpenes and triterpenoids.20 also stated that ekstrak fruits and seeds B. racemosa pointing right their flavonoids and terpenoids. Compounds terpene is a group of compounds of organic hydrocarbons produced in abundance by various types of plants. The compound is in general gives the smell that strong and can protect plants from herbivores and predators.

 

Terpene compounds are also one of the main building blocks in biosynthesis. The basic structure of terpenes is a residue of 2 methylbutane or more precisely or often referred to as isoprene units, (C5)n. Terpenes are also known as isoprenoids. In nature, terpene compounds are dominated as hydrocarbon groups, alcohols, glycosides, ethers, aldehydes, ketones, carboxylic acids and their esters18.

 

Terpenes, or terpenoids, are the largest class of secondary metabolites and are generally water insoluble. Terpenes are synthesized from acetyl-CoA or glycolytic intermediates and formed by the incorporation of five-carbon isoprene units. The terpenes group is synthesized by mevalonic acid (MVA) and methyleritritol phosphate. All terpenes come from a combination of five-carbon elements which has a carbon backbone that is branched from isopentane. The basic structural elements of terpenes are also called isoprene units because terpenes can decompose at high temperatures to produce isoprene, so they are sometimes referred to as isoprenoids45.

 

Terpenes are classified based on the number of constituent units being five-carbon, although extensive modifications sometimes make it difficult to sort out the original five-carbon residues. The typical structure of terpenes is that they contain a carbon skeleton (C5) n, and are classified as hemiterpenes (C5), monoterpenes (C10), sesquiterpenes (C15), terpenes (C20), sesterterpenes (C25), triterpenes (C30), and tetraters (C40)33. The terpenoid compounds have antimicrobial, antifungal, antiviral, antiparasitic, antihyperglycemic, anti-allergenic, anti-inflammatory, antipasmodic, immunomodulatory, and chemotherapeutic properties, varying depending on the type. Terpenes are poisons and food deterrents against a number of insect and herbivorous mammals, so they play an important role in the defense of the plant kingdom46.

 

CONCLUSION:

Plant secondary metabolites have a primary function as a defense system. Although they do not play an important role in plant growth and development, secondary ethabolites play a role in the survival of an organism, both from pests and diseases, as well as for successful reproduction and spread. The results of B.racemosa phytochemical screening originating from the Bireun, Aceh area contain glycosides, flavonoids, saponins, tannins and triterpenoids/ steroids.

 

ACKNOWLEDGEMENT:

This research was funded by the Universitas Sumatera Utara research institute in accordance with the USU TALENTA implementation contract for the 2019, fiscal year number: 439/UN5.2.3.1/PPM/KP-TALENTA USU/2019 dated April 1, 2019 for that I thank the Chancellor of USU

 

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

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Received on 22.04.2021           Modified on 20.07.2021

Accepted on 16.09.2021         © RJPT All right reserved

Research J. Pharm. and Tech. 2022; 15(6):2727-2732.

DOI: 10.52711/0974-360X.2022.00456