Bioactive compounds screening, antimicrobial activities of leave extract from two palatable plants: Piper betle and Murraya koenigii (Curry leaves)

 

Khin Than Yee¹*, Mohamad Iswandy bin Ibrahim¹, Tin Moe Nwe¹, Mya Mya Thwin²,

Mar Mar Lwin¹, Khin Cho Aung¹, Ma Saung Oo², Siti Zaleha binti Raduan¹, Myat San Yi¹

¹Faculty of Medicine and Health Sciences, Universiti Malaysia Sarawak, Malaysia.

² Medical Faculty, University Sultan Zainal Abidin, Kuala Terengganu, Malaysia.

 

ABSTRACT:

Introduction: Piper betle Linn is one of the most commonly used compounding plants for ethno-medical purposes, with its extract generally used in modern products to enrich their functional efficacy. The extraction methods always lead to differences in the antimicrobial efficacy of methanol extracts of bioactive compounds. Purpose: The study was conducted to screen for bioactive compounds and determine their antimicrobial efficacy in a methanol extract of Piper betle and Murraya koenigi leaves from five different regions. Methodology: The phytochemical screening done according to the procedure that is implied in from Patil, et.al, with minor modifications by the researchers of the current study. Antimicrobial activity was determined; efficacy was measured by disc diffusion analysis. Results: Phytochemical screening revealed the presence of saponin, tannin, terpenoids, alkaloids and flavonoids in the extracts. The methanolic extract of betel leaves from all the selected regions except from Bau exhibited antimicrobial activities. Among them, extract from Kuching and Simunjan have no effect on E. coli. The methanolic extract of curry leaves from Kuching, Balai ringai and Bau have antimicrobial activities against Staph aureus and those from Balai ringai is also active against E.coli. Conclusion: Although there were previous reports of phytochemical screening and antimicrobial activities from the extract of these plants, there were still lack of research conducted on the specimens especially from our local community (Sarawak). The outcome of this study will help us to identify the bioactive compounds of the local samples and give us some pictures of their activities on how to ensure these plants can be brought forward based on the origin of the sample.

 

 

 

INTRODUCTION: 

Plants play a significant role in providing nutrients for living organisms; they balance the ecosystem by means of releasing oxygen and absorbing carbon dioxide, thereby improving the quality of the air. Plants play an imperative role in the ecosystem between humans and animals, like carrying out photosynthesis to produce oxygen for the use of other Earth organisms. There are varieties of plants being used in various human cultures around the world for medicinal purposes. It has therapeutic effects on health and wellbeing, which are fulfilled by raw eating, drying, and using ingredients in food, and so on. Plants contain useful bioactive (phytochemical) compounds, which have the potential to be developed into more effective modern medicine. Plants contain useful bioactive (phytochemical) compounds, which have the potential to be developed into more effective modern medicine. Despite great progress in western medicine, the use of herbal medicines is still popular. Nowadays, scientists are increasingly focusing on natural products in order to look for new leads to develop better drugs with fewer side effects. The primary benefits of using plant-derived medicines are that they are relatively safer than synthetic chemical-based compounds and also more cost effective.

The current study, inspired by the Patil et al. (2015) study, was conducted to find out the phytochemical extract from the leaves of Piper betle and to determine the in vitro antimicrobial activity of Piper betle leaves, which go against various microorganisms.¹ In the current context phytochemical potential and in vitro antimicrobial activity of Piper betle Linn leaf extracts Antimicrobial activity observed in the literature study can be determined by measuring the disc diffusion assay observed in Shahid-Ud-Daula et al.'s 2015 study². Betel Leaf has the photochemistry, traditional uses, and pharmacology. A pan-Asian species found in most Asian countries, the medicinal plant Piper betle of the Piperaceae family is a plant abundantly found and grown in Southeast Asia. ³. It is suggested that the plant originates from the Indonesian archipelago⁴. It is a native plant in central and eastern Malaysia. It has also been cultivated in Malaysia and tropical Asia for more than 2500 years⁵.

 

It is a dioecious root climbing plant with green, heart-shaped leaves with 2–3 pairs of secondary veins and an acuminate apex that likes to grow in partially shady environments. Its stems are swollen at the nodes with adventitious roots for climbing adherence⁶. According to Guha (2006) ⁷, the plant is particularly famous in India due to its importance in Indian religious and cultural activities, where it is typically served as mouth fresheners in the form of betel quid. It is also widely used as a post-meal mouth freshener in Sri Lanka, Malaysia, Thailand, and other Southeast Asian countries, including Myanmar.

 

The plant leaves that have pungent and aromatic flavours are used as a mouth freshener³. Betel leaf juice is used as an adjuvant and mixed with other medicines in Ayurveda for better results of treatment. Plant variety, season and climate are the factors that affect the concentration of various biologically active compounds in betel leaf⁹ According to Dwivedi and Tripathi (2014)⁸ , it is applied in a variety of decoction, in healing wounds, impetigo, burns, eczema and lymphangitis. Lee et al. (2015) ¹⁰ stated that the extraction of betel leaves has antimicrobial effects because of the presence of phytochemicals like phenolic compounds. Some studies revealed that betel leaf extract has an antibacterial effect against S. pyogenes¹². Based on A. Syahidah, et.al (2017) ¹², methanolic extract of betel leaves has antibacterial activity against some Gram- positive (S.aureus, S. agalactiae, Bacillus sp.) and Gram-negative bacteria (E.coli, K. pneumonia and P.aeruginosa). 

 

Murraya koenigii, also known as curry leaf, is a green, leafy vegetable that belongs to the family Rutaceae. It is widely used in very small amounts for flavouring foods in Asian cuisines. It has a slightly pungent, bitter taste, and it retains its flavour even after drying. The plant is a medicinal plant native to India, Sri Lanka, and other South Asian countries¹³. It is usually found in tropical and sub-tropical regions and cultivated in China, Australia, Nigeria, etc.

 

Curry leaves contain proteins, carbohydrates, fiber, minerals, carotene, nicotinic acid, vitamins A and C, carbazole alkaloids, koenigine, and mahanimbine. The medicinal use of plants is a result of the phytoconstituents present in them. Some of these chemicals are bioactive and produce definite physiological and biochemical actions in humans and animals. They are known as secondary metabolites or phytochemicals and comprise alkaloids, flavonoids, tannins, phenols, saponins, steroids, glycosides, terpenes, etc. ¹⁴. Murraya koenigii (curry leaves) has medicinal values that are numerous and beneficial to humans; hence, this work seeks to evaluate the phytochemical compositions present in the plant. The plant is highly valued for its use in many traditional cultures, namely in Indian Ayurvedic and Unani prescriptions¹⁵. The curry leaf is believed to have several medicinal properties, such as anti-diabetic, antioxidant, antimicrobial, anti-inflammatory, anti-carcinogenic, and hepato-protective properties¹⁶.

 

The use of plants in traditional medicine is of global interest. Traditional and herbal medicines have been promoted as a source of less expensive and more comprehensive healthcare, especially in developing countries. Some of these medicines are easily available, cheaper, and safer than these modern synthetic drugs. This led to the replacement of medicinal plants and herbs for the use of conventional antibiotics and encouraged an increase in their use and demand for medicinal plants as they play roles in the prevention and management of some health ailments, such as diabetes, cancer, arthritis, and degenerative disorders like Parkinson's and Alzheimer's disease¹⁷.

 

The medicinal values of many plants remain unexploited. Investigations are ongoing to discover novel drugs for the development of new therapeutic agents with a safety index. Although these plants have been investigated before regarding their antimicrobial activities, a report on a comparative study of the antimicrobial efficacy of these leaves from various regions of Sarawak is still lacking. The emphasis is placed on the different degrees of antimicrobial activity investigated and whether they vary with the regions cultivated.

 

MATERIALS AND METHODS:

Plant material: Leaves of the selected plants will be obtained from local residents at Simunjan, Serian, Kuching, Bau, and Balai Ringin area, Sarawak. The plant materials will be washed with tap water followed by distilled water to remove dirt. They will be air dried completely at room temperature and subsequently pulverised into powder using an electric blender. The powder will be kept in a dried and sterile 200-mL conical flask until further use.

 

Extraction of plant material: The dried, ground samples will then be soaked in methanol (500 ml) for 3 days at room temperature. The solvent-containing extracts will then be filtered. The extractions of the ground samples will further be repeated (2x) with methanol (500 ml each cycle). The filtrate from each extraction will be combined and the excess solvent will evaporate under reduced pressure using a rotary evaporator to give crude methanol extracts.

 

Phytochemical screening

Test for Phlobatannins

Deposition of a red precipitate when an aqueous extract (0.2gm) of the plants past was boiled with 5ml 1% HCl (aq) boiled was taken as evidence for the presence of Phlobatannins.

Method for Trepenoids:

0.5gm of the extract will be dissolved with 2.0ml of chloroform.

 

Sulfuric acid is then carefully added to form a lower layer of reddish brown color of the interface indicates the presence of Trepenoids.

Test for Tannin: -

Lead acetate test: 2 ml test extract will be added to 1% lead acetate and observed for yellowish precipitate which indicates the presence of tannin.

 

FeCl₃ test: 4 ml test extract will be treated with 4 ml of FeCl₃. Formation of green colour indicates presence of condensed tannin.

Test for Saponin: Foam test

5 ml test extract will be mixed with 20 ml of distilled water and agitated in graduated cylinder for 15 minutes. The formation of foam will be taken as an evidence for saponin.

Test for Flavonoid: -

Alkaline reagent test: Test extract will be treated with 10 % NaOH solution; formation of the intense yellow colour evidence for presence of flavonoid. NH₄OH test: 10 %

 

NH₄OH will be used to treat the 3 ml of test extract, development of yellow fluorescence indicates positive test.

Mg turning test: Test extract will be treated with Mg followed by few drops of conc. HCl and finally 5 ml of 95 % ethanol will be added. Formation of crimson red colour indicates presence of flavonoid.

Test for Alkaloids: Wagner’s reagent test:

Filtrate (1 ml HCl will be added into a test tube containing 3 ml conc. test extract. The mixture will be heated gently for 20 minutes and filtered after cooling) will be treated with Wagner’s reagent, the formation of reddish precipitate shows presence of alkaloids.

Method for Fehling’s Test:

0.2gm of methanolic crude extract were shaken with 10.0 ml of water, then filtered and the filtrate will be concentrated.

1.0ml each Fehling’s Solution A and B were added and boiled for about 5 minutes.   

 

Antimicrobial testing

Determination of antimicrobial activity

One species of Gram-negative bacteria (Escherichia coli ATCC 11775) and one species of Gram-positive bacteria (Staphylococus aureus ATCC 700698) were used as test microorganism in the antibacterial activity assay.

 

Disc Diffusion Assay (Shahid-Ud-Daula et.al, 2015) ²

The disc diffusion will be used to evaluate the antimicrobial activity of the methanolic extracts. Kirby Bauer method will be used for bacteria inoculum preparation. Blood agar plates will be prepared for fresh bacteria stock which will be obtained from Faculty of Medicine and Health Sciences, UNIMAS to grow for 24 hours in an incubator at 35ºC. A sterile disposable inoculation loop will be used to touch the top of each of 3 to 5 similar colonies on the blood agar. The loop will be transferred into 9 mL of sterile Mueller Hinton broth. The broth will be incubated at 37 ºC for 5 hours and subsequently will be compared with McFarland turbidity standard (10^8 CFU/ml). A sterile cotton swab will be dipped into the broth and streaked on Mueller Hinton agar plate. The agar plate will be rotated automatically using a plate rotator during streaking. The streaking will be started at the centre point of agar plate to the periphery of the agar plate. The streaking process will be repeated about three times to ensure an even distribution of test microorganism. The agar plate will be dried for 5 minutes under room temperature with the lid closed. Penicillin and streptomycin will be used as control for gram positive bacteria while ampicillin, tetracycline and streptomycin will be used as control for gram negative. The impregnated discs with extracts, positive control disc and negative control disc will be placed using sterile plain forceps on the agar in a way that distance of each discs will not nearer than 24 mm. The agar will be kept at refrigeration temperature for 30 minutes for better absorption and subsequently will be Mincubated at 37 ºC for 24 hours in an upright position. A ruler will be used to measure them diameter of inhibition zone. The diameter will be recorded in millimetres.

 

RESULTS:

As shown in Table 1.1, the phytochemical constituents of a methanol extract of betel leaves from some regions contain a variety of amounts of phlobatannin, trepenoids, tannin, saponin, flavonoids, reducing sugar, and alkaloid content. However, the extracts from Serian, Bau, and Balai ring with a lack of phlobatannin.

 

The phytochemical analysis (table-1.2) of methanol extracts from curry leaves revealed that there was saponin, reducing sugar, and alkaloid in all extracts of the regions, and tannin was found in the extract of Bau. Phlobatannin, trepenoids, and flavonoids were absent in all extracts.

 

Regarding the table-1.3, zones of inhibition of methanolic extract of betel leaves from different regions were shown. All the extracts from the region except for Bau have antimicrobial properties. Among them, extracts from Kuching and Simunjan have no effect on E. coli. The extract of betel leaves from Serian was the most potent against Staph aureus, showing the maximum zone of inhibition (15.0 mm), seen at the concentration of 1000 mcg/ml. The minimum zone of inhibition was 6.0 mm, which is effective against Staph aureus with 250 mcg/ml of extract from Serian and 500 mcg/ml of extract from Balai Ringin. 1000 mcg/ml of extract from Serian and Balai ringin have antimicrobial properties against E. coli, and their zone of inhibition was 8.0mm.

 

Table 1.1. Phytochemical screening of betel leaves (B) from some regions

Region	Samples	Phlobatannin	Trepenoids	Tannin	Saponin	Flavonoid	Reducing sugar (Fehling’s test)	Alkaloid (Wagner’s test)
Kuching	KCH(B)	+	+	+++	+++	+	+	+
Simunjan	SMJ (B)	++	+	+++	+++	++	+++	+
Serian	SR (B)	-	++	+++	+	++	++	++
Bau	BAU (B)	-	++	++	+	+	++	++
Balai Ringin	BR (B)	-	++	++	+	++	++	++

Key: (+) Positive test, (-) Negative test, ‘+’ low; ‘++’ moderate; ‘+++’ high

 

Table 1.2. Phytochemical screening of curry leaves (C) from some regions

Region	Samples	Phlobatannin	Trepenoids	Tannin	Saponin	Flavonoid	Reducing sugar (Fehling’s test)	Alkaloid (Wagner’s test)
Kuching	KCH(C)	-	-	-	+	-	+++	+
Simunjan	SMJ (C)	-	-	-	++	-	++	+
Serian	SR (C)	-	-	-	++	-	++	++
Bau	BAU (C)	-	-	+	+	-	++	++
Balai Ringin	BR (C)	-	-	-	++	-	++	+

Key: (+) Positive test, (-) Negative test, ‘+’ low; ‘++’ moderate; ‘+++’ high

 

 

Table 1.3. Zone of inhibition for methanolic extract of leaves of Piper  be

Key: SA – Staphlococcus aureus; EC – Escherichia coli                                                                                       

 

Table 1.4. Zone of inhibition for methanolic extract of leaves of Murraya koenigii (Curry).

Key: SA – ; EC – Escherichia coli

 

Table 1.5. Zone of inhibitions of the methanol extract of betel leaves and curry leaves against Staph aureus

 

Table 1.6. Zone of inhibitions of the methanol extract of betel leaves and curry leaves against E.coli

 

 

Zone of inhibition of methonolic extract of curry leaves (Murraya koenigii) from different regions were shown in the above table. The maximum zone of inhibition for Staph aureus was 10.0 mm with 1000mcg/ml of methanolic extract from Balai ringin. The minimum zone of inhibition which is 7.0 mm, effective against Staph aureus was seen with 1000 mcg/ml of extract from Kuching and Bau. There was no zone of inhibition on E.coli. (Table-1.5).

 

The table-1.5 exhibited the antimicrobial effect against Staph. aureus. by the methanol extract of betel (B) and curry (C) leaves. The extract of betel leaves from Serian has the most potent against   Staph. aureus. compared to those of curry leaves with the concentration of 1000mcg/ml

 

According to table-1.6, methanol extract of betel leaves from Serian and Balai Ringin have the activity against E.coli seen at the concentration of 1000mcg/ml.

 

The zone of inhibition was 8.0 mm which is less than antibiotics control.

 

DISCUSSION:

The current study was carried on methanolic extracts of betel leaves and curry leaves from some regions in Sarawak to investigate the presence of medicinally important phytochemical constituents. The various phytochemicals such as tannins, saponins, flavonoids, alkaloids, trapezoids, phlobatannin, and reducing sugars are revealed in most of the extracts. These biologically active compounds, whose concentration depends on the variety of the plant (which may have different chemo types for the same species), result from growing environmental factors like season and climate, soil fertility, or developmental or other factors. Flavonoids, another constituent of the extracts, exhibited a wide range of biological activities like antimicrobial, anti-inflammatory, anti-angionic, analgesic, anti-allergic, cytostatic and antioxidant properties¹⁸. Several reports can be found on the antimicrobial activity of flavonoids^19-22. The insights drawn from literature studies indicate the inhibition of the growth of various fungi, yeasts, bacteria, and viruses by tannins²³. Terpenoids and tannins are attributed to analgesic and anti-inflammatory activities. Literature studies also reveal that the contribution of tannin astringency speeds up the healing process of wounds and inflamed mucous membranes²⁴. The literature study by Harborne reveals that tannin may be toxic to bacteria, yeast, and filamentous fungi. ²⁵ Moreover, it also shows potential antiviral as well as antibacterial activity. Saponins have been extensively used as detergents, piscicides, and molluscicides in addition to their industrial applications as foaming and surface active agents and also have beneficial health effects²⁶. The presence of bioactive compounds indicates the medicinal value of the plants. By assessing the status of phytochemical properties in the two edible plants from this study, it may improve the health status of people and be valuable for use in pharmaceutical and nutraceutical products of commercial importance. Shitut et al. (1999) confirm the antimicrobial property of betel leaf on some pathogenic bacteria and fungi in their report. ²⁹ Researchers in this study evaluated the antibacterial activity against Gram positive bacteria, Staph aureus, and Gram-negative bacteria, E. coli, using a methanolic extract of betel leaves and curry leaves. Penicillin, streptomycin, ampicillin, and tetracycline are used as standards for antimicrobial assay. The two plants showed different antimicrobial activities against the tested strains. The methanolic extract of betel leaves is more active than that of curry leaves, and they are more effective against Staph aureus than E. coli.

 

This outcome of Gram-negative bacteria has been found to be more resistant than Gram positive bacteria, which is in contrast to the previous reports. ^27-28 Moreover, there have been previous reports showing that betel leaf extract has strong antimicrobial effects. ^30-33

 

The literature studies have shown that the leaf extract of Murraya koenigii possesses antimicrobial activity against urinary tract infections caused by E. coli, K. pneumoniae, and P. aeruginosa. Bioactive compounds in its extract revealed the presence of significant components responsible for its effective antibacterial activity³⁴. Hanan A.H. et al. ³⁵ mentioned in their study that curry leaf extract showed a broad spectrum of very significant antibacterial activity by producing a clear zone of inhibition against Staphylococcus, E. coli, Streptococcus, and Proteus. Mathur A, et al. (2010) deliberated that the methanolic extract of curry leaves inhibited Staphylococcus aureus and Staphylococcus epidermidis, Pseudomonas aeruginosa, and Escherichia coli³⁶. Compared to the present study, a methanolic extract of curry leaves is effective against S. aureus and has no effect on E.coli. This variety may depend on the packaging materials, the storage atmosphere, and the processes of laboratory procedures like washing, drying, extraction, and storage of the extract.

 

CONCLUSION:

As antibiotic resistance is emerging at an alarming rate, it becomes difficult to treat many infectious diseases. Plant-derived medicines have made an enormous contribution towards the improvement of human health and act as a source of motivation for the development of novel drug compounds. The current study concludes that those plants comprise diverse chemical constituents that have the potential to be used in the area of pharmacology. Due to the presence of various compounds that are essential for good health, it can also be used to improve the health status of society. The methanolic extracts of both plants showed moderate antibacterial activity against the tested microorganisms. The outcome is not satisfactory as compared control antibiotics and other studies. Extensive research, such as that on the antimicrobial activity of aqueous and ethanol extracts, should be encouraging in the development of novel antimicrobial new compounds in the near future. More studies are needed to understand and reveal the mechanism of action of those plants.

 

ACKNOWLEDGEMENT:

By using this opportunity, we would like to express our earnest gratitude to everybody at University Malaysia Sarawak who supported to accomplish this study.

 

CONFLICT OF INTEREST:   

There is no conflict of interest between authors.

 

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Accepted on 24.03.2023           © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(3):1452-1458.