Phytochemical and antimicrobial analyses of Plectranthus amboinicus leaf extracts

 

M.S. Sindhu, M. Poonkothai

Department of Zoology, Avinashilingam Institute for Home Science and Higher Education for Women Coimbatore – 641 043, Tamil Nadu, India.

*Corresponding Author E-mail: poonkothaiadu@gmail.com

 

ABSTRACT:

The present study is to evaluate the phytochemical constituents and antimicrobial activity of Plectranthus amboinicus leaf extract using petroleum ether, methanol and water as solvents. The antimicrobial activity of
P. amboinicus leaf extracts were examined against the selected bacterial and fungal isolates namely Escherichia coli, Staphylococcus aureus and Aspergillus niger, Candida albicans using agar well diffusion method. FT –IR analysis was performed to identify the presence of functional groups in the methanol extract. The results of the study revealed the presence of distinctive active secondary metabolites in the tested leaf extract. The results revealed that the methanol extract exhibited maximum antimicrobial activity against the tested bacterial and fungal isolates when compared with aqueous and petroleum ether extracts. The FT-IR analysis reveals the presence of functional groups such as hydroxyl, amines, alkynes, ketones and carboxylic acid in the methanolic extract of P. amboinicus. Thus, P. amboinicus leaves are effective against the selected microbes and recommends that the plant derived phytochemicals are comparatively safer than synthetic alternative, thereby contributing insightful remedial benefits for the treatment of diseases.

 

KEYWORDS: Plectranthus amboinicus, Phytochemicals, FT – IR analysis, Antimicrobial activity.

 

 


INTRODUCTION:

Antibiotics are one of the most important weapon in combating bacterial infections and have significantly benefited the health associated quality of human life since their introduction. However, uncertainty of controlling the infections is the main problem commonly referred as “antibiotic resistance” which is found in several allopathic therapies. Therefore, it is essential to investigate newer drugs with lesser resistance. The multiple drug resistance has developed due to the indiscriminate use of antimicrobials and reemergence of diseases1. Adverse drug reactions and the high cost of antimicrobials have been key contributors to ineffective management of infectious diseases in many developing countries2.

 

Systematic studies among various pharmacological compounds have revealed that any drug may have the possibility of possessing diverse functions and thus may have useful activity in completely different spheres of medicine3. In the recent past, about 61% of new drugs were developed based on natural products and they have been very successful especially in the areas of infectious disease and cancer4. On contrary, 2020 is more into the integration of machine learning networks like Artificial Intelligence which is time consuming and accurate in the identification of diseases. Hence it is clear that research towards discovery rate of active novel chemical entities is declining5. Certainly there is a need to bioprospect new sources and if possible from less explored regions and habitats to maximize the discovery of novel bioactive metabolites.

 

Plectranthus amboinicus (Lour.) known as Indian Borage belongs to the Lamiaceae family, and it is a commonly available aromatic pubescent medicinal herb with essential oil producing capability. It is extensively used in folk medicine to cure conditions such as respiratory ailments, constipation, headache, fever and skin diseases. The leaves are frequently consumed and used as flavoring agents, or integrated as a constituent in the preparation of traditional food6. Hence, the present study is attempted to evaluate the phytochemical constituents and antimicrobial activity of the leaf extracts of P. amboinicus.

 

MATERIAL AND METHODS:

Collection and identification of the plant material:

Fresh leaves of Plectranthus amboinicus was collected from Malappuram, Kerala, India. The sample was authentically identified from Kerala Forest Research Institute, Peechi, Thrissur, Kerala. The leaves were thoroughly rinsed with tap water and ensured that it was devoid of contaminants. The leaves were air dried at room temperature and pulverized to fine powder and stored in air tight container at room temperature for further analysis.

 

Preparation of leaf extracts:

The extract was prepared using three solvents of increasing order of polarity viz., petroleum ether, methanol and water (aqueous). For the preparation of aqueous extract, about 10g of the leaves were homogenised with 10ml of hot water using mortar and pestle, 90ml of hot water was mixed with the residue and stirred for 30min. The finely pooled extract was centrifuged at 10,000rpm for 15 min at 4°C. The collected supernatant was concentrated using rotary evaporator and used for further analysis. While, for the methanol and petroleum ether extraction, 10g of the dried leaf powder was added to 100ml of the respective solvent and extraction was performed by cold maceration method for 72h. After extraction, it was filtered using Whatman filter paper and the solvent was evaporated to dryness under vacuum using a rotary evaporator. The crude extract was weighed and dissolved in a known volume of dimethyl sulphoxide (DMSO). The extraction yield was expressed as

 

       Weight of the dry extract (g)

Extraction yield (%) = -------------------------------------- X 100

                        Weight of the sample used

                             for the extraction (g)

 

Phytochemical analysis:

The petroleum ether, methanol and aqueous extracts of P. amboinicus leaves were subjected to preliminary phytochemical tests namely carbohydrates, proteins and aminoacids, phenols, sterols, glycosides, quinones/anthroquinones, alkaloids, tannins, anthocyanin, flavonoids, terpenoids, saponins and leucoanthocyanin as per the standard methods7.

 

FT- IR analysis:

In FT-IR study, methanolic extract (10mg) of
P. amboinicus leaf powder was taken in a mortar and pestle, grounded with 2.5mg of dry potassium bromide (KBr), filled in a 2mm internal diameter micro-cup and loaded onto FT-IR set at 26ºC±1ºC. The sample was scanned using infrared in the range of 4000-750cm-1 using Fourier Transform Spectrometer (Shimadzu, IR Affinity 1, Japan). The spectral data obtained was compared with the reference chart to identify the functional groups present in the sample8.

 

Antimicrobial analysis:

Agar well diffusion method was employed to determine the antimicrobial activity of the leaf extract of P. amboinicus. The bacterial strains viz., Escherichia coli, and Staphylococcus aureus were selected for evaluation. These bacteria were cultured on the nutrient broth (Himedia, Mumbai, India) at 37±1°C. While, the fungal strains Aspergillus niger and Candida albicans were chosen to evaluate the antifungal property of leaf extracts of P. amboinicus. The fungi were cultured in Sabouraud dextrose broth (Himedia, Mumbai, India) at 28 ± 1°C. Five wells were bored on each Petri plate and the well grown bacterial and fungal cultures were spread plated or swabbed on sterile Muller Hinton and Rose Bengal chloramphenicol agar medium separately. To each well 20µl of the leaf extracts were added and incubated at 37°C for 24hrs (bacteria) and at room temperature for 5 days (fungi) respectively. In addition, standard antibiotic discs namely chloramphenicol (Bacteria) and flucanozole (Fungi) were used as positive control and DMSO served as negative control. After incubation period the diameter of the inhibition zone was measured and expressed in millimetre9,10.

 

RESULTS AND DISCUSSION:

Plant-based medicines are widely employed in various public health practices throughout the globe as they are safe and cost-effective, and efficiently combat various deadly diseases and help in maintaining good health. Amongst the wide number of existing medicinal plants, Plectranthus amboinicus (Loureiro) Sprengel is one of the most documented species in the family Lamiaceae11.World Health Organisation stated that about three-fourth of the world population is still dependent on traditional herb-based medications due to their low cost, easy accessibility and likely negligible side effects in comparison to allopathic medicines12. Undoubtedly, many allopathic medicines have their integral active metabolites from the plants, which itself explains the importance of the practice of traditional medicine.

 

Phytochemical analysis:

The yield of the extracts varied according to the character of solvent used for extraction. Accordingly, the quantification of the dried extracts was 3.86g, 5.42g and 4.37g for petroleum ether, methanol and distilled water respectively. Several methods of extraction have been employed and the net yield varies. For instance it was reported that hexane extraction method produced the highest oil yield (6.52%) when compared to steam distillation (0.55%) and supercritical CO2 extraction methods (1.40%)13. It was also noteworthy to mention that that there was a difference in aroma of the extracts which was attributed to qualitative and quantitative dissimilarity in chemical composition. Significantly, the phytochemical composition varies according to the geographic variation which proportionally reflects on the pharmacological properties of the plant.

 

The phytochemical analysis of leaf extracts of
P. amboinicus confirmed the presence of various phytochemicals. Significantly, the methanol extract indicated the presence of carbohydrates, proteins, amino acids, phenols, saponins, flavonoids, alkaloids, sterols, glycosides and the absence of tannins, terpenoids, quinones/ anthroquinones, anthocyanin and leucoanthocyanin. The major constituent in the leaf of P. amboinicus are the phenolic compounds which is mainly due to the natural production of essential oil with high amount of bioactive compounds such as carvacrol, thymol, β-caryophyllene, α-humulene, γ-terpinene,
p-cymene, α-terpineol and β-selinene14,15.

 

On contrary, in the aqueous extract alkaloids, phenols, flavonoids and sterols were present and the rest of the secondary metabolites were absent. The non-polar solvent, petroleum ether indicated the presence of phenols, terpenoids and sterols whereas all the other phytoconstituents were absent (Table 1). A research work reported the bench mark composition that
P. amboinicus oil is rich in oxygenated monoterpenes, monoterpene hydrocarbons, sesquiterpene hydrocarbons and oxygenated sesquiterpenes16. In addition several research findings pointed that essential oil of leaf of
P. amboinicus was particularly rich in phenolic monoterpenes, which are speculated to exert various pharmacological properties17,18,19. The results of phytochemical from Begonia versicolar Irmsch also indicated the presence of alkaloids, flavonoids, phenols, terpenoids, steroids, carbohydrates, saponins and reducing sugar which supported the findings of the present study20. The phytochemical screening of ethanol and aqueous extracts of Barleria cristata revealed the presence of alkaloids, flavonoids, glycosides, saponins, phenols and tannins21. More recently, interest among phytochemists and biologists has focused on the isolation of specific bioactive compounds of Plectranthus amboinicus and comprehending their pharmacological importance.

 

Table 1. Phytochemical screening of leaf extracts of Plectranthus amboinicus

S. No.

Phytochemicals

Petroleum ether extract

Methanol extract

Aqueous extract

1

Carbohydrates

Molisch test

-

+

-

Benedict test

-

+

-

2

Proteins and Aminoacids

Ninhydrin test

-

+

-

Biuret test

-

+

-

3

Saponins

-

+

-

4

Alkaloids

Mayer’s test

-

+

+

Dragendroff’s test

-

+

+

Wagner’s test

-

+

+

5

Tannins

-

-

-

6

Phenols

Ferric chloride test

+

+

+

Lead acetate test

+

+

+

7

Flavonoids

+

+

+

8

Glycosides

Legal’s test

-

+

-

Keller’s-killiani test

-

+

-

9

Terpenoids

+

-

+

10

Sterols

+

+

+

11

Quinones/ Anthroquinones

Borntrager's test

-

-

-

12

Anthocyanin

-

-

-

13

Leucoanthocyanin

-

-

-

 

FT –IR analysis:

The FT-IR spectrum of P. amboinicus was studied to identify the nature of functional groups present. The peaks and functional groups of the spectrum are depicted in Figure 1. The spectrum revealed a strong and broad peak at 3346cm-1 which is attributed to –OH and
–NH stretching vibrations in hydroxyl and phenol groups respectively. The peaks at 2349cm-1 and
2308cm-1 revealed the strong –COO stretching vibrations of carboxylic acids. The band at 2137 cm-1 attributed to –C≡C– stretching vibrations of alkyne groups. The peaks at 1641 cm-1 and 1544 cm-1 are indicative of strong C=O and N=O stretching. The peaks at 1228 cm-1 and 1217 cm-1 represents the strong C-N stretching vibrations of amine groups. Thus the FT-IR analysis suggests the presence of different functional groups such as hydroxyl, amines, alkynes, ketones and carboxylic acid in the methanolic extract of P. amboinicus.


 

Fig. 1. FT- IR spectrum of Plectranthus amboinicus

 


Antimicrobial activity:

P. amboinicus has appreciable pharmacognostic properties such as antioxidant, antibacterial, antimicrobial, anti-inflammatory and fungitoxic activities due to the presence of volatile essential oils in its leaves22. In last two decades the medicinal plants have regained a wide recognition due to an escalating faith in herbal medicine in view of its lesser side effects compared to allopathic medicine23.

 

The leaf extract of P. amboinicus with the selected solvents was evaluated for antimicrobial activity against selected bacterial and fungal isolates and the zone of inhibition was depicted in Table 2. Their antimicrobial property was evaluated by comparing with the positive controls. Comparative study analysis amongst the different solvents limelight the efficacy of the different phytoconstituents extracted based on the nature of solvents. It is significant to mention that there is a definite correlation between the nature of solvents used for extraction and their respective expression of antimicrobial properties.


 

Table 2. Antimicrobial activity of leaf extracts of Plectranthus amboinicus

Microorganisms

Zone of inhibition (mm)

Methanol extract

Petroleum Ether extract

Aqueous extract

Positive control

Negative control

Bacterial isolates

E. coli

17.3±0.98

10.1±1.3

17.7±1.5

20±2

-

S.aureus

11.3±1.2

5±1

9.9±0.85

14.1±1.4

-

Fungal isolates

A. niger

22.2±0.68

13.1±1.02

19±1.25

24.1±0.8

-

C. albicans

19±2

9.1±0.9

12.3±2

21.1±1.15

-

* The values are represented as Mean±SD

Positive control: Chloramphenicol (Bacteria) / Flucanozole (Fungi), Negative control: DMSO

 


In the present study the leaf extracted using methanol invariably exerted a good zone of inhibition against E. coli (17.3±0.98mm), S. aureus (11.3±1.2mm) and A. niger (22.2±0.68mm), C. albicans (19±2mm) respectively. This can be justified by the presence of phenolic and flavonoid compounds that natively has a trait to control the growth of microorganisms. While the aqueous extract inhibited the microbial strains in a better manner than the petroleum ether leaf extracts. When compared with the standard antibiotics chloramphenical (bacteria) and flucanozole (fungi), the methanol extract had a remarkable antibacterial and antifungal activity (Table 2). Coleus aromaticus proved to be a fairly good antibiotic and a very effective antifungal herb. It had inhibitory action against, Candida krusei and Candida albicans with a marginal difference. Whereas, the antibacterial activity was distinctive against the series of bacterial strains and the least susceptibility for C. aromaticus was recorded against Proteus mirabilis, Escherichia coli, Staphylococcus aureus, Enterococcus faecalis, Klebsiella pneumoniae and Neisseria gonorrhoea24,25. Also, Jatropha curcas, Passiflora foetida and Cassia tora leaves possess potent bactericidal activity against Escherichia coli, Staphylococcus aureus, Bacillus spp. and Pseudomonas aeruginosa26,27. The ethanolic leaves extract of Convolvulus arvensis and Thymus capitatus exhibited considerable antibacterial activity against the tested Gram positive bacterial (Staphylococcus epidermidis, Staphylococcus saprophyticus) and Gram negative bacterial (Proteus vulgaris, Escherichia coli, Citrobactor freundii) strains respectively28.

 

The in vitro antimicrobial activity of the essential oils of C. aromaticus and C. zeylanicus against bacteria (Bacillus megaterium, B. subtilis, Escherichia coli, Staphylococcus aureus, Proteus vulgaris, Pseudomonas aeruginosa and Xanthomonas campestris) and fungi (Aspergillus niger, A. parasiticus, Rhizopus oryzae, Rhizoctonia oryzae-sativae, Colletotrichum musae, Fusarium solani, Candida albicans, and Alternaria brassicicola) revealed that the essential oil of C. zeylanicus had slightly higher inhibitory activity against a wide spectrum of bacteria and fungi 29. Muntingia calabura showed higher inhibitory effect against the pathogens like Staphylococcus aureus, Bacillus subtilis, Escherichia coli. Basella rubra showed mild and good inhibitory activity against Staphylococcus aureus and Aspergillus niger30. Thus the phytochemicals present in the plants are acknowledged for their priceless effects and also gained increasing attention among consumers and scientific community as epidemiological studies has indicated that habitual consumption of plants rich in diverse phytoconstituents is related to a lower risk of non-communicable diseases in human31,32.

 

CONCLUSION:

The present study vividly exhibited that methanolic extract of Plectranthus amboinicus exhibited potential antibacterial and antifungal activity due to the presence of active bioactive compounds. It is more necessary to understand that a variety of species of P. amboinicus exist and their phytocomposition varies according to different geographic regions. More research needs to be done to unveil the inhibitory effect of this plant against virus and other novel antigens. The utilization of this plant has always been a tradition and mostly a community based medicine that is passed on from one generation to another. Hence through this study we insist that the traditional medicine has to be streamlined and properly documented and should be available as evidence based therapies for the benefit of humankind against various infectious diseases.

 

ACKNOWLEDGMENT:

The authors thank the authorities of Avinashilingam Institute for Home Science and Higher Education for Women, Coimbatore, Tamil Nadu, India for providing facilities to carry out the research work and DST - CURIE (No.SR/CURIE/PHASE II/01/2014) for the support given to conduct the study successfully.

 

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

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Received on 02.09.2020            Modified on 28.11.2020

Accepted on 03.01.2021           © RJPT All right reserved

Research J. Pharm.and Tech 2021; 14(12):6379-6384.

DOI: 10.52711/0974-360X.2021.01103