Phyto-pharmacological Evaluation of Pimenta dioica (Linn.) Merill, Essential oil for Anthelmintic and Antimicrobial potential

 

Ardra R Nellikunnel1, Sindhu Rani JA3, Julie Joy2, Jinu John1,2*

1Department of Biotechnology, CMS College, Kottayam, Kerala.

2Doctor John’s Biotech Centre for Research and Development, Kottarakara, Kerala.

3Department of Biochemistry, NSS College, Nilamel, Kollam, Kerala.

*Corresponding Author E-mail: jinu@cmscollege.ac.in

 

ABSTRACT:

Pimenta dioica (Linn.) Merill., belongs to the family Myrtaceous, has been used as a spice and is well known for its culinary as well as medicinal values. The aim of the current study was to assess the in vitro antimicrobial, anthelmintic activity and phytochemical composition of the essential oil extracted from Pimenta dioica leaves. The extraction of essential oil was performed with the Clevenger apparatus, by hydro-distillation. Pimenta dioica essential oil yield was 1.03%. The GC-MS analysis of this Pimenta dioica essential oil showed that eugenol (74.72%) was the predominant component. The essential oil showed potent anthelmintic activity assay when analysed by the adult worm’s motility assay. It has shown a comparable paralysis and disintegration profile of the model organism to that of the standard drug albendazole. The essential oil was also found to be effective against common human pathogenic bacteria, including Staphylococcus aureus, Klebsiella, Escherichia coli, Chromobacterium violaceum and Proteus vulgaris. It also showed moderate antifungal activity against Candida albicans and Aspergillus niger. These results proved that Pimenta dioica essential oil could be used as a source of the lead molecule for developing novel anthelmintic or antimicrobial agents.

 

KEYWORDS: Pimenta dioica, Anthelmintic activity, Essential oil, Antimicrobial potential.

 

 

INTRODUCTION: 

Pimenta dioica (Linn.) Merill, known as Allspice is a typical evergreen plant belongs to the Myrtle family. Southern Mexico, the West Indies, and the tropical forests of the Central and South America are the natural habitats of P. dioica. The trees can reach heights of 43 feet and have dark green leaves and light grey bark1. P. dioica and other herbal decoctions are used in Indian Ayurveda to treat gastrointestinal disorders such as dyspepsia. It has recently the used as a natural substitute to pesticide and fungicide. To ease the pain of strained and cramped muscles, it is added to massage oils. It is also used to treat dyspepsia, headaches, deal with stress and depression and get over weariness2.

 

Essential oils are concentrated, highly volatile, hydrophobic, less viscous than oil, less dense than water, and highly aromatic, and have distinctive fragrances. The aromatic essential oil from Allspice finds applications in the industries such as perfumery, candle making, and cosmetic preparations. The antimicrobial, antioxidant, anti-inflammatory, anti-proliferative, and apoptosis-inducing capabilities of essential oil extracted from Pimenta diocia was thoroughly reviewed and reported by Zhang and Lokeshwar (2012). Allspice essential oils are also reported as oxygen scavengers, vasodilators or antihypertensive, and antiproliferative agents with therapeutic potential as a chemo-preventive agent in cancer treatments. In Caribbean culture, Pimenta diocia leaf extract is used as a folk cure for the treatment of high blood pressure, obesity, digestive issues, menstrual cramps, and stomach pain. It is also said to have a hypotensive effect.2

 

The gastrointestinal infection by parasitic worms is a severe health concern for both humans and livestock, especially in underdeveloped nations. Anthelmintics created by chemical synthesis are being used to combat these nematodes. Finding new medications is necessary to control and maintain successful therapy since resistance to current anthelmintics is now rising. Natural products such as phytochemicals can be a significant source of numerous drug-lead compounds with pharmacological activity. Due to their potential to treat condition that results in significant financial loss and lowers animal productivity for livestock owners, phytochemicals' anthelmintic properties have attracted a lot of attention. Natural plant extracts are traditionally used to treat helminthiasis in animals and humans; however, scientific evidences to prove its efficacy and their chemical composition is still lacking. According to the reports by authors, phytochemicals are found to be a promising alternative to synthetic anthelmintic drugs for treating gastrointestinal helminth infections3-6.

 

Antibiotic resistance has emerged as a result of widespread usage of antibiotics, creating a new health issue. The multidrug resistant bacteria like Staphylococcus aureus, Salmonella sp., Shigella, Enterococcus sp., and Escherichia coli, categorized as both community- and hospital-acquired illnesses. Due to this, there is a high demand for novel and effective antibiotics with least side effects. Now the researchers and scientific community has started to show interest in employing herbal medications with antibacterial properties7. Due to their antifungal and antibacterial properties, essential oils have been investigated as potential sources of new antimicrobial agents aiding food preservation and as alternatives to treat infectious disorders8. New antimicrobial compounds derived from various natural sources are being researched and developed in an effort to tackle antibiotic resistance. The approaches for assessing antimicrobial activity screening have therefore drawn more focus. Numerous studies on the antibacterial properties of essential oils and their chemical composition have been undertaken and published9–12. Significant antiseptic, antioxidant, antiparasitic, antibacterial, antiviral, antifungal, and insecticidal properties have been demonstrated for essential oils13. In order to find new molecules that reduce bacterial resistance, it may be useful to explore and evaluate essential oils. The purpose of this study was to investigate the in vitro antibacterial and anthelmintic capabilities of Pimenta dioica essential oil extracted from the leaves as well as to analyse its phytochemical composition.

 

MATERIALS AND METHODS:

Extraction of essential oil:

Pimenta dioica Linn. leaves were freshly collected from the farm fields of Kottarakkara, Kerala. 200g of this chopped leaf sample was then loaded in the RB flask of the Clevenger apparatus and about 350ml of water was added to it. Essential oil was collected by hydro-distillation process using the Clevenger apparatus. For the essential oil extraction, chopped leave samples were mixed with water and boiled to evaporate volatile components for 3hrs. The oil fraction was collected from the condensate.  Separated oil was then collected in glass bottles containing anhydrous sodium sulphate to remove the residual water content.  It was then stored under refrigeration till further studies.

 

Gas Chromatography-Mass spectroscopic Analysis of the Essential oil:

The essential oil was diluted in hexane (1:100) and filtered through 0.22μm syringe filter before the analysis. GC–MS (QP-2020-NX Shimadzu) equipped with capillary column SH-I-5Sil MS(5% phenyl and 95% polydimethyl siloxane) with a specification of length (30m), inner diameter (0.25mm) and film thickness (0.25μm) was used for the analysis. One microlitre of the sample was injected in split mode with split ratio of 75. Electron ionisation mass spectra were measured at 70 eV over the mass range 45-550. Helium was used as carrier gas at an injection temperature of 250°C. The column temperature was maintained at 60°C for 6 min, then gradually increased to 165°C at the rate of 12°C for 6 min, and finally to 250°C with a rate of 25°C and maintained for 6min. Identification of the phytochemical components were done by using the NIST 20 Library, as the main reference database, all integrated peak of the chromatogram was identified automatically by the GC-MS solution by considering the MS spectral similarity and the Linear Retention Indices (LRI).

 

Antibacterial screening:

Antibacterial assay on Muller-Hinton agar was performed by standard well diffusion method. Escherichia coli, Staphylococcus aureus, Proteus vulgaris, Chromobacterium violaceum and Klebsiella pneumoniae were used for the anti-bacterial studies. The culture plates were prepared by the swabbing technique. Using sterile swab buds, pre-cultured microorganisms were swabbed onto the Muller Hinton Agar plate. Pre-cultured microbes were swabbed into the Muller Hinton Agar plate using sterile swab buds. After 5 min., wells were punched into the agar plate and 10µl and 15µl oil samples were applied in separate wells and an antibiotic disc of tetracycline (30µg) (HIMEDIA) was used as the reference standard. The average diameter of zone inhibitions was measured after 24hours of incubation at 37°C in a bacteriological incubator (KEMI).

 

Anthelmintic Bioassays:

The anthelmintic study was performed as per the method reported by Jinu et al., (2009)14. Indian earthworms (Pheretima posthuma) were used as the test model organisms. They are one of the most widely used models in the research of anthelmintic medications because of their similarities to intestinal parasites.  The earthworms used in the anthelmintic investigation were procured from the damp soil of nearby farm fields, washed with normal saline, and then used. Due to their morphological and physiological similarity to human intestinal roundworm parasites, the earthworms, which are 4-6 cm long and 0.1–0.2cm wide, were employed for all the experimental protocols15.

 

The essential oil was diluted with DMSO (1:1) and normal saline to get 50,100 and 200µl/ml of working concentrations. The standard anthelmintic drug Albendazole (100µg/ml) was used as the positive control. The extract and drug solutions were prepared freshly before the experiment. Eight experimental groups consist of four earthworms each were released into 10ml of desired formulations as follows: normal saline (vehicle control), Albendazole (100µg/ml), and three separate groups in duplicate were treated with the extracts of 50, 100 and 200µg/ml accordingly. The duration of time period it took each individual earth worm to become paralyzed and die was observed. When the worms were become unable to respond or move even when shifted to normal saline, paralysis was thought to have occurred. The worms were reported as dead when they stopped moving, and their body colours started to fade.

 

RESULTS AND DISCUSSIONS:

GC-MS analysis of Pimenta dioica essential oil:

Fresh Pimenta dioica leaves were used to extract the essential oil, and after 3hours of distillation, the yield was reported to be 1.03%. Gas chromatographic analysis of the oil reported several peaks representing its component phytochemicals (Fig.1). Mass spectroscopic analysis revealed the presence of several components, with 1,3,4-Eugenol (74.2%) as the major component (Table 1). This observation was in concordance with reports by others. The phytochemical composition of P. dioica essential oil from Jamaica reported the presence of eugenol, caryophyllene, α-pinene, limonene and 1,8-cineole; whereas the analysis of P. dioica essential oil from Sri Lanka revealed a highest percentage composition of eugenol (85.33%) followed by β-caryophyllene (4.36%), cineole (4.19%), linalool (0.83%) and α-humulene (0.76%)16-17.


 

Fig 1: Gas chromatogram of Pimenta dioica essential oil

 

Table 1: Phytochemical constituents of Pimenta dioica essential oil

Peak

R. Time

% Composition

Name

Base m/z

1

5.494

1.00

α –pinene

93.10

2

6.730

3.27

Amyl vinyl carbinol

57.05

3

7.022 

7.14

β-Myrcene

93.10

4

7.481.

1.30

α-Phellandrene

93.10

5

8.150

0.49

Cymene

119.15

6

8.284  

6.43

Limonene

68.10

7

10.919

1.06

β-Linalool

71.10

8

14.087

0.53

1-Terpinen-4-Ol

71.10

9

17.827

4.05

4-Allylphenol

134.15

10

21.979 

74.72

1,3,4-Eugenol

164.15

 

Antimicrobial activity:

Pimenta dioica essential oil showed moderate antimicrobial potential against the tested Gram-positive and Gram-negative bacteria: Escherichia coli, Staphylococcus aureus, Proteus vulgaris, Bacillus subtilis and Chromobacterium violaceum. Antifungal potential was tested against Candida albicans and Aspergillus niger (Fig.-1, Table 2). The essential oil showed highest activity against B. subtilis (diameter of zone of inhibition: 29mm at a concentration of 20µl), while it was least effective against C.violaceum  with a diameter of inhibition zone, 15mm at a concentration of 20µl.

 

Table 2: Antimicrobial assay of Pimenta dioica leaf essential oil

 

Zone of inhibition (mm)

Essential oil

Tetracycline

10µl

20µl

30µg

Bacteria

E. coli

23

25

33

S.aureus 

19

21

38

B. subtilis

28

29

26

E. aerogenes

14

17

31

K.pneumoniae

15

16

20

P.vulgaris

11

17

19

C.violaceum

14

15

19

Fungus

C.albicans

13

16

10

A. niger

18

21

39

 

The mechanism of action behind the antimicrobial effect of the essential oils against these bacteria could be due to the interference in the formation of the peptidoglycan molecule, a basic structural component of the cell wall of bacteria18. A loss of cell selectivity and an osmotic imbalance brought on by this improper cell wall fabrication might result in bacterial cell lysis. Another possible mechanism of action is interference with protein synthesis in the 30s and 50s subunits of the bacterial ribosome19. Clove essential oil contains eugenol, a molecule with significant antibacterial properties20. The antibacterial, antifungal, antiarthritic, and anticancer potential of Ocimum Sanctum essential oil are also reported to be attributed to eugenol, one of the most potent phytochemical constituents of essential oils21. One of the biggest concerns facing doctors and researchers is the worrisome surge in the multidrug resistance of human infections. The hunt for new and effective medications to address this issue has become necessary due to the ineffectiveness of present medical treatments. The phytochemical components of essential oils are reported to have a wide range of bioactivities, such as anti-inflammatory and antimicrobial22–23.

 

Anthelmintic activity:

The essential oils of P. dioica showed anthelmintic property in a concentration-dependent manner, when evaluated by adult earth worm motility assay (Table 3). The anthelmintic property of essential oil was found to be comparable with that of albendazole in terms of time duration needed for paralyzing and death of the worms. The time duration for paralyzing and death of the worms by P. dioica essential oil at a concentration of 100 ppm was 31 min. and 46 min, while that of albendazole was 28 and 42 min respectively.  The phytochemical composition of the essential oil such as euginol could be responsible for the activity. Due to poor sanitation and hygiene, intestinal parasitic infections are one of the major health issues in the underdeveloped nations, especially among children. It is necessary to focus our research on natural medications based on the medicinal properties of the plants because of the occurrence of drug resistance and possible side effects of modern synthetic drugs24-26.

 

Figure 2: Antimicrobial assay of Pimenta dioica essential oil

 

Table 3: Anthelmintic activity of Pimenta dioica essential oil

Test Sample

Concentration (ppm)

Time taken for paralyze (min)

Time taken for death (min)

Solvent Control (DMSO + Saline)

-

-

-

Pimenta dioica essential oil

50

41±2.86

58±1.98

100

31±1.14

46±2.72

200

19±1.32

26±1.02

Albendazole

100

28±1.86

42±0.94

Observations are expressed as mean ± SEM, N=4.

 

CONCLUSION:

Pimenta dioica essential oil may find applications in various industries such as pharmaceuticals, food, cosmetics and perfumery due to its beneficial health effects. The essential oils extracted from P. dioica leaves were evaluated for its antimicrobial and antihelminthic activity. From the chemical analysis, we have identified ten compounds, of which eugenol was the major component. The phytochemicals of this essential oil turn out to be useful as an antimicrobial and antihelminthic agent, in addition to their other biological properties and industrial applications.

 

CONFLICTS OF INTEREST:

The authors hereby declare that we have no financial or other personal stake in the topics raised or conclusions drawn in this work.

 

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Received on 05.02.2023            Modified on 24.06.2023

Accepted on 19.10.2023           © RJPT All right reserved

Research J. Pharm. and Tech 2024; 17(3):1045-1049.

DOI: 10.52711/0974-360X.2024.00162