INTRODUCTION:

Pimenta dioica (L.) Merr (also known as Pimenta Officinalis, family Myrtaceae), are used worldwide as valuable spices. They are commonly known as allspice, Jamaica pepper and Pimento. This spice possesses the characteristic flavor and aroma of clove, nutmeg,cinnamon and black pepper, all combined in this one spice, hence named allspice¹. Pimenta dioica was used as an ingredient in Tibetan remedy PADMA 28 and was found to induce apoptosis in T cell derived lymphocytic leukemia cell line CEM-C7H2 ². Allspice along with other herbal mixtures is used to relieve indigestion. Not only used in the areas where Allspice originated from, its use has been incorporated in the Indian traditional medicine, the Ayurveda. Allspice is used for treating indigestion might be due to the abundance of the common polyphenol Eugenol in Allspice, which is known to stimulate digestive enzymes³.

Eugenol (1-allyl-3-methoxy-4-hydroxy benzene), a colourless or yellowish oily liquid extracted from clove, pimento, bayleaf, cinnamon oils, is used primarily as a flavouring agent and fragrance⁴. Pharmacologically, eugenol has been reported to exhibit antiseptic properties, analgesic action (local and general), spasmolytic and myorelaxant activities, parasympathetic effects (salivary gland secretion), and direct peripheral vasodilation. It is also used in agricultural applications to protect foods from microorganisms such as Listeria monocytogenes and Lactobacillus during storage, as a pesticide and fumigant. Eugenol possess bactericidal activity^5,6. Eugenol may have recovery effects on arthritis and can be useful as a beneficial supplement in the treatment of arthritis⁷. Eugenol is reported to possess antidiabetic activity⁸. Eugenol is typically found in plant aerial parts like leaves, bark, and flowers, which contain a lot of essential oils. Tulsi leaves have a high amount of eugenol, usually between 40 and 71 percent. The content of Eugenol in various portions of plants, however, fluctuates with the season. The present study was aimed in evaluating the bioactive compounds present in Pimenta dioica and analysing its antibacterial efficacy in vitro

MATERIALS AND METHODS:

Preparation of Ethanol extract:

32g of powdered Allspice berries was weighed and mixed with 100ml of Ethanol. It is then kept for 3days. The extract was filtered by using filter paper and the filtrate was evaporated in incubator at 30°c and used for further use.

Determination of Extract Yield

Ethanol Extract-

32g of powdered Allspice berries was weighed and mixed with 100ml of Ethanol in a separate flask. It is then kept for 3 days. The extract was filtered using filter paper and the filtrate was evaporated in incubator at 30°C. The resultant concentrates were weighed to determine the yield of the extract. The percentage of yield obtained was calculated using the formula,

W₂ – W₁

-------------------------- X 100

W₀

where, W₁= Weight of the beaker alone

W₂=Weight of the extract and the beaker

W₀ = Weight of the sample

Preliminary Phytochemical Tests

The extract of the Allspice was subjected to qualitative analysis for the presence of various phytochemical constituents.

Thin Layer Chromatography:

The solvent system was made from hexane and Ethyl acetate 7:3 (v/v). With a pencil, a thin mark is made at the bottom of the plate to apply the sample spots and Capillary tube was used to spot a sample solution on the silica gel TLC plate at 1cm from the edged of the plate and the drop is allowed to dry. The plate was placed in TLC (Chromo tank) and allows ascend the TLC plate by capillary action. The plate was removed and the solvent front was marked then allowed to dry. The iodine was used as the visualizing agent to detect the spot. A meter rule was used to measure the distance moved by the solvent and distance moved by spot, from which the retention factor (Rf values) of the various spots was calculated.

R_f = Distance move by spot front/ Distance move by solvent front

GC-MS analysis of Pimenta dioica:

Gas chromatography mass spectrometry (GC/MS) is an instrumental technique, comprising a gas chromatograph (GC) coupled to a mass spectrometer (MS), by which complex mixtures of chemicals may be separated, identified and quantified. This makes it ideal for the analysis of the hundreds of relatively low molecular weight compounds. In order for a compound to be analysed by GC/MS it must be sufficiently volatile and thermally stable. In addition, functionalised compounds may require chemical modification, prior to analysis, to eliminate undesirable adsorption effects that would otherwise affect the quality of the data obtained. The sample solution is injected into the GC inlet where it is vaporized and swept onto a chromatographic column by the carrier gas (usually helium). The sample flows through the column and the compounds comprising the mixture of interest are separated by virtue of their relative interaction with the coating of the column (stationary phase) and the carrier gas (mobile phase). The latter part of the column passes through a heated transfer line and ends at the entrance to ion source where compounds eluting from the column are converted to ions.

DPPH Radical Scavenging Assay

The DPPH radical scavenging activity was carried out as per the protocol of Brand Williams., et al.,1995⁹.

Procedure:

1. 500µl of DPPH radical solution was added to both the tubes” T”- Test and “C”- Control.

2. 500µl of Methanol was added to the “C” tube.

3. 500µl of Extract was added to the “T” tube.

4. 500µl of methanol alone serves as Blank- “B”

5. The contents of the tubes were mixed well and kept at room temperature for 30 minutes.

6. The absorbance was read at 517nm colorimetrically.

Control- Sample

% Scavenging Activity= --------------------- X 100

Control

Antimicrobial Activity:

Antimicrobial activity was determined against two different of bacteria (gram-positive and gram-negative) by agar diffusion method. Generally, the antibacterial activity of a compound is expressed in terms of its ability to inhibit the growth of bacteria in nutrient broth or agar. The antimicrobial activity of Pimenta dioica berries (extract) was determined using disc diffusion and well puncture method. Glassware and media were sterilized in autoclave at 15psi for 20 minutes, at 120ºC.

Collection of Micro-organism

Pseudomonas aeruginosa, Bacillus anthracis, Klebsiella pneumonia, Streptococcus pneumonia, Escherichia coli, were used for this assay.

Culturing and preservation of bacteria:

Cultures of test bacterial strains- Pseudomonas aeruginosa, Bacillus anthracis, Klebsiella pneumonia, Streptococcus pneumonia, Escherichia coli, were grown and preserved in the culture media following standard procedures.

Media used:

Nutrient broth was used for the preparation of inoculums of the bacteria and nutrient agar was used for screening method.

RESULTS:

Table 1: Phytochemical screening of P.diocia extract

S.NO	PHYTOCHEMICALS	INFERENCE
1	Tannins	+
2	Saponins	++
3	Flavanoids	++
4	Quinones	-
5	Coumarins	+
6	Alkaloids	+
7	Glycosides	-
8	Cardioglycosides	-
9	Terpenoids	+++
10	Phenols	+++
11	Anthocyanin	-
12	Betacyanin	+
13	Sugars	+
14	Sterols	++

+ Present; -Absent

The results showed that, ethanol extract contained terpenoids and phenols Tannins, saponins, flavanoids, coumarins, alkaloids, Betacyanin, sugars.

Thin layer chromatography:

Plate 1: TLC Fingerprints of Pimenta dioica

a)E- Standard Eugenol b) S1- Crude Extract c) S2- Purified Extract

Table 2: Spot detection in TLC

Solvent system Hexane and ethyl acetate (7:3)	Extract	Spots detected	Rf Value
	Eugenol (standard)	3	0.78
	Crude Extract	1	0.78
	Purified Extract	1	0.78

The result of thin layer chromatography of P. dioica extract is presented ins table 2. The TLC studies of the ethanol extract of P.dioica. Solvent system was hexane and ethylacetate 7:3 (v/v), 3 spot detected in standard Eugenol Rf values 0.78, 1 spot detected in crude extract Rf values 0.78 and 1 spot detected in purified extract Rf values 0.78 respectively.

GAS liquid chromatography:

Fig 1: GC MS analysis of P.diocia

The performance of standard eugenol and plant extract were analyzed through gc-ms (Fig 1) which results in the peaks at same retention time 5.3116 and 5.2326 which confirms the presence of eugenol in Pimenta dioica

Table 3: DPPH radical scavenging assay.

Extract	Test	Control	% Scavenging activity
Sample 1	2	1.69	60
Sample 2	1.9	1.69	66
Sample 3	2.1	54	54
		Average	60
		SD	4.898979486
		Mean ± SD	60 ± 4.89

Table 4 : Zone of inhibition by P.dioica plant extract

S. NO	Microorganism	Cont rol	5 mg/ml	10 mg/ml	15 mg/ml	20 mg/ ml
1	P.aeruginosa	-	0.1	0.3	0.4	0.5
2	B.anthracis	-	0.4	0.3	0.3	0.2
3	K.pneumonia	-	0.1	0.2	0.4	0.3
4	S.pneumonia	-	0.2	0.2	0.4	0.3

The zone of inhibition for 5mg/ml was maximum at B.anthracis, 10mg/ml were maximum at P.aeruginosa and B.anthracis, 15mg/ml were maximum at P.aeruginosa, K.pneumonia, S.pneumonia and 20mg/ml was maximum at P.aeruginosa.

DISCUSSION:

The extraction of Allspice berries by Ethanol solvent were used for the extraction of phytochemicals from the Pimenta dioica berries and the yield was found to be 0.790mg. The results of extraction showed that ethanol was the suitable solvent for phytochemical extraction as it recorded highest yield.

Phytochemical study revealed the presence of different phytochemical constituents in ethanol extracts of Pimenta dioica. The results of phytochemical analysis are presented in table 1. It confirms that the presence of Tannins, Saponins, Flavanoids, Coumarins, Alkaloids, Terpenoids, Phenols, Sugars, Steroids, Betacyanin. Secondary metabolites were reported in most of the medicinal plant products and showing several immunopharmacological properties which is totally related with human healthcare^10,11. The extract was found to contain the above biologically active ingredients which possess a wide range of pharmacological properties. Our studies are in par with earlier reports indicating that P. dioica leaf extract contained glycosides, alkaloids, carbohydrates, flavonoids, tannins, and proteins¹². The extract was reported to contain Methyl eugenol, eugenol, myrcene, β-caryophyllene, cineole, and limonene have also been reported^13-15. The result of thin layer chromatography of P. dioica extract indicated the presence of eugenol in purified extract Rf values 0.78 respectively (table 2).

Table 3 depicts the results of DPPH radical scavenging assay. In the present study the result of the DPPH Scavenging assay confirmed the antioxidant ability of the Allspice berries. The antioxidant activity of phenolics is mainly due to their redox properties which allow them to act as reducing agents, hydrogen donors, and singlet oxygen quenchers and may also have a metal chelating potential and the ethanolic extracts of basil seeds has high antioxidant activity which were 60± 4.89%

Hexane and ethyl acetate extract showed the presence of Eugenol (Rf 0.78); in crude Extract (Rf 0.78), and powdered Extract (Rf 0.78) were present. Thin layer chromatography is usually done for a better identification of the bioactive compounds. In the present study the TLC profiling of the plant extracts again revealed the presence of eugenol. In this study the ethanol extract of Allspice berries were analysed by GC-MS. GC-MS Pattern of the ethanolic extract confirms the presence of eugenol a potent phytochemical and qualitative analysis revealed the presence of various phytoconstituents which contribute to the anti-microbial efficacy of Pimenta dioica.

According to the USDA (2012), foodborne illnesses are primarily caused by bacteria. The need for more Natural meals with little or no processing have been growing; as a result, it is advised to apply natural preservatives, including herbal and spice extracts^17,18to lessen the presence of microorganisms. Disease-causing microbes become resistant to drug treatment are a growing public health problem. Many studies are now developing new antimicrobial with the emergence of microbial organisms’ resistant to multiple antibiotics¹⁹. The use of medicinal plants shows a high potential to treat as antimicrobial agents which generally used as phytochemical extract of the medicinal plant^20,21. Numerous Research indicates that adding aromatic plants directly adding food extracts and essential oils to oxidative or antibacterial properties Carvalho-Costa et al (2015). One kind of aromatic plant is pimenta dioica contains appreciable amount of eugenol, a phenolic component with a broad range of antibacterial properties, in its essential oil²². The P. dioica extract also contains glycosides, alkaloids, proteins, carbohydrates, flavonoids, and tannins, among other bioactive substances. The Antibacterial potential of raw and encapsulated Pimenta dioica essential oil was well established²³.

Table (4) shows the result of antibacterial activity of P. dioica against clinical isolates of S. aureus and p.aeruginosa. P. dioica was more effective in inhibiting test bacteria showed more inhibition in S.pneumonia and P.aeruginosa and lesser inhibition in E.coli and K.pneumonia Plants are shown to be important sources of antibacterial agents capable of inhibiting S. aureus including drug resistant strains. A number of studies have been undertaken which report the efficacy of plant extracts and their purified components to inhibit S. aureus. Sortase A is responsible for the virulence of Gram-positive pathogens, including staphylococci and streptococci. The LPETG is the peptide surface anchor signal for Sortase A. The inhibitors of this enzyme shared similar binding pattern with substrate LPETG. Eugenol and its derivatives may act as sortase A inhibitor. The antimicrobial activity of eugenol and its derivatives was tested in vitro against bacterial strains: Staphylococcus aureus, Streptococcus mutans and Escherichia coli. All the tested derivatives demonstrated antimicrobial activity. Differences between derivatives in terms of in vitro activity and interactions between the amino acid residues were correlated in the docking analysis for the same derivatives. According to the relationship observed in this study between the antimicrobial activity of eugenol and the LPETG peptide structure, some of the eugenol derivatives proved to be more active inhibiting sortase A than eugenol against microorganisms when tested at the same concentrations²⁴. Essential oils (EOs) obtained from medicinal plants have become important area in scientific research because of their ability to treat various ailments²⁵. Hence the use of P.diocia containing eugenol could be widely used for its well reported antioxidant as well as antibacterial properties

CONCLUSION:

Pimenta dioica contains a cornucopia of medicinal compounds that have been exploited by native Caribbean population as well as medical systems of distant countries, such as India where it has a known entry in the Ayurveda system of medicine. We have presented a critical evaluation of its medicinal properties with special attention as a chemo-dietary prevention agent for chronic diseases and malignant cancers. Several compounds abundantly found in Allspice, namely Quercetin, Gallic acid and Ericifolin show both in vitro and in vivo antiproliferative and antitumor activities. GC-MS Pattern of the ethanolic extract confirms the presence of eugenol The ethanolic extract of plant proved to have anti-microbial effect on disease causing Pseudomonas aeruginosa, Bacillus anthracis, Klebsiella pneumoniae and Streptococcus pneumoniae with MIC of 5mg of the ethanolic extract which may be due to the different phytocontituents in the plant extract and partly due to eugenol a potent anti microbial compound.

Studies have shown the efficacy of eugenol against a number of different microorganisms of different origin. These findings indicated a possible use of eugenol as therapeutic tool against a wide variety of diseases. The promising numbers on therapeutic potential of phenolic Eugenol is encouraging and further investigations are needed to fully realize the potential of these derivatives in human health and diseases.

ACKNOWLEDGEMENT:

The authors wish to thank the Department and Management of Dwaraka Doss Goverdhan Doss Vaishnav College for having provided the support for carrying out the study.

CONFLICT OF INTEREST:

There is no conflict of interest.

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Received on 13.10.2023 Modified on 22.11.2023

Research J. Pharm. and Tech 2023; 16(12):5878-5883.

DOI: 10.52711/0974-360X.2023.00952