D C Abeysinghe

P B C Panawala

R M Dharmadasa

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World Journal of Agricultural Research, 2016, Vol. 4, No. 5, 143-146 
Available online at http://pubs.sciepub.com/wjar/4/5/3 
©Science and Education Publishing 
DOI:10.12691/wjar-4-5-3 
 
Phytochemical Distribution and Bioactivity of Different 
Parts and Leaf Positions of Pimenta Dioica (L.) Merr 
(Myrtaceae) 
P.B.C. Panawala1, D.C. Abeysinghe1, R.M. Dharmadasa2,* 
1Department of Plantation Management, Faculty of Agriculture and Plantation Management, Wayamba University of Sri Lanka, 
Makandura, Gonawila (NWP), 60170, Sri Lanka 
2Herbal Technology Section, Industrial Technology Institute, Bauddhaloka Mawatha, Colombo 07, 00700, Sri Lanka 
*Corresponding author: dharmadasarm@gmail.com 
Abstract  Pimenta dioica (L.) Merr. (Myrtaceae) is an evergreen aromatic spice widely used in perfumery, food 
and cosmetic industry in many parts of the world. Present study compared Total Antioxidant Capacity (TAC), Total 
Phenolic Content (TPC), Total Flavonoid Content (TFC), leaf area (LA), and Fresh to Dry weight ratio of Pimenta 
dioica leaves at different leaf positions (1st, 2nd, 3rd, 4th and 5th leaf positions) and different plant parts (immature leaf, 
mature leaf and bark). The TAC, TPC and TFC were determined using Ferric Reducing Antioxidant Power Assay 
(FRAP), modified Folin–Ciocalteu colorimetric method and calorimetric method respectively. Significantly higher 
TAC 562.38 ± 9.42 (mg TE/g DW), TPC 279.53±7.02 (mg GAE/g DW) were observed in leaf extract obtained from 
1st leaf position. However the highest TFC 303.48 ±8.87 (mg RE/g DW) was observed in 5th leaf position. 
According to phytochemical distribution pattern, significantly higher TAC [619.84 ±11.98 (mg TE/g DW)], TPC 
[267.53 ± 5.03(mg GAE/g DW)], TFC [305.48 ±8.87 (mg RE/g DW)] were observed in extracts obtained from bud 
region. The potential of Pimenta dioica leaf material and bark as a fabulous raw material for food, perfumery and 
cosmetic industries. Further harvesting of immature leaves could be suggested for better therapeutic benefits. 
Keywords: Pimenta dioica, Total Antioxidant Capacity, Total Flavonoid Content, Total Phenolic Content 
Cite This Article: P.B.C. Panawala, D.C. Abeysinghe, and R.M. Dharmadasa, “Phytochemical Distribution 
and Bioactivity of Different Parts and Leaf Positions of Pimenta Dioica (L.) Merr (Myrtaceae).” World Journal of 
Agricultural Research, vol. 4, no. 5 (2016): 143-146. doi: 10.12691/wjar-4-5-3. 
1. Introduction 
Pimenta dioica (L.) Merr. (Myrtaceae) is well known 
for its characteristic aroma, therapeutic and culinary 
qualities which resembles to aroma and flavor of clove, 
nutmeg cinnamon and hence it is called allspice [1]. Plant 
is known as, Jamaica pepper, Myrtle pepper and new spice 
in English [2]. Further, P. dioica is small (7-10 m) 
evergreen tree which is cultivated in Jamaica, Cuba, Haiti, 
Brazil, Central America, West Indies, Venezuela, Mexico, 
Honduras, Guatemala and Grenada [3] . Leaves and seeds 
are widely used as commercial items in traditional 
medicine for the treatment of diverse range of ailments 
including flatulence, diarrhea, neuralgia, rheumatism and 
digestive problems [4]. Moreover, it is valued for its 
secondary metabolites like essential oil with compositions, 
oleoresin which is highly utilized in food industry, 
perfumery and cosmetic industry. These diverse ranges of 
utilities may be due to presence of therapeutically active 
secondary metabolites in both seeds and leaves of P. 
dioica. On the other hand, once important plant source 
found, its commercial planting material production is vital 
important. Allspice is conventionally propagated through 
seeds, but the seeds lost their viability soon after harvest [5].  
Allspice being a polygamodioeous tree, identification 
of the functional male and female trees till they flower is 
difficult and hence, clonal propagation is essential to 
obtain uniformly high yielding trees [6]. The substances, 
traditionally referred to as secondary metabolites or 
phytochemicals, often are differentially distributed among 
limited taxonomic groups within the plant kingdom [7]. 
Phytochemicals are chemical compounds that occur 
naturally in plants. Some are responsible for colour and 
other organoleptic properties. The most important of these 
bioactive constituents of plants are alkaloids, tannins, 
flavonoids, and phenolic compounds [8]. Secondary 
metabolites are those metabolites which are not 
functioning in growth (although they may have survival 
function). Many of these compounds now have been 
shown to have important adaptive significance in 
protection against herbivory and microbial infection, as 
attractants for pollinators and seed dispersing animals, and 
as allelopathic agents (allele chemicals that influence 
competition among plant species). Total phenolic content 
(TPC), total flavonoid content (TFC) and total antioxidant 
capacity (TAC) have been renowned as therapeutically 
active substances which regulate important physiological 
actions on the human body [9]. Since antioxidant function 
as free radical scavengers and quenchers of singlet oxygen 
formation, investigation of natural antioxidants has greatly 
 World Journal of Agricultural Research 144 
 
increased in recent years [10]. Out of the commonly used 
spice crops, allspice is distinguished due to its multi 
aroma and flavor characteristics [2].  
Although there are scattered information available on 
different phytochemicals present in different parts of P. 
dioca, systematic study on phytochemical distribution in 
different parts and leaf positions are scattered. Therefore, 
in the present study we compare the distribution of 
different phytochemicals and bioactivity in different parts 
and different leaf positions of P.dioica in order to 
determine the most suitable parts for harvesting by means 
of phytochemicals and bioactivity. 
2. Materials and Methods 
2.1. Location  
The experimental site was situated in the Low Country 
Intermediate-Zone (IL1a), at an elevation of 25 m above 
mean sea level [11]. The average annual temperature, 
relative humidity and solar intensity of the experimental 
area are 31°C, 78% and 85 kilo lux respectively.  
2.2. Planting Materials  
Planting materials were collected from 20 years old 
well grown, pest and disease free mother plant. Plant 
materials were collected and authenticated with comparing 
institutional herbarium specimens. 
2.3. Measurement of Leaf Area  
Leaf area was measured by a leaf area meter (LI-3100C, 
USA). 
2.4. Extraction of Phytochemicals 
Leaves were cut into pieces and air dried for three days 
at room temperature (28±2°C). Then samples (three 
samples from each category) were powdered using motor 
and pestle and sieved with 0.25 mm mesh. 
Powdered samples (0.1 g) were mixed with 5 mL of  
80% methanol vortexed for 15 min. Then it was placed in 
a water bath at 60°C for 40 min and vortex procedure was 
repeated in 10 min interval. After centrifugation at 4,000 
rpm for 5 min, the supernatant was decanted into a 15 mL 
centrifuge tube and the remaining was re-extracted with 5 
mL of 80% methanol. 
2.4.1 Chemicals and Regents 
6-hydroxy-2,5,7,8-tetramethy-chroman-2-carboxilic 
acid (Trolox), Gallic acid, Rutin, 2,4,6-tripyridyl-2-try-
azine (TPTZ), Folin-Ciocalteu reagent and ferric chloride 
(FeCl3.6H2O) were purchased from Sigma Aldrich 
Chemical Co. (St. Louis, Mo) and all other chemicals used 
were analytical grade. 
2.4.2. The Total Phenolic Content (TPC) 
The total phenolic content was quantified using 
modified Folin-Ciocalteu method [12]. Briefly, 4 mL of 
distilled water and 0.5 mL of properly diluted leaf extract 
were mixed with 0.5 mL of 0.5 N Folin-Ciocalteu reagent 
(FCR) and allowed to react for 3 min. Then 1 mL 
saturated sodium carbonate solution was mixed and 
incubated in a water bath for 2 h at 30 °C. The absorbance 
was measured at 760 nm using UV visible spectrophotometer 
(Shimadzu UV Mini 1240 Japan). Gallic acid was used as 
the standard and data were expressed as mg of Gallic Acid 
Equivalent (GAE) /g DW. 
2.4.3. Quantification of Total Flavonoid (TFC) 
Total flavonoid content (TFC) was determined by a 
colourimetric method, with slight modifications [13]. 
Briefly, 0.5 mL of the plant extract was diluted with 3.5 
mL of distilled water. Then 0.3 mL of a 5% NaNO2 
solution was added to the mixture. After 6 min, 0.3 mL of 
a 10% Al(NO3)3. 6H2O solution was added, and the 
mixture was allowed to stand for another 6 min. Then 2 
mL of 2 M NaOH was added, and top up to 8 mL with 
distilled water. After thoroughly mixing, the absorbance 
was measured at 510 nm using UV visible 
spectrophotometer (Shimadzu UV Mini 1240 Japan). 
Rutin was used as the standard and data were expressed as 
mg of rutin equivalent (RE) /g DW. 
2.4.4. Determination of Total Antioxidant Capacity 
(TAC) 
Total antioxidant capacity was determined using Ferric 
Reducing Antioxidant Power (FRAP) assay [14]. 
Methanolic leaf extract (100 μL) was mixed with 900 μL 
of freshly prepared FRAP reagent of pH 3.6 containing 
2.5 mL of 10 mol/L, TPTZ solution in 40 mmol/L, HCl 
plus 2.5 ml of 20 mmol/L FeCl3 and 25 mL of 300 mol/L 
acetate buffer. Absorbance was measured at 593 nm using 
the spectrophotometer (Shimadzu, UV Mini 1240, Japan ) 
after incubating for 4 min. The trolox was used as the 
standard solution and TAC was expressed as mg Trolox 
Equivalents (TE) /g DW. 
2.5. Statistical Analysis  
Statistical comparison of mean values was performed 
by general linear model (GLM) of ANOVA followed by 
Turkey multiple range test of the SAS software package 
(SAS Institute, 1999).  
3. Results and Discussions 
Spices are products derived from different parts of the 
aromatic plants i.e. leaves, roots, stem, branches, flowers, 
seeds, stigmas and styles or the entire plant tops which are 
heavily used by ethnic groups for their flavor, colour, 
aroma and preservation for foods or beverage worldwide 
[15]. The secondary metabolites (phenolics and flavonoids) 
and bioactivity play an important role in plant therapeutic 
value of medicinal plants. These secondary metabolites 
support mainly for the natural defense system, and mainly 
depend on the development stage of plants [16,17]. In the 
present study attempts were made to compare the variation 
of total antioxidant capacity, total phenolic content and 
total flavonoid content of leaves of Pimenta dioica at 
different maturity stages (Table 1). Total antioxidant 
capacity, was decreased with the maturity of leaves and 
TAC of 1st 2nd, 3rd leaf positions were significantly 
different from 4th and 5th leaf positions. 
As demonstrated in Table 1, Total phenolic content and 
total flavonoid content were decreased up to 3rd leaf 
145 World Journal of Agricultural Research  
 
position. However, they were significantly increased at 5th 
leaf position. Meanwhile leaf area was increased up to 4th 
leaf position and decreased at 5th leaf position (Table 1). 
Presence of higher content of TPC, TFC and TAC in 
immature leaves and bud, might be due to the less 
cellulose and pectin in immature leaves and the active 
components present in tender parts are more soluble than 
those present in mature parts. Observed high TAC, TPC 
and TFC in immature leaf and bud, extracts are in 
agreement with previous studies [3].Who investigated the 
higher TAC, TPC and TFC in immature and mature 
Pimenta dioica leaves and who investigated immature and 
mature tea (Camellia sinensis) leaves [18]. 
Table 2 demonstrates the distribution of total 
antioxidant capacity, total phenolic content, total 
flavonoid content in different parts (leaf bud, immature 
leaf, mature leaf and bark) of P. dioica.  
The results clearly demonstrated that the highest TAC, 
TPC and TFC were contained in leaf bud. Order of 
decrease of total antioxidant capacity was leaf 
bud>immature leaf>mature leaf>bark.  
The highest TAC was observed in bud (619.84±11.98 
mg TE/g DW) and the lowest TAC was observed in bark 
(317.67±13.14 mg TE/g DW). As shown in Table 2, TFC, 
bud potion significantly different from bark potion, and 
bark showed higher significant difference than immature 
and mature leaf.  
The highest TPC and TFC were observed in bud 
(267.53±5.03 mg GAE/g DW and 305.48±8.87 mg RE/g 
DW respectively) whereas the lowest TPC and TFC were 
observed in mature leaf (131.53±3.06 mg GAE/g DW and 
221.90±3.30 mg RE/g DW respectively). The correlation 
value between TAC and TPC was R2=0.9462. 
Table 1. Total antioxidant capacity (TAC), total phenolic content (TPC), total flavonoid content (TFC), leaf area, and dry weight:Fresh weight 
ratio of leaves of Pimenta dioica at different leaf positions 
Leaf  
positions 
TAC 
(mg TE/g DW) 
TPC 
(mg GAE/g DW) 
TFC 
(mg RE/g DW) 
Leaf 
area(cm2) Dry weight:Fresh weight ratio 
1st  562.38±9.42a 279.53±7.02 a 296.43±1.89a 0.6685±0.0002 37.80:100 
2nd  552.36±1.63a 206.87±2.31b 291.90±1.49a 13.8515±0.0090 41.91:100 
3rd  546.12±8.69a 154.20±8.72c 259.52±6.44b 39.209±0.113 49.51:100 
4th  522.82±2.86b 154.20±5.29c 269.53±1.80b 50.4705±0.00305 52.12:100 
5th  501.41±11.04c 152.87±1.15c 303.48±8.87a 42.082±0.01033 53.59:100 
Values within a column followed by different letter are significantly different (P<0.05), GAE-Gallic acid equivalent; TE- Trolox equivalent; RE- Rutin 
equivalent. 
Table 2. Total antioxidant capacity (TAC), total phenolic content (TPC), total flavonoid content (TFC) of Pimenta dioica at different plant 
parts 
Parts TAC (mg TE/g DW) 
TPC 
(mg GAE/g DW) 
TFC 
(mg RE/g DW) 
Bud 619.84±11.98a 267.53±5.03a 305.48±8.87a 
Immature leaf 395.99±11.10b 192.20±8.00b 225.48±5.95c 
Mature leaf 325.80±10.29c 131.53±3.06c 221.90±3.3°C 
Bark 317.67±13.14c 143.53±8.33c 245.71±1.43b 
Values within a column followed by different letter are significantly different (P<0.05); GAE-Gallic acid equivalent; TE-Trolox equivalent; RE-Rutin 
Equivalent. 
4. Conclusions 
Present study investigated phytochemicals distribution 
and bioactivity in different parts and different leaf 
positions of Pimenta dioica. All tested bioactive 
compounds were distributed in all tested parts and the 
highest bioactive compounds were reported from leaf or 
leaf bud positions. Further, present study clearly indicated 
that leaf can be used as raw materials for industrial 
purposes instead of fruits.  
Acknowledgements 
Authors wish to express their gratitude all academic and 
nonacademic members in Department of Plantation 
Management, and Mr. Nawarathna, home garden owner, 
Kundasale, Kandy, Sri Lanka, for their valuable assistance 
given to conduct this research study successfully. 
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Bioactivity of Different Parts and Leaf Positions of Pimenta Dioica (L.) Merr (Myrtaceae)

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Bioactivity of Different Parts and Leaf Positions of Pimenta Dioica (L.) Merr (Myrtaceae)

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