The chemical composition of the essential oil obtained by hydrodistillation from the leaves of Phyllanthus reticulatus Poiret (Euphorbiaceae) growing in Nigeria has been studied. The constituents of the oil were analyzed by means of gas chromatography (GC-FID) and gas chromatography coupled with mass spectrometry (GC-MS). Monoterpenes (64.9%) were the dominant class of compounds, followed by sesquiterpenes (23.0%). The major constituents were α-pinene (6.0%), sabinene (7.6%), β-pinene (18.1%), linalool (6.9%) and camphor (7.7%), among the monoterpenes, and β-caryophyllene (11.9%) and germacrene D (8.6%) among the sesquiterpenes. This is the first report on the volatile constituents of Phyllanthus reticulatus.
Phyllanthus reticulatus Poiret., (Family Euphorbiaceae) is a many branched deciduous shrub or small tree sometimes partially scrambling, usually 1 - 5 m high. P. reticulatus usually has a distinct smell that is emitted by the minute flowers when they open towards the early evening. The bark is light reddish-brown or grey-brown with hairy stems when young, which become smooth with age. The leaves alternate along slender branches. They are up to 25 cm long and appear as leaflets of large pinnate leaves. The leaves are thinly textured, usually hairless. It flowers from September to October, but the flowering season can extend from July onwards. P. reticulatus has very small, roundish berry like fruits that are green at first, turning purple-black, 4 - 6 mm in diameter [1] . Extracts of the plant is known to possess both analgesic and anti-inflammatory activities [2] - [4] . Aqueous extract of P. reticulatus can be utilized for prevention of atherosclerosis in hypercholesterolemic patients [5] . There are reports which describe the antiviral [6] , antibacterial [7] , hepatoprotective 8], antioxidant [9] , potential RNase H inhibition and protection against the viral cytopathic effects of HIV-1 [10] , antidiabetic [11] and hypoglycemic [12] activities.
Some biologically active compound such as 2-α-hydroxyfriedel-4(23)-en-3-one and other triterpenoids [13] , purine, 3-(3-methylbut-2-en-1-yl)isoguanine and cleistanthane-type diterpenoid glucoside, 19-hydroxysprucea- nol 19-O-β-D-glucopyranoside [14] , (5R, 6R)-4,6-Dimethoxycarbonyl-5-[2',3',4'-trihydroxy-6'-(methoxycarbonyl) phenyl]-5,6-dihydro-2H-pyran-2-one, 3,4,3'-tri-O-methylellagic acid and methyl gallate [15] , reticulatusides A and B [16] , cytotoxic scopoletin [17] and flavonoid glycosides [18] [19] have been isolated from this plant. The isolation of lupeol, stigmasterol and lupeol acetate from the plant have been reported [20] . Regarding the volatile constituents, there appears to be no published work.
The objective of the present work was to examine the volatile constituents of this plant for future exploration. Our finding into the volatile oils of some poorly studied Nigerian flora was recently published [21] .
2. Materials and Methods2.1. Plant Sample
Leaves of P. reticulatus were collected from a location in Ibefun, Odogbolu, Ogun State, Nigeria, on March, 2012. Botanical identification was performed by Messrs Ugbogu O.A and Shasanya, O.S., at the Herbarium Headquarters, Forestry Research Institute of Nigeria (FRIN), Ibadan, Nigeria, where voucher specimen (FHI 109586) has been deposited for future reference.
2.2. Extraction of Essential Oil
Aliquots (400 g) of the air-dried and pulverized plant sample were subjected to hydrodistillation for 3 h using a Clevenger-type apparatus in accordance with the British Pharmacopoeia specification [22] to produce a pale yellow essential oil.
2.3. Analysis of the Oil
GC analysis was accomplished with a HP-5890 Series II instrument equipped with a HPWax and HP-5 capillary columns (both 30 m × 0.25 mm, 0.25 μm film thickness), working with the following temperature program: 60˚C for 10 min, rising at 5˚C/min to 220˚C. The injector and detector temperatures were maintained at 250˚C; carrier gas nitrogen (2 mL/min); detector dual, FID; split ratio 1:30. The volume injected was 0.5 μL. The relative proportions of the oil constituents were percentages obtained by FID peak-area normalization without the use of response factor.
GC-EIMS analysis was performed with a Varian CP-3800 gas-chromatograph equipped with a HP-5 capillary column (30 m × 0.25 mm; film thickness 0.25 μm) and a Varian Saturn 2000 ion trap mass detector. Analytical conditions: injector and transfer line temperature 220˚C and 240˚C respectively; oven temperature programmed from 60˚C - 240˚C at 3˚C/min; carrier gas was helium at a flow rate of 1 mL/min; injection of 0.2 μL (10% hexane solution); split ratio 1:30. Mass spectra were recorded at 70 eV. The acquisition mass range was 30 - 300 m/z at a scan rate of 1 scan/sec.
2.4. Identification of the Constituents
Identification of the constituents was based on comparison of the retention times with those of authentic samples, comparing their linear retention indices (LRI) relative to the series of n-hydrocarbons, and on computer matching against commercially available spectral [23] - [25] . Further identifications were also made possible by the use of homemade library mass spectra built up from pure substances and components of known oils and MS literature data. Moreover, the molecular weights of all the identified substances were confirmed by GC-CIMS, using MeOH as CI ionizing gas.
3. Results and Discussion
P. reticulatus yielded low content of essential oil 0.12% (v/w) on a dry weight basis. Table 1 shows the identities of 84 compounds identified in the oil of P. reticulatus, accounting for 99.7% of the total oil contents. Figure 1 depicts the GC chromatogram of the essential oil. The classes of compounds identified in the oil were monoterpene hydrocarbons (42.0%), oxygenated monoterpenoids (23.5%), sesquiterpene hydrocarbons (29.4%), oxygenated sesquiterpenoids (3.6%) and non-terpene derivatives (1.2%). The major oil constituents were α-pi- nene (6.0%), sabinene (7.6%), β-pinene (18.1%), linalool (6.9%) and camphor (7.7%), among the monoterpenes; and β-caryophyllene (11.9%) and germacrene D (8.6%) among the sesquiterpenes. This may represent the first analysis of the oil of this species.
Regardless of Phyllanthus being large family, with about 1000 species, the essential oils of P. reticulatus has not been investigated. However, the volatile constituents of few species grown have been reported. Phytol (21.5%), β-citronellol (17.7%), trans-geraniol (13.5%), cis-3-hexenol (12.6%) and 1-hexanol (11.3%) were the major constituents of Phyllanthus salviaefolius H.B.K. [26] . However, linalool (36.4%) and phytol (13.0%) dominated the oil of Phyllanthus amarus Sch. and Thonn [27] . Volatile compounds have been isolated from P. acidus (L.) Skeels fruits fermented for 1, 3 and 6 months. Among the 46 compounds identified, acids and alcohols dominated the volatiles profile; acids particularly characterized the quantitative profile of the volatile compounds after 6 months of fermentation. Other significant changes were in the sesquiterpenes, with increments of d- and α-cadinene after 3 months of fermentation, and α-cadinol and t-muurolol after 6 months [28] . Phyllanthus arenarius Beille in Lecomte has n-hexadecanoic acid (14.0%), 1,2-benzene dicarboxylic acid, bis (2-me- thylpropyl) ester (12.7%) and di-n-octyl phthalate (10.3%) as its main compounds [29] while Phyllanthus urinata L., was rich in 3,3,5-trimethylcyclohexanone (17.2%) and n-hexadecanoic acid (12.4%). The abundant of 3,3,5-trimethylcyclohexanone (12.4%) and 3,7-dimethyl-1,6-octadien-3-ol (10.2%) was reported in the oil of Phyllanthus niriru L. [29] . The essential oil of Phyllanthus emblica L. contained high amounts of β-caryophyl- lene, β-bourbonene, 1-octen-3-ol, thymol, and methyleugenol [30] . Another investigation reported that β-bour- bonene, heptadecanol, pentadecanone, thymol, β-caryophyllene, β-neoclovene, nerol and borneol were the major compounds were the main oil contents of the plant [31] . (E)-Isoelemicin (36.40%) was the main compounds of Phyllanthus muellerianus (Kuntze) Excel which also showed antimicrobial property [32] .
Gas chromatogram of Phyllanthus reticulates oil on HP-5MS column (30 m × 0.25 mm) with nitrogen carrier
Essential oil constituents of Phyllanthus reticulates
Compoundsa
Retention Timesb
RIc
RId
Percentage (%)
(E)-Hex-3-en-1-ol
3.29
854
850
0.4
Hexan-1-ol
3.48
872
863
Tr
n-Nonane
4.02
900
900
0.3
Tricyclene
4.55
928
921
Tr
α-Thujene
4.64
931
924
1.3
α-Pinene
4.81
940
932
6.0
Camphene
5.16
954
946
1.9
Thuja-2,4(10)-diene
5.29
958
953
Tr
Benzaldehyede
5.45
962
962
Tr
Sabinene
5.78
977
969
7.6
β-Pinene
5.88
980
974
18.1
Octan-3-one
6.11
988
979
Tr
Myrcene
6.23
992
988
2.4
Octan-3-ol
6.34
995
988
Tr
α-Phellandrene
6.64
1005
1002
Tr
α-Terpinenne
7.01
1018
1014
0.4
p-Cymene
7.26
1027
1020
0.2
Limonene
7.39
1031
1024
2.6
1,8-Cineole
7.48
1034
1026
2.7
(Z)-β-Ocimene
7.69
1041
1032
Tr
(Z)-Oct-3-en-1-ol
7.97
1047
1047
Tr
(E)-β-Ocimene
8.04
1051
1044
Tr
γ-Terpinene
8.41
1062
1054
0.6
cis-Sabinene hydrate
8.70
1070
1065
0.2
cis-Linalool oxide (furanoid)
8.89
1075
1067
Tr
Non-1-en-3-ol
9.16
1083
1088
Tr
Terpinolene
9.47
1089
1086
0.9
Linalool
9.90
1099
1095
6.9
n-Nonanal
10.07
1103
1100
0.2
Octen-1-ol acetate
10.39
1107
1110
0.3
cis-p-Menth-2-en-1-ol
10.72
1122
1118
Tr
trans-p-Menth-2-en-1-ol
11.43
1125
1136
Tr
Camphor
11.62
1144
1141
7.7
Menthone
12.01
1155
1158
0.3
Pinocarvone
12.37
1163
1160
0.2
Borneol
12.49
1166
1165
0.5
Menthol
12.79
1174
1167
1.9
Terpinen-4-ol
12.97
1178
1174
1.1
(Z)-Hex-3-enyl butanoate
13.41
1186
1184
Tr
α-Terpineol
13.54
1190
1186
1.0
Methyl salicylate
13.70
1192
1190
0.6
n-Decanal
14.21
1205
12.01
Tr
Verbenone
14.34
1208
1204
0.3
β-Cyclocitral
14.80
1218
1217
Tr
Nerol
15.18
1228
1227
Tr
3-Methyl-hex-3-en-1-yl butanoate
15.36
1236
1232
Tr
Piperitone
16.24
1253
1249
Tr
Menthyl acetate
18.01
1294
1294
0.1
δ-Elemene
19.85
1340
1335
0.5
α-Cubebene
20.36
1351
1345
Tr
Eugenol
20.74
1358
1356
Tr
α-Copaene
21.48
1376
1374
1.5
β-Bourbonene
21.58
1384
1387
1.0
β-Cubebene
22.11
1390
1387
Tr
β-Elemene
22.28
1392
1389
0.2
Cyperene
22.46
1398
1398
0.2
Isocaryophyllene
22.79
1405
1408
Tr
Dodecanal
22.97
1408
1408
Tr
β-Caryophyllene
23.32
1418
1417
11.9
β-Copaene
23.73
1429
1430
0.2
α-Guaiene
24.08
1439
1437
0.2
Aromadendrene
24.35
1441
1439
0.2
α-Humulene
24.75
1455
1452
1.8
allo-Aromadendrene
25.05
1461
1458
0.5
cis-Muurola-4(14),5-diene
25.29
1463
1465
Tr
γ-Muurolene
25.74
1477
1479
1.8
Germacrene D
25.92
1480
1484
8.6
(E)-β-Ionone
26.18
1485
1487
Tr
cis-β-Guaiene
26.31
1490
1492
Tr
epi-Cubebol
26.50
1494
1493
0.4
trans-β-Guaiene
26.75
1500
1502
Tr
α-Bulnesene
26.90
1505
1509
0.3
δ-Amorphene
27.02
1512
1511
Tr
Cubebol
27.35
1515
1514
0.5
δ-Cadinene
27.69
1524
1522
0.4
Germacrene B
28.83
1556
1559
0.1
Spathulenol
28.99
1576
1577
0.4
Caryophyllene oxide
30.04
1581
1582
1.2
Humulene epoxide II
31.07
1606
1608
Tr
1,10-di-epi-Cubenol
31.54
1614
1618
0.3
t-cadinol
32.39
1641
1639
0.2
Cubenol
32.56
1647
1645
Tr
α-Cadinol
32.86
1652
1652
0.2
α-Eudesmol
32.98
1654
1652
0.4
TOTAL
99.7
Monoterpene hydrocarbons
42.0
Oxygenated monoterpenes
23.5
Sesquiterpene hydrocarbons
29.4
Oxygenated sesquiterpenes
3.6
Others
1.2
aElution order on HP-5MS column; bRetention time in order with respect to the chromatogram (Figure 1); cRetention indices on HP-5 MS capillary column; dLiterature retention indices (References 23 - 25); Tr, Trace amounts < 0.1%.
The main compounds of P. salviaefolius could not be identified in P. reticulatus. Except for linalool, the quantitatively significant compounds of P. amarus were conspicuously absent from P. reticulatus. Also, the low contents of d-cadinene and α-cadinol, and the absence of the sesquiterpenoid compounds such as α-cadinene and t-muurolol in this study makes the composition differ from that found in P. acidus. Also, the major compounds in the oils of P. arenarius, P. urinate and P. niriru were absent in P. reticulatus. In addition, the oil of P. reticulatus could be distinguished from those of P. embelica by its lack of thymol, methyleugenol, heptadecanol, pentadecanone and nerol. Notably, (E)-isoelemicin, the main compound of P. muellerianus was not identified in P. reticulatus.
4. Conclusion
The chemical constituents of essential oil obtained from P. reticulatus grown in Nigeria are being reported for the first time. In addition, a comparison of the chemical composition was made with the other known essential oils from Phyllanthus plants. It could be seen that the essential oils of Phyllanthus plants exhibit high chemical variability. Each species has its own compositional pattern different from other. The very high content of compounds identified in the oil of P. reticulatus may be an important chemical and economic characteristic of the oil sample.
Acknowledgements
Authors are grateful to Messrs Ugbogu and Shasanya for the botanical identification of the plant.
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