Chemical composition, antimicrobial, and cytotoxicity studies on S. erianthum and S. macranthum essential oils

Abstract

Context: Solanum erianthum D. Don and Solanum macranthum Dunal (Solanaceae) are widely used in traditional medicine. The leaves act as an abortifacient and in particular to treat leucorrhoea, sores, and skin irritations.

Objective: This study was undertaken to characterize the volatile constituents of the leaf and fruit essential oils of S. erianthum and S. macranthum; their antimicrobial and in vitro cytotoxic bioassay against human breast and prostate tumor cells.

Methods: The volatile oils were obtained by hydrodistillation and analyzed for their constituents by gas chromatography-mass spectrometry (GC-MS). Minimum Inhibitory Concentrations (MIC) were determined using the microbroth dilution technique while the cytotoxic potentials were evaluated using the Cell Titre 96^(R) AQ_ueous Non-Radioactive Cell Proliferation Assay method.

Results: Solanum erianthum essential oils were characterized by the abundance of α-terpinolene (17.8%), α-phellandrene (17.5%), p-cymene (15.7%) and β-pinene (11.7%) in the leaves; α-humulene (23.1%), humulene epoxide II (20.0%), caryophyllene oxide (16.5%), methyl salicylate (11.8%) and β-caryophyllene (10.9%) in the fruits. The leaf oil of S. macranthum consisted of (E)-phytol (29.0%), pentadecanal (28.1%) and pentadecane (7.7%) while the major fruit oil constituents were α-humulene (36.5%), β-caryophyllene (17.8%), ethyl palmitate (9.4%), and methyl salicylate (8.2%). Solanum erianthum leaf volatile oil demonstrated potent inhibitory activity against Hs 578T and PC-3 human breast and prostate tumor cells respectively. In addition, the Solanum essential oils exhibited significant antimicrobial activity (19.5–625 µg/mL) on pathogens employed in the assay.

Conclusion: The Solanum essential oils possess strong antimicrobial activity in addition to the potent cytotoxic potential of S. erianthum leaf oil against Hs 578T and PC-3 cells.

Keywords::

• Solanaceae
• essential oils
• terpenoids
• GC-MS
• antimicrobial activity
• cytotoxicity

Introduction

The genus Solanum (Solanaceae) comprises of 1700 species commonly found in the temperate and tropical regions of the world (Burkill, 2000). The genus is represented by some 25 species in Nigeria including five introductions: S. wrightii Benth, S. melongena L., S. tuberosum L., S. mammorum L. and S. seaforthianum Andr. (var. disjunctum) (Gbile & Adesina, 1988). Solanum erianthum D. Don is a shrub or small tree about 6 m high with dense soft stellate hairs. The leaves act as an abortifacient and are considered a potent medicine for expelling all impurities through the urine and in particular to treat leucorrhoea (Burkill, 2000). The plant is also used to treat stomachache, sores in the mouth and applied externally to skin irritations and rashes. Solanum macranthum Dunal (syn. S. wrightii Benth) or ‘Giant Potato tree’ is a shrub and an ornamental plant. The leaves are large, lobed and prickly. The fruits are small and oval shaped (Oliver, 1960; Blomqvist & Nguyen, 1999; Burkill, 2000).

There have been sustained increases in the incidences of breast and prostate cancer in developing countries in recent years and intensive scientific research on this subject matter is imperative. Sixty percent of currently used anticancer drugs are derived one way or the other from natural sources (Houghton, 1995; Cragg & Newmann, 2001).

The use of Solanum nigrum L. as one of the herbal ingredients in prescriptions of Traditional Chinese Medicine to treat liver cancer, mammary cancer, uterine cervix cancer, gastric cancer and other cancers (Son et al., 2003) further stimulated the interest to assess the essential oils of S. erianthum and S. macranthum for anticancer activity; and the folkloric claims for the antimicrobial potential of these plants.. Essential oils have been shown to possess antimicrobial (Zu et al., 2010) and cytotoxic properties on breast and prostate carcinoma cells (Monajemi et al., 2005; Zu et al., 2010). The essential oil constituents of S. aculeastrum Dunal (Srinivas et al., 2006) and S. pseudocapsicum L. (Aliero et al., 2006, 2007) have been reported. The phytochemical and antimicrobial studies (Ajaiyeoba, 1999) of other Nigerian Solanum species have also been investigated. As far as we know, there are no literature reports on the chemical constituents and biological activities of the essential oils from S. erianthum and S. macranthum. This paper reports for the first time the volatile oils composition from the leaves and fruits of these plants, as well as their in vitro cytotoxicity and antimicrobial activities.

Methods

Plant

The fresh leaves and fruits of S. erianthum and S. macranthum were collected in the month of July, 2009 within the University of Ibadan, Nigeria. Plant samples were authenticated by Mr. F. Usang of the Herbarium Headquarters, Forest Research Institute of Nigeria (FRIN), Ibadan, Nigeria, where voucher specimens (FHI 106923 and FHI 106921, respectively) were deposited. The plant materials were air-dried under a laboratory shade prior to extraction.

Extraction of the volatile oils

Essential oils were obtained by hydrodistillation of the pulverized air-dried plant samples (500 g) in an all glass Clevenger-type apparatus in accordance with the British Pharmacopoeia specifications (1980). The distillation time was 4 h in all experiments. The oils obtained were stored under refrigeration until moment of analyses.

Gas chromatography-mass spectrometryanalyses (GC/MS)

The volatile oils were subjected to GC-MS analyses on an Agilent system consisting of a model 6890 Gas chromatograph, a model 5973 Mass Selective Detector (MSD) and an Agilent ChemStation Data system. The GC Column was an HP-5ms fused silica capillary coated with (5% phenyl)-methylpolysiloxane (equivalent to USP phase G27) stationary phase, film thickness of 0.25 µm, a length of 30 m, and an internal diameter of 0.25 mm. The carrier gas was helium with a column head pressure of 8.28 psi and flow rate of 1.0 mL/min. The inlet and MSD detector temperatures were maintained at 200°C and 230°C, respectively, while the MS transfer-line temperature was 280°C. The GC oven temperature was programmed as follows: 40°C initial temperature held for 10 min; increased at 3°/min to 200°C; increased 2°/min to 220°C. The samples were dissolved in CH₂Cl₂ to give a 1% w/v solution and a split injection technique was used. The split ratio was 1:30. Mass spectra were recorded at 70 eV.

Identification of each individual constituent of the essential oils was achieved based on their retention indices (determined with reference to a homologous series of normal alkanes), and by comparison of their mass spectral fragmentation patterns (NIST database/ChemStation data system) (Adams, 2001) and with reference to previously reported data in the literature (Roussis et al., 2000; Kobiasy et al., 2002; Dural et al., 2003; Skaltsa et al., 2003; Bertea et al., 2005). The carbon numbers of n-alkanes used for the RI were from C₉-C₃₆.

Cell culture media

Human Hs 578T breast ductal carcinoma cells (ATCC. No. HTB-129) (Hackett et al., 1977) were grown in 3% CO₂ environment at 37°C in DMEM with 4500 mg glucose per litre of medium, supplemented with 10% fetal bovine serum, 10 µg bovine insulin, 100,000 units penicillin and 100 mg streptomycin per liter of medium, and buffered with 44 mM NaHCO₃, pH 7.35. Human PC-3 prostatic carcinoma cells (ATCC No. CRL-1435) (Kaighn et al., 1979) were grown in 3% CO₂ environment at 37°C in RPMI-1640 medium with l-glutamine, supplemented with 10% fetal bovine serum, 100,000 units penicillin and 10.0 mg streptomycin per liter of medium, and buffered with 15 mM Hepes and 23.6 mM NaHCO₃.

Cytotoxicity screening

Hs 578T cells were plated into 96-well cell culture plates at 1.0 × 10⁵ cells per well and PC-3 cells at 1.9 × 10⁴ cells per well. The volume in each well was 100 µL for both cell types. After 48 h, supernatant fluid was removed by suction and replaced with 100 µL growth medium containing 2.5 or 1.0 µL of DMSO solution of oil (1% w/w in DMSO), giving a final concentration of 250 or 100 µg/mL respectively for each oil. Hs 578T cells were tested at final concentration at 250 µg/mL and PC-3 cells at final concentration of 100 µg/mL. Solutions were added to wells in four replicates. Medium controls and DMSO controls (25 and 10 µL DMSO/mL) were used. Tingenone (250 and 100 µg/mL) was used as a positive control (Setzer et al., 1998). After the addition of compounds, plates were incubated for 48 h at 37°C. The medium was then removed by suction and 100 µL of fresh medium was added to each well. In order to establish percent kill rates, the cell Titer 96^® AQ_ueous Non-Radioactive Cell Proliferation Assay was performed (Promega Technical Bulletin, 1996). After colorimetric readings were recorded in triplicates (using a molecular Devices SpectraMAX Plus microplate reader, 490 nm), average absorbances, standard deviations, and percent kill ratios (% kill_cmpd/% kill_DMSO) were calculated.

Antimicrobial screening

Essential oils were screened for antibacterial activity against the Gram-positive bacteria, Bacillus cereus (ATCC No. 14579) and Staphylococcus aureus (ATCC No 29213) and the Gram-negative bacteria, Pseudomonas aeruginosa (ATCC No 27853) and Escherichia coli (ATCC No 254922). Minimum inhibitory concentration (MIC) was determined using microbroth dilution technique (Sahm & Washington, 1991). Dilutions of the oils were prepared in cation-adjusted Mueller Hinton Broth (CAMHB) beginning with 50 μL of 1% w/w solutions of essential oils in DMSO plus 50 μL CAMHB. The oil solutions were serially diluted (1:1) in CAMHB in 96-well plates. Organisms at a concentration of approximately 1.5 × 10⁸ colony forming units (CFU)/mL were added to each well. Plates were incubated at 37°C for 24 h; the final minimum inhibitory concentration (MIC) was determined as the lowest concentration without turbidity. Gentamicin was used as a positive control while DMSO was used as a negative control.

Antifungal activity was determined as described above using Candida albicans (ATCC No. 10231) in yeast nitrogen base growth medium with approximately 7.5 × 10⁷ CFU/mL. Antifungal activity against Aspergillus niger (ATCC No 16401) was determined as above using potato dextrose broth inoculated with A. niger hyphal culture diluted to a McFarland turbidity of 1.0. Amphotericin B was the positive control. Both C. albicans and A. niger were maintained at 32°C and 25°C respectively for 24 h.

Results

The percentage yields of essential oils of S. erianthum were 0.13 and 0.27% w/w, respectively, for the leaves and fruits, while S. macranthum oils were obtained in 0.10% yield. The volatile oils composition is displayed in Table 1. In the leaves and fruits oils of S. erianthum, 35 and 36 compounds accounting for 97.4 and 96.0% of the total oil contents respectively were identified. The major components of the leaf oil were the hydrocarbon monoterpenes: α-terpinolene (17.8%), α-phellandrene (17.5%), p-cymene (15.7%) and β-pinene (11.7%). However, the fruit counterpart was dominated by the sesquiterpenoids comprising of α-humulene (23.1%), humulene epoxide II (20.0%), caryophyllene oxide (16.5%) and β-caryophyllene (10.9%). Methyl salicylate (11.8%), an aromatic ester also constituted a sizeable proportion of the oil sample.

Table 1.  Volatile constituents of the essential oils from the leaves and fruits of Solanum erianthum and Solanum macranthum

RI ^a	Compound ^b, c	Percentage composition
		S. mac leaves	S. mac fruits	S. eri leaves	S. eri fruits
854	(E)-2-Hexenal	0.4	0.1	–	Tr
943	α-Pinene	–	–	3.5	0.2
954	Camphene	–	–	0.1	Tr
966	Benzaldehyde	–	–	–	0.1
979	β-Pinene	–	0.1	11.7	0.3
996	Δ-2-Carene	1.5	–	–	–
1003	α-Phellandrene	0.2	–	17.5	–
1009	Δ -3-Carene	–	–	–	Tr
1023	p-cymene	2.1	1.2	15.7	1.3
1031	1,8-Cineole	1.5	0.3	4.1	0.3
1057	(E)-β- Ocimene	–	–	0.3	–
1067	γ-Terpinene	–	–	1.4	–
1092	α- Terpinolene	–	–	17.8	–
1102	Linalool	–	–	2.4	0.1
1118	1,3,8-p-Menthatriene	–	–	0.2	–
1130	α- Campholene aldehyde	–	–	Tr	–
1142	(E)-Verbenol	–	–	0.6	–
1164	Pinocarvone	–	–	0.1	–
1182	Terpinene-4-ol	–	–	0.9	–
1186	p-Methylacetophenone	–	–	1.5	–
1191	p-Cymene-8-ol	–	–	4.4	–
1195	Methyl salicylate	–	8.1	1.5	11.8
1204	Myrtenol	–	–	0.3	–
1207	α-Phellandrene Epoxide	–	–	2.0	–
1257	Piperitone	–	–	1.3	–
1268	Ethyl salicylate	–	–	–	0.4
1301	Carvacrol	–	–	1.0	–
1320	(E,E)-2,4-Decadienal	–	0.7	–	0.6
1350	α-Cubebene	–	–	–	0.3
1379	α-Copaene	–	2.2	–	2.9
1380	E-β-Damascenone	0.3	–	–	–
1386	β-Bourbonene	–	0.4	–	0.4
1395	β-Elemene	–	1.5	–	1.1
1400	Tetradecane	0.6	–	–	–
1409	γ-Caryophyllene	–	0.2	–	0.4
1419	β- Caryophyllene	0.5	17.8	0.3	10.9
1421	α-(E)-Ionone	0.2	–	–	–
1432	β-Gurjunene	–	–	–	0.1
1453	α-Humulene	–	36.0	0.1	23.1
1457	Precocene 1	1.3	–	–	–
1459	(E)-β-Farnesene	–	–	1.9	–
1480	γ-Muurolene	–	–	–	0.2
1482	Germacrene D	–	3.5	–	0.2
1485	ar-Curcumene	–	–	0.5	–
1488	β-lonone	2.5	0.3	–	0.2
1494	Valencene	–	–	–	0.2
1496	α-Selinene	–	0.4	–	–
1498	α-Zingiberene	–	–	0.5	–
1499	α-Muurolene	–	–	–	0.2
1501	Pentadecane	7.7	0.6	–	–
1506	Germacrene A	–	0.5	–	0.1
1508	(E,E)-α-Faenesene	–	–	0.4	–
1511	β-Bisabolene	–	–	–	Tr
1512	Tridecanal ^d	0.6	–	–	–
1515	γ-Cadinene	–	0.4	–	0.3
1526	δ-Cadiene	–	1.7	–	0.7
1527	β-Sesquiphellandrene	–	–	0.4	–
1533	(E)-γ-Bisabolene	–	–	Tr	–
1536	(Z)-Nerolidol	–	–	0.1	–
1566	(E)-Nerolidol	0.5	0.3	0.4	–
1581	ar- Turmerol	–	–	0.3	–
1582	Caryophyllene oxide	–	5.0	–	16.5
1590	Dihydro-ar-turmerone	–	–	0.2	–
1599	Hexadecane	0.2	–	–	–
1608	Humulene epoxide II	–	5.5	–	20.0
1611	Tetradecanal	1.2	–	–	–
1641	τ-Cadinol	–	0.5	–	–
1643	Caryophylla-4 (12),8(13)-dien-5β-ol	–	–	–	0.3
1647	Cubenol	–	–	–	0.6
1651	Torreyol	–	–	–	0.4
1661	α-cadinol	–	0.9	–	1.5
1669	β-Turmerone	–	–	4.7	–
1677	Cadalene	–	–	–	0.7
1705	α-Turmerone	–	–	1.3	–
1713	Pentadecanal ^e	28.1	–	–	0.1
1799	Octadecane	0.2	–	–	–
1811	Hexadecanal	0.5	–	–	–
1840	Hexahydrofarnesylacetone ^f	1.1	–	–	–
1899	Nonadecane	0.3	0.1	–	–
1913	Heptadecanal	1.0	–	–	–
1921	Methyl palmitate	0.2	0.4	–	–
1942	Isophytol	0.3	–	–	–
1990	Ethyl palmitate	5.7	9.3	–	–
1999	Eicosane	0.8	–	–	–
2090	Methyl lineolate	–	0.1	–	–
2100	n-Heneicosane	0.6	0.3	–	–
2116	(E)-Phytol ^g	29.0	–	–	–
2123	Methy stearate	–	0.1	–	–
2152	Linoleic acid ^h	–	0.8	–	–
2199	Docosane	0.5	0.1	–	–
2298	Tricosane	0.4	0.1	–	–
2400	Tetracosane	0.3	–	–	–
2499	Pentacosane	0.2	–	–	–
Total		90.5	99.5	99.4	98.5
Monoterpene hydrocarbons		3.8	1.3	68.2	1.8
Oxygen containing monoterpenes		4.2	8.7	20.2	12.8
Sesquiterpene hydrocarbons		0.5	64.6	4.0	41.8
Oxygen containing sesquiterpenes		0.8	12.2	7.0	40.9
Fatty acids		49.1	11.9	–	0.1
Aliphatics		1.5	0.8	–	0.6
Diterpenoids		29.3	–	–	0.4
Others		1.3	–		0.1
% Yield of the oil (w/w)		0.10	0.10	0.13	0.27

^aRetention indices on HP-5ms capillary column; ^bIdentification by comparison of the mass spectral and retention index data; ^cClasses of compounds as found in the oils; ^dRI value from Skaltsa et al., 2003; ^eRI value from Dural et al., 2003; ^fRI from Roussis et al., 2000; ^gRI from Kobiasy et al., 2002; ^hRI from Bertea et al., 2005; -, not detected.

Tr, trace <0.1%; S. mac- Solanum macranthum; S. eri- Solanum erianthum.

Thirty-four components each were identified in both the leaf and fruit oils of S. macranthum representing 91.1 and 99.4% of the total oil composition, respectively. The quantitatively significant constituents of its leaf oil were the diterpenoid, (E)-phytol (29.0%) and the fatty acids of pentadecanal (28.1%), pentadecane (7.7%) and ethyl palmitate (5.7%). Sesquiterpene hydrocarbons made up the bulk of the fruit oil. These are α-humulene (36.5%) and (β)-caryophyllene (17.8%). Ethyl palmitate (9.4%) and methyl salicylate (8.2%) were also observed in significant quantities in the fruit oil.

The cytotoxic activities of both volatile oils are presented in Table 2. The leaf oil of S. erianthum exhibited an in vitro inhibitory effect against Hs 578T (human breast ductal carcinoma cells) and PC-3 (human prostate carcinoma cells). On the other hand, the results revealed that S. macranthum leaf oil was not cytotoxic to both cell lines at the tested concentration. However, S. macranthum fruit oil displayed considerable cytotoxic activity on Hs 578T cell line (79.39%). The Hs 578T cells were more sensitive to the lethal activity of the Solanum oils than the PC-3 cell lines (Table 2).

Table 2.  Cytotoxic activities of S. erianthum and S. macranthum essential oils^a

Sample	Hs 578T ^b	PC-3 ^c
S. erianthum leaf oil	98.86 (0.63)	97.94 (2.05)
S. erianthum fruit oil	13.70 (20.24)	0
S. macranthum leaf oil	2.20 (2.23)	0
S. macranthum fruit oil	79.39 (9.40)	47.43 (3.68)
Tingenone	100.00	100.00

^a% kill at tested concentrations in triplicate (standard deviations in parentheses); negative controls, medium and DMSO had no effects in the assay; ^bHuman breast (ductual) tumor cells (% kill at 250 μg/mL); ^cHuman prostate tumor cells (% kill at 100 μg/mL).

Reference compound; tingenone at concentration of 250 or 100 ppm.

Table 3 reports the antibacterial and antifungal activities of the essential oils. Solanum erianthum leaf volatile oil demonstrated broad spectrum of antimicrobial activities; strongly active against Aspergillus niger (19.5 µg/mL) and exerted least activity on Pseudomonas aeruginosa (625 µg/mL). The fruit essential oil of S. macranthum also showed promising antibacterial and antifungal activities as indicated by its potent inhibitory effect on both Staphylococcus aureus and A. niger (39 and 19.5 µg/mL, respectively). In addition, the results revealed that S. macranthum leaf oil possessed relatively the least antimicrobial activities in the study.

Table 3.  Antimicrobial activities of S. erianthum and S. macranthum volatile oils (MIC μg/mL).

Sample	B.c	S.a	E.c	P.a	C.a	A.n
S. erianthum leaf oil	312	156	625	1250	625	312
S. erianthum fruit oil	78	156	625	625	625	156
S. macranthum leaf oil	156	156	625	625	625	39
S. macranthum fruit oil	312	39	625	625	625	19.5
Positive control	1.22^a	0.61^a	2.44^a	1.22^a	0.61^b	0.61^b

B.c, Bacillus cereus (ATCC No. 14579); S.a, Staphylococcus aureus (ATCC No 29213); E.c, Escherichia coli (ATCC No. 25922); P.a, Pseudomonas aeroginosa (ATCC No. 27853); C.a, Candida albicans (ATCC No. 10231); A.n, Aspergillus niger (ATCC No. 16401).

^aGentamicin sulphate; ^bAmphotericin B; Negative control, DMSO had zero effect in the assay.

Discussion

A comparison of the compositional pattern of the investigated oils revealed some qualitative and quantitative variations. The leaf oil of S. erianthum was characterized by the abundance of monoterpenes (86.3%) while its fruit essential oil consisted of sesquiterpenoids (80.8%). On the other hand, sesquiterpenoid compounds (77.5%), monoterpenes (10.1%) and fatty acids (11.9%) were classified in the fruit oil of S. macranthum. as can be seen in Table 1 that each oil sample has its own compositional profile. While a total of 93 constituents were identified in the chemical analyses of the oils, only p-cymene, 1, 8-cineole and β-caryophyllene were common to all the oils. Several of the fatty acids reported for both oils of S. macranthum were conspicuously not detected in S. erianthum oils. The root essential oil of S. pseudocapsicum was reported to contain high proportions of fatty acids (26.8%) and implicated to contribute to its medicinal properties. It is not uncommon to find diverse chemical profiles of essential oils from different plant parts. Cinnamon leaves, bark, flowers, fruits and roots yield different essential oils (Jayaprakasha et al., 1997, 2003; Kaul et al., 2003).

The essential oil from the unripe berries of S. pseudocapsicum was found to be predominated by decane (41.06%), undecane (29.26%), monoterpenoids (14.79%), sesquiterpenoids (3.21%) and a diterpene phytol (5.94%) (Aliero et al., 2006). Srinivas et al. (2006) reported that the leaf oil of S. aculeastrum consisted of n-nonane (12.4%), o-phthalic acid (11.8%) and 1,2-dimethyl benzene (8.8%) as the main compounds while the hexane extract had undecane (21.7%), o-phthalic acid (14.9%), tetradecane (10.8%) and tridecane (10.0%) in abundance. These compositional patterns were quite different from those of the oils under present study.

Furthermore, in vitro cytotoxic and antimicrobial assays carried out in order to evaluate the biological potentials of the investigated oils revealed the potent inhibitory cytotoxic effects of S. erianthum leaf essential oil against Hs578T and PC-3 cell lines. The compositional pattern of the leaf oil may have influenced such notable biological activities. Potent cytotoxic activities were considered at ≥90% lethality at tested concentrations of 250 and 100 ppm for both breast and prostate carcinoma cells respectively. It should be noted, however, that prominent constituents of the oil such as p-cymene, β-pinene, (E)-phytol, α-humulene, β-caryophyllene, caryophyllene oxide and long chain aldeydes have exhibited a variety of biological activities, including anticancer (Legault et al., 2003; Legault & Pichette, 2007) and antimicrobial activity (Rajab et al., 1998; Griffin et al., 1999; Dorman & Dean, 2000; Shunying et al., 2005; Yoshihiro et al., 2005).

Conclusion

The various medicinal properties and biological activity of S. erianthum and S. macranthum are a function of their distinct chemical profiles. This study indicates that S. erianthum leaf essential oil possesses notable cytotoxic activity against Hs 578T and PC-3 carcinoma cells. Furthermore, the Solanum oils also exhibit in vitro antimicrobial activity against medicinally important pathogens. There is no information on the traditional use of S. erianthum in cancer therapy; however, present findings would enhance the further exploitation of the oils for other biological and therapeutic purposes.

Acknowledgements

The authors acknowledge generous financial support from an anonymous private donor to WNS and to late Mr Felix Usang of the Herbarium, FRIN, Ibadan, Nigeria for the identification of the plant samples.

Declaration of interest

There are no conflicts of interest.