Volatile constituents and biological activities of Pycnostachys abyssinica and Pycnostachys eminii extracts

Abstract

Context: Pycnostachys abyssinica Fresen and Pycnostachys eminii Gürke (Lamiaceae) are used in traditional Ethiopian medicine against eye and skin infections, “Mitch disease”, and dysentery.

Objective: Our study was aimed at characterizing essential oil (EO), phytochemical groups, and antimicrobial and anthelmintic activity of extracts to underscore the species’ indigenous medicinal use.

Materials and methods: Plant organs of Pycnostachys species were subjected to hydrodistillation, and essential oils (EO) analyzed by GC-MS. Phytochemical compounds, antimicrobial (diffusion assay) and anthelmintic activity (bioassay) of gradient solvent extracts of different polarity were studied.

Results: In the stem and root EO of P. abyssinica, 25 (99%) and 30 (99.79%) compounds were detected respectively, with estragole (70.4%) (stem) and exo-fenchyl acetate (30.6%) (root) as the most abundant compounds. In leaf, stem and root EO of P. eminii, 30 (90.66%), 27 (90.59%) and 27 (99.96%) compounds were detected, respectively, with high levels of β-caryophyllene (from 18.08% to 28.85%) and germacrene D (from 15.1% to 22.06%). Alkaloids, saponins, phytosterols, flavonoids, polyphenols, diterpenoids and carotenoids were detected in Pycnostachys. Petroleum ether, chloroform and methanol extracts showed distinct antimicrobial effects with generally higher potential activity of lipophilic and semi-lipophilic fractions. Leaf and root methanol extracts of both species showed lethal activity against earthworms.

Discussion: Identified EO constituents and phytochemical groups underscore the observed antifungal, antibacterial and anthelmintic activity of Pycnostachys gradient solvent extracts.

Conclusion: EO analysis, phytochemical screening, and antimicrobial and anthelmintic assays indicate the biological potential of Pycnostachys species from Ethiopia, and emphasize their pharmacological and indigenous applications.

Keywords::

• Agar-well diffusion method
• antibacterial
• antifungal
• anthelmintic
• essential oil
• GC-MS
• Lamiaceae
• phytochemical screening

Introduction

The Lamiaceae is a relatively commonly encountered family, especially in the temperate regions of the world. It comprises about 3500 species distributed among more than 200 genera, represented by 41 genera in Ethiopia (Ryding, 2006). The genus Pycnostachys (Lamiaceae) is native to tropical Africa and South Africa with extension to Madagascar. It is represented by about 40 species, of which six species occur in Ethiopia. Both Pycnostachys abyssinica Fresen., Pycnostachys recurvata Ryding and Pycnostachys sp. Mesfin & Kagnew (code no. GY 2249) are recognized as endemic species (Burger, 1967). Aromatic medicinal plants, including Pycnostachys, have been widely used in Ethiopian herbal medicine, as anthelmintic, antidiabetic, antimicrobial, antiviral, antioxidant, insect repellant, and molluscicidal drugs (Abebe & Ayehu, 1993). The phytochemical composition, biological and ethnopharmacological activities of Pycnostachys in general and the Ethiopian species in particular, have only been minimally investigated. An ethnobotanical survey conducted locally, however, showed the medicinal properties of P. abyssinica. The leaves of the plant are boiled in water and the vapor is inhaled for the treatment of “Mitch disease”, an infectious and inflammatory disorder. After warming, the leaves are pressed and the juice is used in the treatment of skin and eye infections. Leaves are used as a tea for the treatment of dysentery, and the aerial parts of this plant are also applied as termite repellant (Kloose et al., 1978). P. abyssinica is known to produce essential oil capable of repelling insects, thus underscoring the plants’ use as live fences (Asfaw, 2002) and natural pesticide in grain storage in Ethiopia (Blum & Bekele, 2000). Furthermore, the plant’s methanolic extracts exhibited antimicrobial activity against common bacterial and fungal strains, in particular Staphylococcus aureus (Messele et al., 2004). Only a few scientific reports on other members of the genus Pycnostachys exist; they indicate their phytochemical potential due to the content of biologically active compounds. Decoctions of Pycnostachys eminii are applied both as a tea and a bath against malaria in Uganda (Ssegawa & Kasenene, 2007). Moreover, phenolic structures have been identified such as rosmarinic acid in Pycnostachys meyeri and Pycnostachys coerulea (Pedersen, 2000), and caffeic acid esters (nepetoidins) in Pycnostachys umbrosa (Grayer et al., 2003). More recent studies reported the anti-inflammatory properties of Pycnostachys reticulata from South Africa (Fawole et al., 2010), antistaphylococcal activities based on diterpenes identified in Pycnostachys urticifolia and P. reticulata (Bascombe & Gibbons, 2008a, 2008b), and the antituberculosis potential of Pycnostachys erici-rosenii (Tabuti et al., 2010). However, to the authors’ knowledge, no scientific reports regarding essential oil profiles and bioassays of gradient solvent extracts of P. abyssinica and P. eminii are currently available. Results from the present study are expected to add useful knowledge about biologically active compounds in these species regarding their pharmacological and indigenous applications.

Materials and methods

Plant material

The whole plants were collected from Jimma Town, southwestern Ethiopia (7° 40′ 0″ N, 36° 50′ 0″ E), in June 2006. Voucher specimens, JH001 and JH002 for P. eminii and P. abyssinica, respectively, were identified by Melaku Wondafrash (National Herbarium, Department of Biology, Addis Ababa University), and deposited with the National Herbarium. The freshly collected plant parts were used for extraction of the EOs, while the air dried powdered plant parts were used for gradient solvent extraction.

Hydrodistillation of essential oils

Freshly collected leaf, stem and root of the two species were subjected to hydrodistillation in a Clevenger-type apparatus for 3 h after the mixture started boiling. The distillation apparatus consisted of a heating mantle, a 5 L round-bottom extraction flask, a 3 mL graduated receiver (Dean and Stark) and a condenser (jacketed coil). The volume of EO collected in the receiver was measured and percentage yield was calculated on a fresh weight basis. Portions of the EOs dissolved in xylene (10 µL in 1 mL xylene) were subjected to GC-MS analysis.

Preparation of gradient extracts

Powdered plant parts (100 g each) packed in a thimble were successively extracted in a Soxhlet apparatus using petroleum ether (60–80°C), chloroform and methanol as solvents until the last portions of the extractive became colorless. Each fraction collected separately was concentrated in vacuo. The resulting semi-solid mass was dried in an oven (Gallenkamp, England) at 40°C. The dried mass was weighed and percentage yield calculated on a dry weight basis.

GC-MS analysis

A Varian Star 3400 CX gas chromatograph (Walnut Creek, CA) coupled with a Varian Saturn 3 mass spectrometer were used for the analysis. EO samples (1 µL) were injected into the GC, using an automatic injector in split mode. The following capillary columns were used in the analysis: Chrompack CP-Wax 52 CB (Palo Alto, CA; fused silica, 30 m × 0.32 mm i.d., film thickness 0.25 µm) and Chrompack WCOT fused silica CP-Sil 5 (30 m × 0.25 mm i.d., film thickness 0.25 μm). The carrier gas was He (5 and 12 psi for the Chrompack CP-Wax and CP-Sil 5 columns, respectively) at 50 mL/min through the injector and 30 cm/s through the column. The injector temperature was 220°C for all analyses. The GC temperature program for both columns was held at 40°C for 1 min, ramped from 40°C to 220°C at a rate of 3°C/min, and then held at 220°C for 2 min. The MS detector was set at 220°C and a mass range of m/z 40–300 was recorded. All mass spectra were acquired in EI mode. The compounds in the EOs were identified by the use of a combination of mass spectrum data base search (IMS Terpene Library 1989; NIST05 MS Database), relative retention indices and comparison of mass spectra (Adams, 1995). Relative retention indices on Chrompack CP-Wax 52 CB and CP-Sil 5 columns were calculated by co-injecting a series of aliphatic hydrocarbons (C₈-C₂₄). Quantitative analysis was performed by peak area normalization (%) measurements (TIC, total ion current) of GC-MS chromatograms.

Phytochemical screening

Residue from the gradient solvent extracts or the powdered plant material, respectively, were used for chemical screening to detect for the presence or absence of alkaloids, saponins, carotenoids, phytosterols, polyphenols (tannins, flavonoids, and coumarins) and anthraquinones following standard procedures (Debela, 1993).

Test organisms

The bacterial strains used for the in vitro antibacterial tests in this study were Staphylococcus aureus ATCC 25923 (Gram positive), Escherichia coli ATCC 25922 (Gram negative) and Pseudomonas aeruginosa ATCC 27853 (Gram negative). Aspergillus niger ATCC 10535 and Aspergillus flavus ATCC 11489 (both moulds) and clinical isolates of yeasts (Candida albicans and Cryptococcus neoformans) were used for the in vitro antifungal tests. All organisms were obtained from the Department of Infectious Diseases, Ethiopian Health and Nutrition Research Institute (EHNRI). Earthworms collected from Shegole gorge, Addis Ababa, were used for the in vitro anthelmintic activity test.

Antimicrobial activity test

Muller-Hinton Agar (MHA) (Lot No 418600, Oxoid, Hampshire, England), Nutrient broth (DIFCO Laboratories, Detroit, MI) and Sabouraud Dextrose Agar, SDA (Lot No.408142, Oxoid) were employed for antimicrobial activity screening. All culture media were prepared and treated according to the manufacturer guidelines. Antimicrobial activity assays of the gradient solvent extracts against the selected microorganisms was performed by the agar-well diffusion method in triplicates using standard procedures (CLSI, 2003; Hymete et al., 2005).

Anthelmintic activity test

The anthelmintic activity of the gradient extracts was tested against earthworms using a reported standard method (Hymete et al., 2005).

Results

Hydrodistillation of the leaves of P. abyssinica yielded golden-orange colored oil (0.014% v/w), stems a light yellow oil (0.0026% v/w) and roots a reddish brown oil (0.0043% v/w). Similarly, fresh leaves, stems and roots of P. eminii afforded 0.125% v/w (light yellow), 0.003% v/w (light orange) and 0.022% v/w (reddish brown) of EO, respectively. Highest EO yield (0.125% v/w) was obtained from the leaves of P. eminii. On gradient solvent extraction using petroleum ether, P. abyssinica yielded 7.42, 2.78 and 2.07% residues from leaf, stem and root samples, while chloroform yielded 4.57, 1.39, and 1.21%, and methanol 18.29, 10, and 6.14%, respectively. P. eminii yielded 9.67, 1.58, and 1.34% residue on petroleum ether extraction of leaf, stem and root samples, respectively. Extraction with chloroform yielded 4.75, 1.27, 0.81%, and extraction with methanol afforded 20, 6.55 and 3.69% for leaves, stems and roots, respectively.

Results of the GC-MS analysis of the EOs of the various plant parts of the study plants are presented in Table 1. Twenty-five (99%) and thirty (99.79%) compounds were detected in the essential oils of P. abyssinica stem and root, respectively. The stem EO of P. abyssinica (PAS) contained mainly monoterpenes (98.66%), oxygenated monoterpenes accounting for 78.52% of the total oil, with estragole (70.4%) being the major component followed by exo-fenchyl acetate (5.18%). Limonene (8%) was the major component among the hydrocarbon monoterpenes with smaller amounts of terpinolene (4.06%) and γ-terpinene (3.8%). The EO of P. abyssinica root (PAR) contained monoterpenes (39.73%) and sesquiterpenes (60.06%). Oxygenated monoterpenes accounted for the larger part of the monoterpenes (34.16%) with exo-fenchyl acetate (30.6%) being the major component. Similarly, oxygenated sesquiterpenes represented a higher amount of the sesquiterpenes (44.72%). The major components among oxygenated sesquiterpenes were (E)-nerolidol (16.80%) and β-eudesmol (11.09%), while α-eudesmol (5.38%), τ-cadinol (4.61%) and caryophyllene oxide (4.05%) occurred in lower amounts. β-caryophyllene (5.33%) and α-caryophyllene (6.61%) were the most abundant components of the sesquiterpene hydrocarbons.

Table 1.  Constituents of the essential oils of P. abyssinica and P. eminii.

RRI*	Compound	EO composition (% peak area)
		PAS	PAR	PEL	PES	PER
939	α-Pinene	0.16	0.27	0.39	0.87	1.88
954	Camphene	0.07	0.26	0.61	1.49	1.03
975	Sabinene	0.07	tr	0.12	0.34	–
979	β-Pinene	–	0.24	1.1	0.72	2.35
991	β-Myrcene	0.49	–	–	–	–
1003	α-Phellandrene	0.05	–	–	–	–
1017	α-Terpinene	0.03	–	–	–	–
1025	p-Cymene	0.27	0.23	–	–	tr
1026	ο-Cymene	–	–	–	–	0.3
1029	Limonene	8.0	1.21	0.14	0.47	1.53
1037	(Z)-Ocimene	2.94	0.13	–	–	–
1050	(E)-Ocimene	0.2	0.1	–	–	–
1060	γ-Terpinene	3.8	1.74	–	–	–
1087	Fenchone	2.37	–	–	–	–
1089	Terpinolene	4.06	1.39	–	0.16	0.38
1097	Linalool	–	–	0.74	–
1117	endo-Fenchol	0.05	0.18	–	–	–
1122	exo-Fenchol	0.11	–	–	–	–
1146	Camphor	0.06	–	–	–	–
1169	Borneol	tr	–	–	–	–
1177	4-Terpineol	0.06	–	–	–	–
1196	Estragole	70.4	tr	–	–	0.66
1220	endo-Fenchyl acetate	0.18	–	–	–	13.76
1223	exo-Fenchyl acetate	5.18	30.6	–	–	–
1235	Thymol methyl ether	–	0.56	–	–	–
1285	(E)-Anethole	0.11	–	–	–	–
1289	Bornyl acetate	–	2.82	–	–	4.34
1351	α-Cubebene	–	–	0.39	–	–
1375	α-Ylangene	–	–	0.29	0.34	–
1377	α-Copaene	–	–	1.63	0.69	–
1388	β-Bourbonene	–	–	0.46	0.92	–
1391	β-Elemene	–	–	0.2	0.36	0.38
1410	α-Gurjunene	–	0.35	0.22	–	–
1413	(Z,Z)-α-Bergamotene	–	–	–	–	0.35
1419	β-Caryophyllene	–	5.33	21.64	28.85	18.08
1434	β-Gurjunene	–	0.3	0.33	0.26	–
1435	(Z,E)-α-Bergamotene	–	0.67	–	–	0.35
1440	α-Guaiene	–	–	0.1	0.21	0.57
1455	α-Caryophyllene	–	6.61	1.81	2.72	7.1
1457	(E)-β-Farnesene	0.22	–	0.15	–	tr
1480	γ-Muurolene	–	–	1.92	0.44	10.54
1485	Germacrene D	–	0.36	21.97	22.06	15.1
1490	β-Selinene	–	0.46	0.38	0.25	3.0
1496	Viridiflorene	–	–	–	–	2.89
1498	α-Selinene	–	–	0.56	1.2	1.53
1506	(E,E)-α-Farnesene	–	–	4.59	3.68	–
1506	β-Bisabolene	0.04	–	–	–	–
1514	γ-Cadinene	–	–	4.99	5.43	–
1523	δ-Cadinene	–	–	4.07	1.85	–
1557	Elemicin	0.08	1.26	0.2	0.77	1.12
1563	(E)-Nerolidol	–	16.8	10.95	3.79	1.5
1578	Spathulenol	–	1.56	–	–	–
1583	Caryophyllene oxide	–	4.05	6.55	9.34	5.05
1601	Guaiol	–	1.23	–	–	–
1640	τ-Muurolol	–	–	1.29	0.98	–
1642	τ-Cadinol	–	4.61	0.93	0.47	–
1651	β-Eudesmol	–	11.09	–	–	1.31
1654	α-Eudesmol	–	5.38	1.94	1.93	4.86
	Total identified (%)	99.0	99.79	90.66	90.59	99.96

*Relative retention indices on an apolar column (CP-Sil 5); tr, trace (<0.01%); –, not detected; PAS, P. abyssinica stem; PAR, P. abyssinica root; PEL, P. eminii leaf; PES, P. eminii stem; PER, P. eminii root.

In the leaf, stem and root EO of P. eminii, 30 (90.66%), 27 (90.59%) and 27 (99.96%) compounds were detected, respectively. Sesquiterpenes were the dominant constituents of these EOs accounting for 87.56% (leaf), 86.54% (stem) and 73.73% (root). The major components of the oxygenated sesquiterpenes of P. eminii leaf (PEL) were (E)-nerolidol (10.95%) and caryophyllene oxide (6.55%). Similarly, germacrene D (21.97%) and β-caryophyllene (21.64%) were the most abundant sesquiterpene hydrocarbons of the leaf EO with smaller amounts of γ-cadinene (4.99%), (E, E)-α-farnesene (4.59%) and δ-cadinene (4.07%). The stem EO of P. eminii (PES) dominated by sesquiterpene hydrocarbons (70.03%) contained β-caryophyllene (28.85%) and germacrene D (22.06%). Oxygenated sesquiterpenes accounted for 16.51%, and caryophyllene oxide (9.34%) and (E)-nerolidol (3.79%) were the major oxygenated sesquiterpenes. Major sesquiterpene hydrocarbons in the root EO of P. eminii (PER) were β-caryophyllene (18.08%), germacrene D (15.1%), γ-muurolene (10.54%) and α-caryophyllene (7.1%). Similarly, α-eudesmol (4.86%) and caryophyllene oxide (5.05%) were the major oxygenated sesquiterpenes detected. Among the monoterpenes, endo-fenchyl acetate (13.76%) was the major constituent of P. eminii root.

Phytosterols and diterpenoids were detected in the petroleum ether and chloroform fractions of all parts of the studied plants. Carotenoids were detected in the leaf petroleum ether and chloroform fractions of both species. Saponins were detected in the methanol fraction of all parts of both plants, and tannins in the methanol fractions of the leaves and roots. Polyphenols were detected in the methanol fractions of all parts of both species except the stem of P. abyssinica. Alkaloids were present in chloroform extracts of the leaves of both plants and roots of P. abyssinica.

Petroleum ether fractions of P. abyssinica roots showed antibacterial activity against Staphylococcus aureus and Pseudomonas aeroginosa, while both petroleum ether and chloroform root extracts exhibited activity against Candida albicans and Cryptococcus neoformans (Table 2). Leaf petroleum ether extract of P. eminii showed antistaphylococcal activity, and both leaf and root petroleum extracts were active against Cryptococcus (Table 3). Petroleum ether extracts of all morphological parts of P. eminii exhibited antifungal activity against Candida. In Table 4, the lethal activity of methanol fractions of the leaves and roots of the studied plants against earthworms are presented, while petroleum ether and chloroform fractions were inactive. At 250 μg/mL, the leaf and root extracts of P. eminii killed 13.3 ± 0.5% and 23.3 ± 0.5% of the test animals, respectively. At the highest concentration tested (1500 μg/mL), methanol extracts from the leaf and root of P. eminii killed 63.3 ± 1.1% and 73.3 ± 0.5% of the test animals as against 100% of niclosamide at 84.5 μg/mL. P. abyssinica root methanol extract killed 13.3 ± 0.5% and 50.0 ± 1.0% of the worms at 250 and 1500 μg/mL doses, respectively. The leaf extract of P. abyssinica showed the lowest activity (13.3 ± 0.5%) at 500 μg/mL and a better activity (56.7 ± 0.5%) at the highest dose tested (1500 μg/mL).

Table 2.  Zone of inhibition produced by petroleum ether (PE), chloroform (CHCl₃) and methanol (MeOH) extracts of P. abyssinica against bacterial and fungal strains (n = 3, average ± SD).

Plant part	Fraction	Concentration (µg/µL)	Zone of inhibition (mm)
			Sa^s	Pa^s	Ca^i	Cn^i
Leaf	PE	12.5	–	–	6.3 ± 0.5	11.3 ± 1.5
		25	6.0 ± 2.0	–	8.0 ± 1.0	12.7 ± 1.1
		50	7.0 ± 1.0	–	10.3 ± 1.1	14.3 ± 1.5
	CHCl3	12.5	–	–	–	5.7 ± 0.5
		25	–	4.3 ± 0.5	–	10.0 ± 1.0
		50	–	5.7 ± 1.1	–	11.3 ± 1.1
	MeOH	12.5	–	–	–	–
		25	4.0 ± 1.0	–	–	–
		50	8.0 ± 2.6	–	–	–
Stem	PE	12.5	–	–	–	–
		25	6.3 ± 1.5	–	–	–
		50	8.7 ± 2.1	–	–	–
	CHCl3	12.5	–	–	–	–
		25	–	–	–	–
		50	5.3 ± 2.1	–	–	–
	MeOH	12.5	–	–	–	–
		25	–	–	–	–
		50	–	–	–	–
Root	PE	12.5	7.3 ± 1.5	8.0 ± 2.0	–	–
		25	10.3 ± 2.0	12.3 ± 0.5	–	–
		50	12.0 ± 1.7	13.3 ± 1.5	–	–
	CHCl3	12.5	–	–	9.3 ± 1.1	–
		25	4.0 ± 1.0	–	11.3 ± 0.5	–
		50	7.3 ± 1.1	–	12.7 ± 0.5	–
	MeOH	12.5	–	–	–	–
		25	6.7 ± 2.0	–	–	–
		50	11.0 ± 2.6	–	–	–
Gentamycin sulfate		0.1	27.0 ± 1.1	18.0 ± 0.5	x	–
Ketoconazole		0.3	x	x	23.3 ± 0.5	29.0 ± 1.1
Dimethyl sulfoxide		50% (v/v)90% (v/v)	x-	x-	-x	-x
Methanol		80% (v/v)			–	–

Sa^s, Staphylococcus aureus (standard); Pa^s, Pseudomonas aeroginosa (standard); Caⁱ, Candida albicans (clinical isolate); Cnⁱ, Cryptococcus neoformans (clinical isolate); –, no inhibition zone; x, not tested.

Table 3.  Zone of inhibition produced by petroleum ether (PE), chloroform (CHCl₃) and methanol (MeOH) extracts of P. eminii against bacterial and fungal strains (n = 3, average ± SD).

Plant part	Fraction	Concentration (µg/µL)	Zone of inhibition (mm)
			Sa^s	Ca^i	Cn^i
Leaf	PE	12.5	8.3 ± 1.1	13.3 ± 0.5	–
		25.0	10.3 ± 0.5	14.3 ± 1.5	–
		50.0	12.3 ± 1.5	15.0 ± 1.0	–
	CHCl3	12.5	–	–	7.7 ± 2.0
		25.0	–	–	12.3 ± 1.1
		50.0	–	–	14.3 ± 1.1
	MeOH	12.5	–	–	–
		25.0	5.3 ± 0.5	–	–
		50.0	9.7 ± 2.0	–	–
Stem	PE	12.5	–	8.3 ± 1.0	7.3 ± 0.5
		25.0	–	9.0 ± 2.0	8.0 ± 1.0
		50.0	–	11.3 ± 0.5	9.7 ± 1.1
	CHCl3	12.5	–	–	–
		25.0	–	–	–
		50.0	–	–	–
	MeOH	12.5	–	–	–
		25.0	–	–	–
		50.0	–	–	–
Root	PE	12.5	–	9.7 ± 0.5	11.7 ± 1.1
		25.0	5.3 ± 0.5	11.7 ± 1.1	14.7 ± 1.5
		50.0	6.0 ± 1.0	14.3 ± 1.5	15.3 ± 1.5
	CHCl3	12.5	–	–	6.7 ± 0.5
		25.0	–	–	9.7 ± 1.5
		50.0	5.7 ± 1.1	–	11.3 ± 1.1
	MeOH	12.5	–	–	–
		25.0	–	–	–
		50.0	–	–	–
Gentamycin sulfate		0.1	27.0 ± 1.1	x	x
Ketoconazole		0.3	x	29.3 ± 0.5	29.0 ± 1.1
Dimethyl sulfoxide		50% (v/v)	x	–	–
		90% (v/v)	–	x	x
Methanol		80% (v/v)	x	–	–

Caⁱ, Candida albicans (clinical isolate); Cnⁱ, Cryptococcus neoformans (clinical isolate); Sa^s, Staphylococcus aureus (standard); –, no inhibition zone; x, not tested.

Table 4.  In vitro mortality of earthworms (in %) observed upon treatment with methanol fractions of P. abyssinica and P. eminii (n = 3, average ± SD).

	Fraction	P. abyssinica		P. eminii
		Leaf MeOH	Root MeOH	Leaf MeOH	Leaf MeOH
Test solutions (µg/mL)	125	–	–	–	–
	250	–	13.3 ± 0.5	13.3 ± 0.5	23.3 ± 0.5
	500	13.3 ± 0.5	23.3 ± 0.5	36.7 ± 0.5	50.0 ± 1.0
	1000	36.7 ± 0.5	43.3 ± 0.5	53.3 ± 0.5	63.3 ± 0.5
	1500	56.7 ± 0.5	50.0 ± 1.0	63.3 ± 1.1	73.3 ± 0.5
Control	Niclosamide (84.5 µg/mL)	100 ± 0.0
	Dechlorinated tap water	–
	Tween 80 (0.5% v/v)	–

–, no death recorded.

Discussion

The antimicrobial activities of gradient solvent extracts of P. abyssinica and P. eminii are summarized in Tables 2 and 3. Data present the zone of inhibition (excluding the well diameter) at concentrations of 12.5, 25 and 50 µg/µL and their comparison with standard antibacterial and antifungal agents. Antimicrobial tests revealed that the antimicrobial activity of the extracts resides in the non-polar and/or semi-non-polar petroleum ether and chloroform fractions, in which the presence of phytosterols, diterpenoids and carotenoids was ascertained. Minimum inhibitory concentrations (MIC) of the antimicrobially active extracts of the various parts of the studied plants were determined using the agar-well diffusion assay. Accordingly, petroleum ether fraction of the leaf of P. abyssinica, petroleum ether extract of leaf and root and chloroform extract of root of P. eminii had an MIC of 2.5 µg/µL against C. neoformans, while petroleum ether extract of the root of P. abyssinica had a MIC of 10 μg/μL against S. aureus and P. aeruginosa. The leaf petroleum ether extract of P. eminii had a MIC of 2.5 and 5 μg/μL against C. albicans and S. aureus, respectively. Chloroform root extract of P. abyssinica inhibited the growth of C. albicans with a minimum concentration of 10 μg/μL.

Although antimicrobial activity of methanol extracts of P. abyssinica against S. aureus have been reported (Messele et al., 2004), scientific studies on biological activity and phytochemical composition of various species of Pycnostachys are lacking. The family Lamiaceae is generally known to be a rich source of diterpenoids of many structural types which possess antibacterial and antifungal properties (Diaz et al., 1988; Gonzalez et al., 1989; Cole et al., 1991; Cole, 1992; Batista et al., 1994; Kilic, 2006). Similarly, the antimicrobial activity of plant steroids has also been demonstrated in many studies (Tanaka et al., 1969; Subhisha & Subramoniam, 2005; Rojas et al., 2006).

Accordingly, diterpenoids and steroids detected in P. abyssinica and P. eminii might be responsible for the inhibitory effect observed against the sensitive microorganisms. The active principles in the extracts, being lipophilic and/or semi-lipophilic, are likely to act on the cell membrane or intracellularly. The observation that the gradient solvent extracts of P. abyssinica showed activity against some of the bacterial strains and the yeasts may be the basis for their claimed ethnomedical use for a variety of respiratory, eye and skin infections.

Conclusions

The stem essential oil of P. abyssinica is dominated by the relatively more biologically active oxygenated monoterpenes with estragole as the most abundant constituent. The root EO of this species contained high amounts of both oxygenated monoterpenes and oxygenated sesquiterpenes. The EOs from all morphological parts of P. eminii contained mainly sesquiterpenes, with hydrocarbon sesquiterpenes being the dominant constituents such as germacrene D and β-caryophyllene representing the larger proportion in all parts. Most of the gradient extracts showed varying levels of activity against the tested microorganisms. Petroleum ether and chloroform extracts of P. abyssinica and P. eminii were found to be more active than the methanol extracts. Among the four test fungi, the yeasts were found to be more susceptible than the filamentous fungi, while Gram negative bacteria were relatively resistant to the extracts of both species. Higher antimicrobial activity was observed in the leaf extracts from both plants. Additionally, the leaf and root methanol extracts of both plants showed lethal activity against earthworms. The presented results from essential oil analysis, antimicrobial, and anthelmintic assays with gradient extracts, together with phytochemical screening, indicate the biological potential of Pycnostachys species from Ethiopia, and emphasize the pharmacological and indigenous applications of these plants.

Declaration of interest

J. Hussien and A. Hymete would like to acknowledge Ethiopian Health and Nutrition Research Institute (ENHRI) for providing the test organisms. J. Hussien and A. Hymete would like to acknowledge Addis Ababa University for financial assistance.