Ngassapa et al Tropical Journal of Pharmaceutical Research January 2016; 15 (1): 107-113 ISSN: 1596-5996 (print); 1596-9827 (electronic) © Pharmacotherapy Group, Faculty of Pharmacy, University of Benin, Benin City, 300001 Nigeria. All rights reserved. Available online at http://www.tjpr.org http://dx.doi.org/10.4314/tjpr.v15i1.15 Original Research Article Chemical Composition and Antimicrobial Activity of Geniosporum rotundifolium Briq and Haumaniastrum villosum (Bene) AJ Paton (Lamiaceae) Essential Oils from Tanzania Olipa D Ngassapa1*, Deborah KB Runyoro1, Konstantinos Vagionas2, Konstantia Graikou2 and Ioanna B Chinou2 1 Department of Pharmacognosy, School of Pharmacy, Muhimbili University of Health and Allied Sciences (MUHAS), PO Box 2 65013, Dar es Salaam, Tanzania, Division of Pharmacognosy & Chemistry of Natural Products, School of Pharmacy, University of Athens, University Campus of Zografou, 157 71 Athens, Greece *For correspondence: Email: ongassapa@muhas.ac.tz; o_ngassapa@yahoo.co.uk; Tel: +255-713-246 227; Fax: +255-222150465 Received: 9 May 2015 Revised accepted: 12 November 2015 Abstract Purpose: To determine the chemical composition and antimicrobial potential of essential oils from two aromatic plants of Tanzania, Geniosporum rotundifolium Briq. and Haumaniastrum villosum (Benè) A.J. Paton (Lamiaceae). Method: Essential oils from the aerial parts of the plants were extracted by hydro-distillation for 3 h using a Clevenger type of apparatus. The constituents were analyzed by gas chromatography – mass spectrometry (GC/MS).The minimum inhibitory concentrations of the essential oils were determined for eight bacterial strains and three pathogenic fungi using agar dilution method. Results: The constituents of G. rotundifolium oil were mainly oxygenated derivatives of mono- and sesquiterpenes; spathulenol (12.46 %), α-terpineol (4.65 %) and germacrene-D (3.71 %) were the most abundant. Those of H. villosum oil were predominantly sesquiterpenes (72.61 %) with caryophyllene oxide (19.01 %), humulene epoxide II (11.95 %), β-bourbonene (5.7 %), α-humulene (5.63 %) and βcaryophyllene (5.39 %) being more abundant. The oil of G. rotundifolium exhibited weak to moderate activity against the bacterial species but showed no activity against the test fungi. However, H. villosum oil showed very promising activity against all the test microorganisms (MIC 0.08 – 10.34 mg/mL). Conclusion: The major components of G. rotundifolium essential oil were oxygenated derivatives of mono- and sesquiterpenes whereas those of H. villosum were sesquiterpenes. All tested microorganisms were susceptible to H. villosum oil. Keywords: Geniosporum rotundifolium, Haumaniastrum villosum, Essential oils, Chemical composition, Antimicrobial activity Tropical Journal of Pharmaceutical Research is indexed by Science Citation Index (SciSearch), Scopus, International Pharmaceutical Abstract, Chemical Abstracts, Embase, Index Copernicus, EBSCO, African Index Medicus, JournalSeek, Journal Citation Reports/Science Edition, Directory of Open Access Journals (DOAJ), African Journal Online, Bioline International, Open-J-Gate and Pharmacy Abstracts INTRODUCTION Geniosporum rotundifolium Briq. and Haumaniastrum villosum (Benè) A.J. Paton (Lamiaceae) are known as “Nkulilo” in the Nyakyusa dialect of Rungwe District, Mbeya Region, Southwestern Tanzania. Geniosporum rotundifolium (syn. G. paludosum Bak) [1], is a stout, erect, perennial herb which grows in damp grassland at high altitude [2]. It is confined to Trop J Pharm Res, January 2016; 15(1): 107 Ngassapa et al several African countries including Tanzania [3]. Its leaves, stems and essential oils are given in combination with leaves of other plants for a number of medical uses. In Burundi it is used as an enema, cough remedy, laxative and antiabortion while in Uganda it is used against fungal and bacterial infections [4]. A previous study on G. rotundifolium growing in Cameroon indicated that the essential oil from this plant possessed significant antifungal activities against Fusarium moniliforme and Rhizopus stolonifera. Furthermore, its chemical composition was determined with sesquiterpene hydrocarbons constituting more than 90 % of the oil [5]. Haumaniastrum villosum is an annual or shortlived perennial herb confined to the African continent and Madagascar, in the sub-humid climate [6]. There is scanty information on the medicinal uses and biological activities of H. villosum and to our knowledge there is no information on its phytochemical studies. Its synonym H. galeopsifolium, has been reported to be used traditionally in Burundi, alone or in combination for a number of health problems including urogenital infections [1]. It has also been reported to be used in controlling crop pests in the Democratic Republic of Congo [7]. In the current study, chemical compositions and antimicrobial activities of the essential oils of Geniosporum rotundifolium and Haumaniastrum villosum from Tanzania are reported for the first time. EXPERIMENTAL Plant material Aerial parts (leaves and flowering tops) of G. rotundifolium and H. villosum were collected from the wild, in Rungwe district, Mbeya region, Tanzania in June, 2000. The plants were authenticated by Mr. H. Selemani of the Department of Botany, University of Dar es Salaam. Voucher specimen Nos. ODN/DBR 001 for G. rotundifolium and ODN/DBR 002 for H. villosum, respectively, were deposited in the herbarium of the Department of Pharmacognosy, School of Pharmacy, Muhimbili University of Health and Allied Sciences. Isolation of essential oil All materials were air-dried in the shade, prior to hydro-distillation of essential oils for 3 h in a Clevenger-type apparatus. The essential oils collected over water were separated, dried over anhydrous sodium sulfate and stored at 4–6 oC until chemical screening. analysis and antimicrobial Gas chromatography Gas chromatography (GC) analysis was carried out on a Perkin-Elmer 8500 gas chromatograph with a flame ionization detector (FID), fitted with a Supelcowax-10 fused silica capillary column (30 m x 0.32 mm, 0.25 µm film-thickness). The column temperature was programmed from 75 to 200 oC at a rate of 2.5 oC/min. The injector and detector temperatures were programmed at 230 o C and 300 oC, respectively. Helium was used as the carrier gas, at a flow rate of 1 mL/min. Gas chromatography-mass spectrometry Gas chromatography-mass spectrometry (GCMS) analysis was carried out using a Hewlett Packard 5973-6890 GC-MS system operating on EI mode (equipped with a HP 5MS 30 m x 0.25 mm x 0.25 µm film thickness capillary column). Helium (2 mL/min) was used as the carrier gas. The temperature of the column was programmed from 60to 280 oC, at a rate of 3 °C/min. Split ratio, 1:10. Identification of components The compounds were identified by comparison of their retention indices (RI) [8] retention times (RT) and mass spectra with those of authentic samples, viz, 1,8-cineole, camphor, pulegone, piperitone, bornyl acetate, spathulenol, βcaryophyllene and β-caryophyllene oxide (Extrasynthese), borneol, linalool, limonene (Fluka AG), α –pinene, β –pinene (Aldrich) and/or the NIST/NBS, Wiley libraries spectra and the literature [9]. The percentage composition of the essential oil is based on computer calculated peak areas without correction for FID response factor. Evaluation of antimicrobial activity Antimicrobial activity of the essential oils against bacteria and fungi was determined using the agar dilution technique. The microorganisms included four Gram-positive bacteria: Staphylococcus aureus (ATCC 25923), Staphylococcus epidermidis (ATCC 12228); Streptococcus mutans and Streptococcus viridian, with the last two being clinical isolates and oral pathogens; four Gram-negative bacteria: Escherichia coli (ATCC 25922), Enterobacter cloacae (ATCC 13047), Klebsiella pneumoniae (ATCC 13883) and Pseudomonas aeruginosa (ATCC 227853); and three species of Candida, namely, C. albicans (ATCC 10231), C. tropicalis Trop J Pharm Res, January 2016; 15(1): 108 Ngassapa et al (ATCC 13801) and C. glabrata (ATCC 28838). Standard antibiotics (netilmicin and amoxicillin) were used as positive controls. Technical data have been described previously [10]. Briefly, stock solutions of the tested samples were prepared at 10 mg/mL in dichloromethane. Serial dilutions of the stock solutions in broth medium (100 μL of MüllerHinton broth or on Sabouraud broth for the fungi) were prepared in a microtiter plate (96 wells). Then 1 μL of the microbial suspension (the inoculum, in sterile distilled water) was added to each well. For each strain, the growth conditions and the sterility of the medium were checked and the plates were incubated as referred above. Standard antibiotics, netilmicin and amoxicillin (at concentrations 4-88 μg/ml), were used as positive controls. For each experiment, the pure solvent, dichloromethane, was also applied as negative control. The experiments were repeated three times and the results were expressed as average values. Minimum inhibitory concentrations (MICs) were determined for all the samples and the standard pure compounds, under the same conditions, for comparison purposes. The MICs were taken as the lowest concentrations preventing visible growth. RESULTS The oils obtained from both plant species were pale yellow liquids with slight aromatic smell. The yield was 0.06 % v/w for G. rotundifolium and 0.12 % v/w for H. villossum. A total of 59 components, comprising 91.15 % of the oil got separated in the GC of G. rotundifolium, of which 54 constituents were identified (Table 1(a), 1(b) and 1(c). A 44.89 % of the oil was composed of oxygenated derivatives, while mono and sesquiterpene hydrocarbons constituted 36.67 % of the oil. The major compounds identified were spathulenol (12.46 %), α-terpineol (4.65 %) and germacrene-D (3.71 %). In a previous study on plants growing in Cameroon, it was found that sesquiterpene hydrocarbons constituted 90.1 % of the oil with germacrene D, β-caryophyllene and β-gurjunene being the major components [5]. The difference in the composition could be attributed to differences in the geographical location, climate, season and age at which the plants were collected. In the essential oil of Haumaniastrum villosum, a total of 44 components were identified, representing 85.6 % of the oil (Table 2(a) and 2(b)); oxygenated derivatives were again the most abundant chemical category (44.48 % followed by monoand sesquiterpene hydrocarbons (34.24 %) The most abundant components were caryophyllene oxide (19.01 %), humulene epoxide II (11.95 %), βbourbonene (5.7 %), α-humulene (5.63 %) and β-caryophyllene (5.39 %). The oils as well as pure reference compounds were tested for antimicrobial activity against eight bacterial species and three species of Candida. The antimicrobial activity as minimum growth inhibitory concentrations of the essential oils, some pure components and the reference antimicrobial agents, are shown in Table 3(a) and (b). Both oils exhibited different levels of antimicrobial activity against the tested microorganisms. The G. rotundifolium oil showed moderate activity against Staphylococcus aureus and Staphylococcus epidermidis and weak activity against E. coli and had no activity at tested concentrations against Pseudomonas aeruginosa, Klebsiella pneumoniae and Enterobacter cloacae. On the other hand, H. villosum oil showed very promising antimicrobial activity against all the tested microorganisms (bacteria and fungi) with minimum inhibitory concentrations ranging from 0.08 to 10.34 mg/mL. Among the microorganisms, S. aureus was the most sensitive (MIC 0.08 mg/mL) and E. coli was the least sensitive (MIC 10.34 mg/mL). DISCUSSION The major compounds identified for the essential oil of G. rotundifolium were different from those identified previously for plants growing in Cameroon in which sesquiterpene hydrocarbons constituted 90.1 % of the oil with germacrene D, β-caryophyllene and β-gurjunene being the major components [5]. The difference in the composition could be attributed to differences in the geographical location, climate, season and age at which the plants were collected. It would be worth reporting that H. villosum oil was strongly active against S. mutans, S. viridis, Candida albicans, C. tropicalis and C. glabrata (with MIC’s 0.14-0.94 mg/mL), which were resistant to oils from G. rotundifolium and other plants growing in Tanzania, as reported previously [10-12]. In addition, the essential from G. rotundifolium was devoid of antifungal activity against the tested Candida species unlike the essential oil growing in Cameroon which was previously reported to have shown significant antifungal activity against Fusarium moniliforme and Rhizopus stolonifera [15]. Trop J Pharm Res, January 2016; 15(1): 109 Ngassapa et al Table 1: Chemical composition of the essential oil of Geniosporum rotundifolium No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 Constituent α-Pinene Camphene β-Pinene 1-Octen-3-ol 3-Octanol p-Cymene Limonene Eucalyptol Cis-ocimene Trans-β-Ocimene γ-Terpinene cis-Sabinene hydrate α-Terpinolene Linalool α-Thujone α-Camphonelal Trans-pinocarveol Camphor ε-Myroxide Borneol Terpinen-4-ol α-Terpineol Myrtenal Unknown Verbenone Trans-carveol Carvone Hexyl tiglate α-Cubebene Eugenol α-Copaene β-Bourbonene trans-β-Damascenone β-Elemene Methyl eugenol β-Caryophyllene β-Gurjunene α-Bergamotene α-Humulene Alloaromadendrene α-Amorphene Germacrene-d Ar-curcumene β-Ionone Epibicyclosesquiphellandrene α-Muurolene Γ-Cadinene Δ-Cadinene α-Calacorene Cerolidol Spathulenol Caryophyllene oxide Salvial-4(14)-en-1-one Unknown Unknown α-Cadinol Cadalene Unknown Unknown Total % 2.49 1.10 1.82 0.73 0.65 1.48 2.65 1.10 0.49 0.30 0.32 0.61 0.28 2.43 0.74 0.39 1.17 2.28 1.14 1.26 2.85 4.65 tr 2.10 0.40 0.72 0.42 0.56 0.60 2.09 2.83 2.91 0.60 1.66 1.34 2.09 0.91 0.77 0.52 1.15 1.62 3.71 0.31 0.74 1.17 ΚΙ(α) 936 951 978 983 988 1027 1031 1033 1042 1052 1061 1069 1088 1102 1104 1127 1139 1144 1146 1166 1178 1191 1193 1196 1206 1220 1244 1333 1347 1359 1372 1379 1382 1387 1406 1411 1423 1432 1447 1453 1472 1475 1479 1482 1488 0.75 0.74 2.68 0.54 0.42 12.46 2.6 0.85 1.58 1.78 1.69 0.78 1.25 2.88 91.15 1494 1506 1519 1537 1564 1574 1575 1585 1602 1648 1652 1671 1686 KI(α1) 935 949 976 981 995 1025 1029 1031 1040 1068 1103 1125 1138 1143 1145 1169 1177 1190 1194 1206 1219 1244 1331 1347 1358 1373 1381 1382 1387 1404 1414 1425 1432 1449 1456 1472 1476 1482 1486 1490 1509 1520 1539 1576 1579 1588 1604 1653 1655 1676 1692 2168 ΚΙ(β) 939 951 979 979 991 1025 1029 1031 1037 1050 1060 1070 1089 1097 1102 1126 1139 1146 1145 1165 1177 1189 1196 1205 1217 1243 1333 1351 1359 1377 1388 1385 1391 1404 1419 1434 1435 1455 1460 1485 1485 1481 1489 1494 1500 1514 1523 1546 1563 1578 1583 1595 1654 1677 Trop J Pharm Res, January 2016; 15(1): 110 Ngassapa et al Table 2: Chemical composition of the essential oil of Haumaniastrum villosum No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 Constituent α-Pinene β-Pinene 1-Octen-3-ol p-Cymene Limonene Eucalyptol Trans-pinocarveol Camphor Menthone Isomenthone Neomenthol α-Terpineol Linalool Pulegone Piperitone α-Cubebene Cycloisosativene α-Ylangene α-Copaene β-Bourbonene β-Cubebene β-Elemene β-Caryophyllene α- Humulene Trans- β-Farnesene Alloaromadendrene α-Amorphene Germacrene-d β-Selinene α-Muurolene β-Bisabolene γ-Cadinene Trans-calamenene δ-Cadinene α-Cadinene Elemol Caryophyllene oxide Salvial-4(14)-en-1-one Humuleneepoxide II Unknown Unknown β-Eudesmol α-Cadinol Total % 0.14 0.11 0.33 0.14 0.27 0.29 0.17 0.33 0.63 1.32 1.16 0.10 1.26 0.55 0.40 0.45 0.70 3.32 1.35 5.70 0.81 1.00 5.39 5.63 0.32 0.17 1.34 0.70 0.74 0.57 0.15 3.61 0.97 0.42 0.24 2.79 19.01 1.24 11.95 3.28 2.60 1.00 2.95 85.60 ΚΙ(α) 937 978 983 1027 1031 1033 1141 1145 1156 1166 1166 1192 1101 1241 1256 1350 1366 1371 1375 1384 1389 1391 1417 1453 1457 1475 1475 1479 1484 1497 1508 1512 1523 1530 1537 1551 1583 1592 1609 1615 1622 1652 1656 KI(β) 939 979 979 1025 1029 1031 1139 1146 1163 1163 1166 1189 1097 1237 1253 1351 1364 1375 1377 1388 1388 1391 1419 1455 1457 1485 1485 1485 1490 1500 1506 1514 1529 1523 1539 1550 1583 1595 1608 1651 1654 Trop J Pharm Res, January 2016; 15(1): 111 Ngassapa et al Table 3(a): Antimicrobial activity (MIC, mg/mL) of the essential oils and identified pure compounds S. aureus S. epidermidis P. aeruginosa K. pneumoniae E. cloaceae E. coli S. mutans S. viridans C. albicans C. tropicalis C. grabrata Essential oil/compound G. rotundifolium H. villosum 1,8- Cineole Limonene Linalool Camphor Pulegone Piperitone Bornyl acetate Borneol Spathulenol α-Pinene β- Pinene 3.25 0.08 9.50 >20 0.25 2.70 1.20 1.50 1.95 1.25 1.35 7.50 12.00 3.50 0.95 9.50 >20 0.25 1.95 0.95 2.25 1.75 1.57 1.50 9.50 16.00 >20 1.25 2.75 >25 >20 2.80 1.45 0.60 2.30 2.50 >20 6.00 >20 >20 1.37 2.35 >25 >20 3.24 1.76 0.80 3.25 3.75 >20 15.00 >20 >20 2.50 3.00 >25 1.75 2.75 1.37 1.10 3.75 4.20 >20 8.00 >20 18.50 10.34 2.00 >20 1.25 1.33 1.45 0.95 4.88 4.50 8.50 2.00 9.75 0.14 0.37 1.75 - 0.39 0.45 1.26 - 0.94 4.85 4.00 - 0.74 3.76 4.00 - 0.82 3.56 2.00 - Table 3(b): Antimicrobial activity (MIC, mg/mL) of the essential oils and identified pure compounds (contd) - - C. grabrata 0.75 - C. tropicalis C. albicans 0.25 - S. viridans >20 >6.40 10-2 -3 2x10 S. mutans >20 2.43 -3 8x10 -3 2.8 x10 E. coli >20 1.23 -3 8x10 -3 2.2x10 E. cloaceae >20 0.87 8.8 x10-3 -3 2.4x10 K. pneumoniae >20 0.90 -3 4x 10 -3 2x10 P. aeruginosa >20 0.073 4x10-3 -3 2x10 S. epidermidis S. aureus Essential oil/compound β- Caryphylene β- Caryphyleneoxide Netilmicin Amoxycillin - Trop J Pharm Res, January 2016; 15(1): 112 Ngassapa et al The observed antimicrobial activity in the studied essential oils could be attributed to their major components. In the case of G. rotundifolium, the activity could be mainly, due to the oxygenated sesquiterpene spathulenol, which showed two to three times more activity than the oil, while the activity of H. villosum oil compared well with that of β-caryophyllene oxide. The antimicrobial activity of these oils could also be attributed to the major and minor constituents of the oils, constituents with the known antimicrobial activity such as spathulenol [11], linalool [13] and camphor [14], and their synergistic effects. 14]. [EOL.org Available from http://www.gwannon.com/species/Geniosporumrotundifolium. 4. Kamatenesi-Mugisha M, Oryem-Origa1 H, Odyek O, Makawiti DW. Medicinal plants used in the treatment of fungal and bacterial infections in and around Queen Elizabeth Biosphere Reserve, western Uganda. Afr .J. Ecol.2008; 46 (Suppl. 1: 90-97. 5. François T, Michel JDP, Vyry WNA, Fabrice FB, Lambert SM, Henri AZP, Chantal M. Composition and Antifungal Properties of Essential Oils from Five Plants Growing in the Mountainous Area of the West Cameroon, Journal of Bulletin 1997; 52: 293-378. 7. Bin Mushambanyi TM. Local inhabitants’ control strategies of crop pests in Eastern Democratic Republic of Congo, by exploiting the local plant diversity species. Delpinoan.s, 2002; 44: 65-74. 8. Massada Y. Analysis of Essential Oil by Gas Chromatography and Spectrometry; New York, John Wiley & Sons, 1976. 9. Adams, R.P. Identification of Essential Oil Components by Gas Chromatography/Mass Spectrometry, 4th ed.; Carol Stream, IL, USA, Allured Publishing Co, 2007. Composition and antimicrobial activity of the essential The authors are grateful for the funding provided by Muhimbili University of Health and Allied Sciences (MUHAS) through Sida Research Capacity Strengthening, which enabled us to accomplish this work. The assistance provided by technical staff at University of Athens, Athens, Greece and MUHAS for access to laboratory facilities, as well as the support received from late Rev Moses Mbila Mwakyendelwa during the collection of plant materials, are also highly appreciated. oils of two different populations of Lippia javanica growing in Tanzania. Flav. Frag. J.2003; 18: 221-224. 11. Bougatsos C, Ngassapa O,Runyoro DKB, Chinou IB. Chemical Composition and in vitro Antimicrobial Activity of the essential oils of two Helichrysum species from Tanzania. Zeitschr Naturforsch 2004; 59c: 368-372. 12. Vagionas K, Graikou K, Chinou IB, Runyoro D, Ngassapa O. Chemical analysis and antimicrobial activity of essential oils from the aromatic plants Artemisia afra Jacq. And Leonotis ocymifolia (Burm. F.) Iwarsson var raineriana (Vision1) Iwarsson growing in Tanzania. J. Essent. Oil Res. 2007; 19: 396-400. 13. Koutsoudaki REFERENCES C, Krsek, M, Rodger, A. 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