Chemical Composition of the Essential Oils of Cyperus rotundus L. from South Africa

The essential oils from the rhizomes of Cyperus rotundus L. collected from two different locations (Empangeni-A and KwaDlangezwa-B; both in the Kwa-Zulu Natal Province of South Africa) were obtained by hydrodistillation and analyzed by capillary GC and GC/MS. Forty-one and 43 components were identified, representing 89.9% and 92.0% of sample A and sample B, respectively. α-Cyperone (11.0%), myrtenol (7.9%), caryophyllene oxide (5.4%) and β-pinene (5.3%) were major compounds in the oil of sample A. The main constituents of the oil of sample B were β-pinene (11.3%), α-pinene (10.8%), α- cyperone (7.9%), myrtenol (7.1%) and α-selinene (6.6%).


Introduction
Cyperus rotundus L., (family Cyperaceae), also known as purple nutsedge or nutgrass, is a common perennial weed with slender, scaly creeping rhizomes, bulbous at the base and arising singly from the tubers which are about 1-3 cm long. The tubers are externally blackish in colour and reddish white inside, with a characteristic odour. The stems grow to about 25 cm tall and the leaves are linear, dark green and grooved on the upper surface. Inflorescences are small, with 2-4 bracts, consisting of tiny flowers with a red-brown husk. The nut is three-angled, oblong-ovate, yellow in colour and black when ripe. C. rotundus is indigenous to India, but are now found in tropical, subtropical and temperate regions [1,2].
As part of our on-going research on the chemical composition of the essential oils of South African medicinal and aromatic plants from the family Cyperaceae, the present investigation reports on the essential oils from the rhizomes of Cyperus rotundus found in South Africa.

Results and Discussion
Hydrodistillation of the fresh rhizomes of C. rotundus collected from the two different locations yielded 0.20% and 0.16% pale yellowish oils for Empangeni (sample A) and KwaDlangezwa (sample B), respectively. The compositions of the two oils are displayed in Table 1, where constituents are listed in order of their elution on the (DB-5) column. A total 58 components were detected, 41 and 43 of which were identified, accounting for 88.9% and 92.0% of the oil of Empangeni and KwaDlangezwa samples, respectively. The oil of the Empangeni sample was characterized by larger amounts of sesquiterpenes (59.8%) than monoterperenes (29.1%), while the KwaDlangezwa oil sample had a relatively similar content of sesquiterpenes (45.9%) and monoterperenes (46.1%). The sesquiterpenic composition of the oil of Empangeni is dominated by α-cyperone (11.0%), caryophyllene oxide (5.4%) and β-selinene (5.1%), and the compounds: myrtenol (7.9%), β-pinene (5.3%) and trans-pinocarveol (4.0%) were the major representative of monoterpenoids. In the oil of KwaDlangezwa, β-pinene (11.3%), α-pinene (10.8%), α-cyperone (7.9%), myrtenol (7.1%), α-selinene (6.6%), limonene (5.7%) and β-selinene (4.6%) were the main constituents. Although the compositions of the oil from each location varied, certain similarities were evident. The composition profile of the oil of Empangeni sample shows that it is richer in sesquiterpenes and is therefore, similar to other reported essential oils compositions of C. rotundus from different countries [14,[30][31][32][33][34][35]. However, the chemical pattern of the oil of KwaDlangezwa sample is completely different from the sesquiterpenoid dominating types.  In all the previous reports on the chemical composition of essential oils of C. rotundus from around the world, α-cyperone, cyperene, cyperotundone and β-selinene were found to be the major compounds identified in higher concentrations, along with other constituents such as, α-copaene, valerenal, caryophyllene oxide, patchoulenyl acetate and sugeonyl acetate ( Table 2). In addition, some reports have had the occurrence of α-pinene, β-pinene, limonene and 1,8-cineole as minor components of the essential oils of C. rotundus [14,15]. However, in our results (Tables 1 and 2), cyperene, αcyperone and β-selinene were present only in small concentrations, while cyperotundone, valerenal, petchoulenyl and sugeonyl acetates were not detected at all in our oil samples. Interestingly, the major constituents of the KwaDlangezwa oil sample, α-pinene (10.8%) and β-pinene (11.3%) have not been previously reported in dominant quantities among the major compounds from the oils of C. rotundus (Table 2). Table 2. Comparison of major constituents of C. rotundus rhizomes oils from different countries. Constituents  1  2  3  4  5  6  7  8  9 10 Comparing the present results with those previously reported in the literature on the essential oil compositions of C. rotundus from different countries [14,[30][31][32][33][34][35], it is apparent that, there are many differences regarding the major constituents of the oils of C. rotundus, which further suggests the existence of more chemical diversity within the C. rotundus species [33]. However, cyperene and αcyperone are the two major compounds similar in the essential oils of C. rotundus from Africa (Nigeria, Tunisia and South Africa). The observed compositional difference between C. rotundus found in South Africa and the rest of the world could be due to climactic and environmental conditions, chemotypes, nutritional status of the plants, and other factors, which can influence essential oil composition [14,30,31,[37][38][39][40][41]. In conclusion, the essential oils of C. rotundus from South Africa can be related to the M-type due to the presence of cyperene, β-selinene and α-cyperone.

Oil isolation
Finely chopped fresh rhizomes of each sample (500 g) were separately subjected to hydrodistillation in a Clevenger-type apparatus for 4 h in accordance with the British Pharmacopoeia specification [42]. The sample was added to distilled deionized water (1.5 L) in a 2-5 L roundbottomed flask and heated to boiling, after which the essential oil was evaporated together with water vapour and finally collected in a condenser. The upper phase that contained the essential oil was separated from the lower one and the distillate isolated was preserved in a sealed sample tube and stored under refrigeration until analysis [43,44].

GC analyses
GC analysis was carried out on a Hewlett Packard HP 6820 Gas Chromatograph equipped with a FID detector and DB-5 column (60 m x 0.25 mm id), film thickness was 0.25 µm and the split ratio was 1:25. The oven temperature was programmed from 50 °C (after 2 min) to 240 °C at 5 °C/min and the final temperature was held for 10 min. Injection and detector temperatures were 200 °C and 240 °C, respectively. Hydrogen was the carrier gas. An aliquot (0.5 µL of the diluted oil) was injected into the GC. Peaks were measured by electronic integration. A homologous series of n-alkanes were run under the same conditions for determination of retention indices.

GC-M S analyses
GC-MS analyses of the oils were performed on a Hewlett Packard HP 6890 Gas Chromatography interfaced with Hewlett Packard 5973 mass spectrometer system equipped with a DB-5 capillary column (30 m x 0.25 mm id, film thickness 0.25 µm). The oven temperature was programmed from 70-240 °C at the rate of 5 °C/min. The ion source was set at 240 °C and electron ionization at 70 eV. Helium was used as the carrier gas at a flow rate of 1 mL/min. Scanning range was 35 to 425 amu. 1.0 µL of diluted oil in hexane was injected into the GC/MS.

Identification of components
The components of the oils were identified base on the comparison of their retention indices and mass spectra with those standards, Wiley 275 library mass spectra databased of the GC/MS system and published data [45][46][47].