2455 Journal of Applied Sciences Research, 7(12): 2455-2461, 2011 ISSN ISSN 1819-544X This is a refereed journal and all articles are professionally screened and reviewed

ORIGINAL ARTICLES

Solvent-Free Microwave Extraction and Hepatoprotective Activity of esculentus L. and Essential Oils

1H.D. Hassanein, 1N.M. Nazif, 2E.A. Aboutabl and 1F.M. Hammouda

1Phytochemistry Department, National Research Center, 12311, Cairo, Egypt. 2Pharmacognosy Department, Faculty of Pharmacy, 11562, Cairo University, Egypt.

ABSTRACT

Solvent-free microwave extraction (SFME) has been applied to the extraction of essential oils from and Cyperus articulatus tubers. The compositions of the essential oils were identified by GC-MS. The obtained with SFME contained substantially higher amounts of oxygenated compounds and lower amounts of monoterpenes. Essential oils which were obtained from C. articulatus and C.esculentus were characterized by much larger amounts of sesquiterpenes (67.44% and 64.6% respectively) than monoterpenes (0.81% and 20.68% respectively). C. articulatus essential oil also was characterized by higher contents of none terpenoid compounds than C.esculentus (25.53 % and 12.09%) for tubers respectively. Culture of monolayer of rat hepatocyte is an in vitro method for the investigation of the hepatoprotective and hepatotoxic effect of essential oils. C.esculentus and C.articulatus tubers essential oils, showed no hepatotoxic effect till a concentration of 1000 μgmL-1. The hepatoprotective effects of C.esculentus and C.articulatus tubers against the toxic effect of 25 mM paracetamol are 18.5 and 40 g mL-1 respectively.

Key words: Cyperus esculentus, Cyperus articulatus, Solvent-free microwave extraction, Essential oil, GC/MS.

Introduction

In recent years, the use of microwave for extraction of constituents from material has shown tremendous research interest and potential. Conventional techniques for the extraction of active constituents are time and solvent consuming, thermally unsafe and the analysis of numerous constituents in plant material is limited by the extraction step. This highlights the importance of extraction step in setting up respectable standards for herbal medicine worldwide (Mandal et al., 2007). The method of solvent-free microwave extraction at atmospheric pressure of essential oil in plant material involves placing the sample in the microwave reactor, without any added solvent or water. The internal heating of the water within the sample distends its cells and leads to rupture of the glands and oleiferous receptacles. This process thus frees essential oil, which is evaporated by the in-situ water of the plant material. A cooling system outside the microwave oven continuously condenses the vapours which are collected on specific glassware. The excess of water is refluxed back to the extraction vessel in order to restore the in-situ water to the sample. Once the essential oils have been extracted they can be analyzed by GC-MS. The genus Cyperus is also well-known for its essential oil (Couchman et al., 1964). The essential oils of Cyperus species have long been used in folk medicine and as raw material for perfumes. Cyperus species, are becoming a traditional medicinal plant in China, and , and in tropical, subtropical and temperate regions; it is used against spasms and stomach disorders (Pharmacopeia Commission of PRC, 2005). The essential oil of Cyperus possesses analgesic, anti-inflammatory, antipyretic (Liu et al., 1989) and antifungal activity, insecticidal (Khater and Shalaby, 2008). Several sesquiterpenes including cyperone, cyperene and patchoulenone are thought to be the biological active ingredients in the essential oil (Jin et al., 2011). There are 19 species of Cyperus grown wild in Egypt at different habitats, two of the 19 species were found to have some commercial values as being nutritional supplemental foods, herbal medicine and perfume industry. The tubers of Cyperus esculentus are edible and known as "habb el aziz", chufas or tiger nuts. Many people use parts of the plant as remedies for a wide variety of ailments, sometimes in conjunction with other . Cyperus articulatus L. is known as “priprioca” or “piriprioca,” the forming tubers, which possess a very pleasant and strong smell. In the Amazonian region, “piripiri” has been used to treat many diseases (Plowman et al., 1990). C. articulatus was also found to be effective against Pseudomonas aeruginosa and Staphylococus aureus (Desmarchelier et al., 1996) and active to epileptiform discharges (Bum et al., 1996).

Coresponding Author: Faiza M. Hammouda, Phytochemistry Department, National Research Center, 12311, Cairo, Egypt. E-mail: [email protected] 2456 J. Appl. Sci. Res., 7(12): 2455-2461, 2011

Liver diseases are one of the major national health problems in Egypt. Hence the need for the development of hepatoprotective agents has increased. Many years ago, the tubers of Cyperus species have been used as remedy for the treatment of several diseases such as hepatotoxicity (Mehta et al., 1999) and as an antioxidative agent (Satoh et al., 2004). The effect of oral administration of C.esculentus oily extract was studied for its hepatoprotective and hepatocurative activities against CCl4-induced hepatic damage in male albino rats. C.esculentus significantly lowered the serum levels of Alanine transaminase (ALT), Aspartate transaminase -1 (AST) and Alkaline phosphatase (ALP) (p≤0.005) at a dose of 200 mg Kg (b.wt.) as compared to CCl4- treated animals (Ameen et al., 1999). In this paper we present the results of performing a comparative evaluation of the essential oil of both tubers obtained by free-solvent microwave extraction and the study of the hepatoprotective and hepatotoxic effect of the essential oil.

Experimental:

Plant Material:

Tubers of Cyperus esculentus L. (Family ) were collected from Rashid area (Borg Rashid), Egypt; October 2008. The plant was kindly brought by Prof. Dr. Salah Zarad; Water Relation and Field Irrigation Department-National Research Center, Cairo, Egypt and identified by Prof. Dr. Kamal Zaid, Botany Department, Faculty of Science, Cairo University. A voucher specimen by the number of 222, is deposited at the Faculty of Science’s herbarium, Cairo University.

Methods:

Sample Preparation:

Fresh tubers (100 g) were grinded and distillated at 800 W for 30 min using an adapted microwave distillation apparatus which consists of a microwave oven connected to a Clevenger-type apparatus. The yield of essential oil from C.esculentus is 3.2 gm and for C.articulatus 1.7 gm.

Equipment:

GC/MS was carried out using an HP5890 Series II Gas Chromatography, HP 5972 Mass Selective Detector and Agilent 6890 Series Autosampler. A Supelco MDN-5S 30 m by 0.25mm capillary column with a 0.5 µm film thickness was used with helium as the carrier gas at a flow rate of 1.0 ml/min. The GC oven temperature was programmed at an initial temperature of 40˚C for 5 minutes, then heated up to 140˚C at 5˚C/min and held at 140˚C for 5 min, then heated to 280˚C at 9˚C /min and held for 1 additional minute. Injector and detector temperatures were set at 280˚C. Mass spectrometry was run in the electron impact (EI) at 70eV. The identification of the chemical constituents were determined by their GC retention times, interpretation of their mass spectra and confirmed by mass spectral library search using the National Institute of Standards and Technology (NIST) database.

Isolation and Culture of Rat Hepatocytes Monolayer:

Primary culture of rat hepatocytes was prepared according to Seglen method, 1976, modified by Kiso et al., 1983, using a waster male rat (250-300 gm), obtained from the animal house of the NRC (National Research Center, Cairo). Animal procedures were performed in accordance with the Ethics Committee of the National Research Centre and followed the recommendations of the National Institutes of Health Guide for Care and Use of Laboratory Animals (Publication No. 85-23, revised 1985).

IC50 Determination on Hepatocytes:

After 22-24 hours, rat hepatocyte monolayer washed twice with Phosphate Buffer Saline (PBS). In order to -1 determine IC50, different concentrations were prepared for each of the samples (100 – 1000 µg mL ). After two hours of cells incubation with the extracts, cell viability was determined using MTT assay, which was performed according to the method of Mosmann, 1983 modified by Carmichael et al., 1986. Absorbance of formasan crystals produced by viable cells were read at 540 and 630 nm dual wavelength using automatic kinetic microplate reader (Labsystems Multiskan RC reader). Each experiment repeated three times and the mean absorption of each concentration was calculated. A graph plotted with x-axis showing different concentrations of extracts used, y-axis showing the absorbance percentage of viable cells. IC50 was graphically

2457 J. Appl. Sci. Res., 7(12): 2455-2461, 2011 determined from the concentration that yielded an absorption coinciding with 50% of cells that received no extract.

Evaluation of Hepatprotective Activity:

Monolayer primary cultures of rat hepatocytes performed in the 96-well plate, incubated for 22-24 hrs, and then washed twice with PBS. Different concentrations were prepared from the essential oils (12.5-100 µg mL-1) using serial dilutions. For each concentration, three replicates were carried out; the plate was then incubated for 2 hrs, and washed twice with PBS. The concentration of the extract that was able to protect the cells from the hepatotoxic effect of paracetamol considered hepatoprotective.

Results and Discussions

GC/MS Analysis of the Volatile Constituents of C.Articulatus and C.Esculentus Tubers:

Results of GC/MS analysis of the volatile constituents of C.articulatus and C.esculentus tubers are compiled in Tables (1 and 2) and figures (1,2,3,4,5 and 6).

Table 1: Molecular formula, molecular weight (MW), Chemical Abstract Registry Numbers (CAS#), GC retention time (tR), Kovats retention index (KI), Identification numbers (ID#) and Percentage composition of the essential oils from Cyperus species. Name Formula MW CAS # tR KI ID # C.articulatus C.esculentus 1,3,5-Trioxepane C4H8O3 104 6-6-5981 9.54 771 1 1.59 - 7- C H O 108 822-80-0 10.34 742 2 2.14 0.1 hydroxynorbornadiene 7 8

3-Furaldehyde C5H4O2 96 498-60-2 11.32 804 3 2.75 - 2-Ethylfuran C6H8O 96 3208-16-0 12.36 894 4 - 0.12 α-Thujene C10H16 136 5-2-2867 13.88 931 5 - 0.1 5-methyl-2- C H O 110 620-02-0 15.42 954 6 - 0.1 furancarboxaldehyde 6 6 2 Unknown - - - 15.73 - - 1.80 -

Eucalyptol C10H18O 154 470-82-6 20.49 1039 7 0.81 - trans-Pinocarveol C10H16O 152 547-64-5 22.46 1139 8 - 8.73 Myrtenol C10H16O 152 515-00-4 24.01 1194 9 - 6.3 cis-carveol C10H16O 152 1197-06-4 24.67 1242 10 - 5.55 α-Cubebene C15H24 204 17699-14-8 26.81 1351 11 0.65 - Neryl acetate C12H20O2 196 141-12-8 27.97 1365 12 0.7 - Unknown - - - 29.67 - - 1.59 2.38

β-patchoulene C15H24 204 508-55-4 30.43 1380 13 0.41 - Cyperene C15H24 204 2387-78-2 31.15 1398 14 2.4 - β-Caryophyllene C15H24 204 87-44-5 31.52 1404 15 1.12 1.51 Aromadendrene C15H24 204 109119-91-7 31.82 1439 16 0.76 - Rotundene C15H24 204 65128-08-7 32.05 1468 17 0.27 - γ-Muurolene C15H24 204 30021-74-0 32.26 1477 18 0.59 - Germacrene D C15H24 204 23986-74-5 32.72 1482 19 0.65 - β-selinene C15H24 204 17066-67-0 33.12 1485 20 0.44 - α-selinene C15H24 204 473-13-2 33.37 1494 21 1.88 2.09 α-muurolene C15H24 204 31983-22-9 33.68 1501 22 - 1.28 Amorphene C15H24 204 483-75-0 33.94 1506 23 15.64 13.87 γ-cadinene C15H24 204 39029-41-9 34.19 1513 24 1.53 1.51 cis-calamenene C15H22 202 483-77-2 34.4 1521 25 20.19 17.68 δ-cadinene C15H24 204 483-76-1 34.62 1524 26 1.27 - trans-calamenene C15H22 202 483-77-2 34.79 1532 27 4.35 5.03 Ledol C15H26O 222 577-27-5 34.97 1565 28 1.19 1.59 Globulol C15H26O 222 51371-47-2 35.08 1583 29 1.86 1.84 Dillapiol C12H14O4 222 484-31-1 35.38 1622 30 9.4 11.02 1-epi-cubenol C15H26O 222 19912-67-5 35.52 1627 31 2.03 4.00 Unknown - - - 35.86 - - 2.17 -

β-bisabolol C15H26O 222 15352-77-9 36.14 1671 32 2.2 7.61 Cedra-8-en-15-ol C15H24O 220 21441-72-5 36.39 1688 33 1.05 3.14 Germacrone C15H22O 218 6902-91-6 36.5 1693 34 1.24 - α-cyperone C15H22O 218 473-08-5 36.65 1727 35 0.53 2.42 Cyclocolorenone C15H22O 218 489-45-2 36.81 1745 36 1.15 1.03 Aristolone C15H22O 218 6831-17-0 37.02 1756 37 0.64 - Unknown - - - 37.19 - - 0.41 -

Ledene oxide C15H24O 222 JJJ45-N 39.25 1890 38 3.4 - Ethylpalmitate C18H36O2 284 628-97-7 40.54 1993 39 - 0.2 Ethyleicosanoate C22H44O2 340 18281-05-5 41.27 1994 40 8.95 0.55 Ethyl-(E)-9- C20H38O2 310 6114-18-7 41.51 2179 41 0.25 0.25

2458 J. Appl. Sci. Res., 7(12): 2455-2461, 2011

Octadecenoate Total 100 100

Table 2: Chemical class composition of the tested essential oil. Groups C.esculentus C.articulatus Monoterpene Hydrocarbon 0.1 - Oxygenated Monoterpene 20.58 0.81 Subtotal 20.68 0.81 Sesquiterpene Hydrocarbon 42.97 52.15 Oxygenated Sesquiterpene 21.63 15.29 Subtotal 64.6 67.44 Oxygenated Diterpene 0.25 0.25 Non terpenoids 12.09 25.53 Unknown 2.38 5.97 Total 100 100

Fig. 1: Chemical Structures of the nonterpenoids with their ID numbers as shown in table 1.

Fig. 2: Chemical Structures of the monoterpenes hydrocarbon with their ID numbers a shown in table 1.

Fig. 3: Chemical Structures of the oxygenated monoterpenes with their ID numbers as shown in table 1.

Fig. 4: Chemical Structures of the oxygenated Diterpene with their ID numbers as shown in table 1.

2459 J. Appl. Sci. Res., 7(12): 2455-2461, 2011

Fig. 5: Chemical Structures of the oxygenated sesquiterpene with their ID numbers as shown in table 1.

Fig. 6: Chemical Structures of the sesquiterpene hydrocarbon with their ID numbers as shown in table 1.

100% Viable cell control 50% Viable cell control Cyperus esculentus Cyperus articulatus

100

80

60

40

Viability PercentageCell 20

0 125 250 500 1000 Sample Concentration µg/ml

Fig. 7: Viability of Monolayer of Rat Hepatocytes after 2 hrs Treatment with Different Concentrations of the Extracts Using MTT Colourimetric Assay. Each Point Represents the Mean (n=3).

In Vitro Monolayer Hepatocyte Culture:

Hepatotoxicity:

IC50 on monolayer of rat hepatocytes was determined for essential oil extract of both C.esculentus and C.articulatus that showed no toxicity on hepatocytes until 1000 g mL-1 concentration (Figure 7).

2460 J. Appl. Sci. Res., 7(12): 2455-2461, 2011

Evaluation of Hepatoprotective Activity Applying Rat Hepatocyte Monolayer:

The essential oil fraction exhibited a hepatoprotective activity for C.esculentus and C.articulatus at 18.5 and 40 g mL-1 respectively (Figure 8).

100% Viable cell control Silymarin 50µg/ml 50% Viable cell control Paracetamol 20 mM Cyperus esculentus Cyperus articulatus

100

80

60

40

Percentage Viability Cell 20

0 12.5 25 50 100 Sample Concentration µg/ml

Fig. 8: Viability of Monolayer of Rat Hepatocyte after 2 hrs Treatment with Different Concentrations of the Extracts Followed by Treatment with 20 mM paracetamol for 1hr. in comparison with 50 µg Silymarin as Control Using MTT Colourimetric Assay. Each Point Represents the Mean (n=3).

Discussion:

GC/MS analysis of the essential oil of the Cyperus species revealed at least 32 terpenoides and 8 nonterpenoid volatile chemicals as shown in Table 1. The tested samples showed that sesquiterpenoids were the predominate constituents of both Cyperus tubers. Percentage composition of each group of the identified compounds is shown in Table 2 for each individual oil sample. Essential oil of both tubers that was obtained from C. articulates and C.esculentus was characterized by much larger amounts of sesquiterpenes (67.44% and 64.6% respectively) than monoterperenes (0.81% and 20.68% respectively). C. articulatus essential oil also was characterized by higher contents of none terpenoid compounds than C.esculentus (25.53 % and 12.09%) for tubers respectively. Essential oil of C. articulatus is dominated by 1,3,5-Trioxepane, 7- hydroxynorbornadiene, 3-Furaldehyde, Cyperene, β-Caryophyllene, α-selinene, Amorphene, γ-cadinene, cis-calamenene, δ-cadinene, trans-calamenene, Ledol, Globulol, Dillapiol, 1-epi-cubenol, β-bisabolol, Cedra-8-en-15-ol, Germacrone, Cyclocolorenone and Ethyleicosanoate. Essential oil of C. esculantus is dominated by trans-Pinocarveol, Myrtenol, cis-carveol, β- Caryophyllene, α-selinene, α-muurolene, Amorphene, γ-cadinene, cis-calamenene, trans-calamenene, Ledol, Globulol, Dillapiol, 1-epi-cubenol, β-bisabolol, Cedra-8-en-15-ol, α-cyperone and Cyclocolorenone. However, the chemical pattern of the two oils is somewhat different from those previously reported in the literature on the essential oil of Cyperus species from different countries, which further suggests the existence of more chemical diversity within the Cyperus species (Zoghbi et al., 2006). This could be due to climactic and environmental conditions, chemotypes, nutritional status of the plants, and other factors, which can influence essential oil composition (Loziene and Venskutonis, 2005). The in vitro study of the hepatotoxicity, indicates that the IC50 of the essential oil content of C.esculentus and C.articulatus tubers on monolayers of rat hepatocytes was >1000 µgmL-1 concentration and exhibited hepatoprotection at 18.5 and 40 µg mL-1 respectively.

Acknowledgment

This work is part of the project titled:"Development of a natural product for the treatment or/and control of hepatitis C virus (HCV) infection" between the national research centre and the industrial modernization centre (industrial modernization program) and the Academy of scientific research and technology, Egypt.

2461 J. Appl. Sci. Res., 7(12): 2455-2461, 2011

References

Ameen, A., W.I. El Eraky and N.A.Z. Yassin, 1999. Hepatoprotective and curative effects of Nigella sativs and Cyperus esculentus oily extracts in liver damage. J. Egypt. Soc. Pharmacol. Exp. Ther., 18(1): 33-41. Boulos, L., 2005. Flora of Egypt, 4: 371-390. Bum, E.N., C.L. Meier, S. Urwyler, Y. Wang and P.L. Herrling, 1996. Extracts from Rhizomes of Cyperus articulatus (Cyperaceae) Displace [3H]CGP39653 and [3H]Glycine binding from Cortical Membranes and Selectively Inhibit NMDA Receptor-Mediated Neurotransmission. J. Ethnopharmacol., 54: 103-111. Carmichael, J., W. DeGraff, A. Gazdar, J. Minna and J. Mitchell, 1987. Evaluation of tetrazolium-based semiautomated colourimetric assay: assessment of chemosensetivity testing. Cancer Res., 47: 936-942. Couchman, F.M., A.R. Pinder and N.H. Bromham, 1964. Studies on the essential oil of Cyperus articulatus L. Tetrahedron, 20: 2037-2045. Desmarchelier, C., E. Mongelli, J. Coussio and G. Ciccia, 1996. Studies on the Cytotoxicity, Antimicrobial and DNA-Binding Activities of Plants used by the Ese’ejas. J. Ethnopharmacol., 50: 91-96. Gupta, M.B., T.K. Palit, N. Singh and K.P. Bhargava, 1971. Pharmacological studies to isolate the active constituents from possessing anti-inflammatory, antipyretic and analgesic activities. Indian J. Med. Res., 59: 76-82. Jin, J.H., D.U. Lee, Y.S. Kim and H.P. Kim, 2011. Anti-allergic activity of sesquiterpenes from the rhizomes of Cyperus rotundus. Arch Pharm Res., 34(2): 223-8. Khater, H.F. and A.A. Shalaby, 2008. Potential of Biologically Active Plant Oils to Control Mosquito Larvae (Culex Pipiens, Diptera: Culicidae) From an Egyptian Locality. Rev Inst Med Trop Sao Paulo., 50(2): 107- 12. Kiso, Y., M. Tohkin and H. Hikino, 1983. Assay method for antihepatotoxic activity using galactosamine- induced cytotoxicity in primary-cultured hepatocytes. J. Nat. Prod., 46(6): 841-847. Liu, G.Q., Q.J. Wang, Z.Q. Xie, 1989. J. Chin. Pharm. Univ., 20: 48-51. Loziene, K. and P.R. Venskutonis, 2005. Influence of environmental and genetic factors on the stability of essential oil composition of Thymus pulegioides. Bio. Syst. Ecol., 33: 517-525. Mandal, V., Y. Yogesh Mohan and S. Hemalatha, 2007. Microwave Assisted Extraction – an Innovative and Promising Extraction Tool for Medicinal Plant Research. Pharmacognosy Reviews, 1(1): 7-18. Mehta, R.S., M.B. Shankar, M. Geetha and A.K. Saluja, 1999. Evaluation of Cyperus rotundus for hepatoprotection activity. Indian J. N. prod., 15(1): 13-17. Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxic assay. J. Imm. Meth., 65: 263-271. NIH Guide for the Care and Use of Laboratory Animals (1985). a) DHEW Publication Number (NIH) 78-23, Revised 1978 and b) NIH Publication Number. 85-23, Revised, U.S. Department of Health, Education and Welfare. Research Triangle Park, North Carolina. Pharmacopeia Commission of PRC, 2005. Pharmacopoeia of the People’s Republic of China, Vol. I, Chemical Industry Press, Beijing, p: 181. Plowman, T.C., A. Leuchtmann, C. Blaney and K. Clay, 1990. Significance of the Fungus Balansia cyperi Infecting Medicinal Species of Cyperus (Cyperaceae) from Amazonia. Econ. Bot., 44: 452-462. Satoh, A., T. Yokozawa, E.J. Cho, T. Okamoto and Y. Sei, 2004. Antioxidative effects related to the potential anti-aging properties of the Chinese prescription. Arch Gerontal Geriatr., 39(1): 69-82. Seglen, P.O., 1976. Preparation of isolated rat liver cells. Methods Cell Biol., 13: 29-83. Zoghbi, M., E. Andrade, J.Oliveira, L. Carreira and G. Guilhon, 2006.Yield and Chemical Composition of the Essential Oil of the Stems and Rhizomes of Cyperus articulatus L. Cultivated in the State of Para, . J. of Essential Oil Res., 18: 10-12.