CHAPTER VI ISOLATION AND PHYTOCHEMICAL CHARACTERIZATION OF BIOACTIVE COMPOUNDS FROM THE RHIZOMES OF Cyperus rotundus. L

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1 CHAPTER VI ISOLATION AND PHYTOCHEMICAL CHARACTERIZATION OF BIOACTIVE COMPOUNDS FROM THE RHIZOMES OF Cyperus rotundus. L 6.1 INTRODUCTION Cyperus rotundus Linn., (Family -Cyperaceae),is a medicinal plant, also known as purple nut edge or nut grass, is a common perennial weed with slender, scaly creeping rhizomes, bulbous at the base and arising 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. Cyperus rotundus is indigenous to India, but are now found in tropical, subtropical and temperate regions (Pooley et al, 1998 and Gordan gray, 1995). The genus Cyperus includes common weeds found in upland and paddy fields in temperature to tropical regions. C. rotundus, which are used as traditional folk medicines for treatment of stomach and inflammatory diseases (Gupta et al 1971; Singh et al., 1970). The Cyperus rotundus L have been reported to contain oils, alkaloids, glycosides, saponins, flavonoids, tannins. The rhizomes of C. rotundus have been used in ancient medicine in India for fever, dysentery, purities, pain, vomiting and various blood disorders (Kirtikar et al., 1944). In particular, plant extracts offer a rich potential source of novel anti platelet agents (Ballabeni et al., 2007). 93

2 C. rotundus has been reported to contain sesquiterpenes, hydrocarbons, epoxides and ketones and also used as anti-inflammatory estrogenic, antipyretic, antiemetic, diuretic, hypotensive agent (Aslam, 2002). There are some reports on the phytochemical analysis of species belonging to Cyperus rotundus found in the literature. These scientific studies on the species of this genus showed the presence of constituents belonging mainly to the groups of sesquiterpenes, flavonoids, tannins sterols, alkaloids, benzoquinones and essential oils (Zargari, 1990 and Lawal and Oyedeji, 2009). A novel norsesquiterpene, named norcyperone, and three known compounds:(-)- clovane-2,9-diol, rosenonolactone, and 5α,8α-epidioxy-(20S,22E,24R)-ergosta-6,22- dien-3β-ol were isolated from the rhizomes of Cyperus rotundus L.(Yan Xu et al.,2008).the rhizome oils of this plant from different countries also showed compositional differences, suggesting the existence of phytochemical varieties. Cyperene ( %) and α-cyperone ( %) were the most abundant constituents of the oils of Nigerian and Tunisian species, but the concentrations of other main components varied (Ekundayo et al., 1991 and Kilani et al., 2005). Root extracts of C. rotundus (CR) showed the presence of β-sitosterol, cyperene, cyperol, flavonoids, sesquiterpenoids, ascorbic acid and polyphenols (Sonwa and Konig, 2001). In this present chapter, the phytochemical evaluations for the various eluted fractions were carried out. 6.2 PLANT MATERIAL The rhizomes of the Cyperus rotundus Linn. Were harvested from outfield near Nagercoil, Kanyakumari District, Tamil Nadu, South India in the month of October. Botanical identification was carried out by the Head of the Department 94

3 of Botany, Sri Paramakalyani College, Alwarkurichi (Ref SPKCZ/17), South India. Fresh plant roots were rinsed severally with clean tap water to make it dust and debris free. Then the roots were dried in the shady condition at the room temperature for 15 days until they become crispy while still retaining the brownish coloration. Dried roots were ground in electric chopper and made into coarse powder, stored in an air tight container in a refrigerator prior to subsequent analysis. 6.3 MATERIALS AND METHODS Preparation of the plant extract Ethyl acetate and methanol extracts were obtained by using soxhlet apparatus. The two extracts, with different polarities were concentrated by evaporating it to dryness under reduced pressure by rotary vaccum evaporator (New Lab, Company) to obtain the respective extracts and each residue was stored at 4 0 C. These two extracts were resuspended in Dimethyl sulfoxide. The extracts were then stored at c until further analysis Column chromatography Methanol extract The column was packed with silica gel G for column chromatography (60 120) and built the column with n-hexane mesh. The slurry of methanol root extract (3g) was introduced in to the column. The 50ml of fractions were collected by eluting hexane: ethyl acetate (H: EA) and also followed by ethyl acetate: methanol (EA: M). The order of elution is H:EA(70: 30),H:EA (60: 40), EA100%, EA:M(98:2),EA : M (95:5),EA : M (90 : 10),EA: M (80:20) EA : M (60 ; 40), EA : M (40 : 60), EA : M (20 : 80), 100% Methanol. The 95

4 collected fractions were then concentrated using rotary vacuum evaporator under reduced pressure was used for further pharmacological and spectral analysis Column chromatography Ethyl acetate extract The column was packed with silica gel G (60-120mesh) for column chromatography and built the column with dichloromethane. The slurry of ethyl acetate root extract (6g) was introduced into the column. The 50ml of fractions were collected by eluting dichloromethane: methanol (DCM: M). The order of elution is DCM : M (80 : 20),DCM : M (70 : 30),DCM : M (60 : 40),DCM : M (50: 50),DCM : M (40 : 60),DCM : M (30 : 70),DCM : M (20 : 80), DCM : M (10 : 90), 100% Methanol. Finally the collected fractions were evaporated and concentrated using rotary vacuum evaporator under reduced pressure and were used for further pharmacological and spectral analysis. 6.4 PHYTOCHEMICAL SCREENING Phytochemical screening of eluted fractions was tested for the presence of various phytochemical constituents.the analyses were carried out by following techniques Preliminary screening The various fractions of methanolic and ethyl acetate root extracts of Cyperus rotundus L.were used to screen for the following the phytochemicals like Triterpenoids, reducing sugars, alkaloids, phenolic compounds, Saponin, xanthoprotein, tannins, aromatic acids, flavonoids, phytosterols small quantities of all the fractions were dissolved separated in distilled water and filtered. The filtrate was subjected to further analysis. 96

5 6.4.2 Detection of Triterpenoids To the filtrate with one or two pieces of tin and three drops of thionyl chloride was added slowly, a violet or purple colour solution indicates the presence of triterpenoids Detection of Reducing sugars a. Fehling s Test: Small portion of the various filtrates were treated with Fehling s solutions I and II the heated on a water bathe. A brick red precipitate indicates presence of reducing sugars. b. Benedict s Test: Small portion of the various filtrates were treated with equal quantities of Benedicts reagent. An yellow precipitate indicates the presence of reducing sugar. c. Detection of Alkaloids Small fraction of various filtrates were separately stirred with few ml and dilute hydrochloric acid and filtered. The filtrate was tested with various alkaloid reagents such as Mayer s, Hager S, Wagner s and Dragendorff s reagent. d. Mayer s Test To small quantity of the various filtrates Mayer s reagents was added the formation of cream coloured precipitate indicates the presence of alkaloids. e. Hager s Test To small quantity of the various filtrates, Hager s reagent was added. Formation of yellow coloured precipitate indicates the presence of alkaloids. 97

6 f. Wagner s Test To small quantity of the various filtrates, Wagner s reagent was added. Formation of reddish brown coloured precipitate indicates the presence of alkaloids. g. Dragendroff s Test: To small quantity of the various filtrates, Dragendroff s reagent was added Formation of reddish brown coloured precipitate indicates the presence of alkaloids DETECTION OF PHENOLIC COMPOUNDS a. Ferric chloride Test: To the filtrate add few drops of neutral ferric chloride was added and the appearance of an intense blue or violet colour were recorded. This indicated the presence of phenolic compounds. b. Detection of saponins Small portion of the various filtrates were diluted with 20 ml of distilled water and it was agitated in a graduated cylinder for 15 minutes. Formation of foamy layer was record, it indicated the presence of saponin. c. Detection of Xanthoproteins To the filtrates add few drops of concentrated nitric acid was added with excess amount of ammonia.no red orange precipitate indicates the absence of xantho proteins in all fractions. d. Detection of Tannins To the filtrate, 2ml of solution of gelatin was added white precipitate was seen which indicates the presence of tannins. 98

7 e. Detection of Aromatic acids To small quantity of the various filtrates add saturated sodium bicarbonate, no brisk effervescence indicates the absence of Aromatic acids. f. Detection of Flavanoids Small portion of the various filtrates were dissolved in alcohol and treated with magnesium metal followed by concentrated hydrochloric acid. Formation of Magenta colour indicates the presence of flavonoids. g. Detection of phytosterols Small quantity of various filtrates were dissolved in 5ml of chloroform solutions were subjected to salkowski s and Liebermann-Burchard s test for the detection of phytosterols. h. Salkowski s Test To the 1ml of above prepared chloroform solution, few drops of concentrated sulfuric acid was added. It gave red colour which indicates the presence of phytosterols. i. Liebermann-Burchard s Test The above prepared chloroform solution was treated with few drops of concentrated sulfuric acid followed by 1ml of acetic anhydride solution. It gave green colour, which shows the presence of phytosterol Determination of total phenolics and tannins The total phenolic content was determined according to the method described by Siddhuraju and Becker (2003). Ten microlitre aliquots of the extracts (10mg/2ml) were taken in test tubes and made up to the volume of 1 ml with distilled water. Then 0.5 ml of Folin-Ciocalteu phenol reagent (1:1 with water) and 2.5 ml of sodium carbonate solution (20%) were added sequentially in each tube. Soon after vortexing the reaction 99

8 mixture, the test tubes were placed in dark for 40 min and the absorbance was recorded at 725 nm against the reagent blank. The analysis was performed in triplicate and the results were expressed as gallic acid equivalents. Using the same extracts the tannins were estimated after treatment with polyvinyl polypyrrolidone (PVPP) (Siddhuraju and Manian, 2007). One hundred milligrams of PVPP was weighed into a mm test tube and to this 1 ml distilled water and then 1 ml of the sample extracts were added. The content was vortexed and kept in the test tube at 4 C for 4h. Then the sample was centrifuged (3000 rpm for 10 min at room temperature) and the supernatant was collected. This supernatant has only simple phenolics other than tannins (the tannins would have been precipitated along with the PVPP). The phenolic content of the supernatant was measured as mentioned above and expressed as the content of non-tannin phenolics (tannic acid equivalents) on a dry matter basis. From the above results, the tannin content of the sample was calculated as follows: Tannin (%) = Total phenolics (%) Non-tannin phenolics (%) Determination of total flavanoid content The flavonoid content was determined by the use of a slightly modified colorimetry methoddescribed previously by Zhishen et al. (1999). A 0.5ml aliquot of appropriately (10mg/2ml) diluted sample solution was mixed with 2ml of distilled water and subsequently with 0.15ml of 5% NaNO2 solution. After 6 min, 0.15 ml of 10% AlCl 3 solution was added and allowed to stand for 6 min, and then 2ml of 4% NaOH solution was added to the mixture. Immediately, water was added to bring the final volume to 5ml, and then the mixture was thoroughly mixed and allowed to stand for another 15min. Absorbance of the mixture was determined at 510 nm versus water blank. 100

9 The analysis was performed in triplicate and the results were expressed as rutin equivalent Phytochemical Separation by using TLC Method Each fractions of the column eluted sample was subjected to TLC to find out the separation of single compound and confirmation from the fraction. Thin Layer Chromatography was performed on prepared plates with Silica gel F254 grade (Merck, Darmstadt, Germany) as stationary phase. A one-dimensional ascending development technique was used to detect the constituents of an extract on TLC plate. Visual detection was done in daylight and under UV light at a wave length of 254 and 344 nm depending on the nature of compounds separated UV-Spectrophotometry analysis The absorbance spectra of the extracted samples were measured using UV visible spectrophotometer 2203 at wavelength range from nm. The detector used was diode array detector with deionized water GC - MS Analysis The Clarus 500 GC-MS used in the analysis employed a fused silica column packed with Elite-1 (100% dimethyl poly siloxane, 30nm X 0.25nm ID X 1 m df) and the components were separated using Helium as carrier gas at a constant flow rate of 1 ml/min. The 2 sample extract injected into the instrument was detected by the Turbo gold mass detector (Perkin Elmer) with the aid of the Turbo mass 5.1 software. During the 36 th minute GC-MS extraction process, the oven was maintained at a temperature of C with 2 minutes holding. The injector temperature was set at C (mass analyzer). 101

10 The different parameters involved in the operation of the clarus 500 GC-MS were also standardized (Inlet temperature: C; Source temperature: C). Mass spectra were taken at 70 ev; a scan interval of 0.5 s and fragments from 45 to 450 Da. The MS detection was completed in 36 minutes. The detection employed the NIST Ver year 2012 library (Fig ) 6.5 RESULTS AND DISCUSSION Phytochemical screening of various fractions of methanol extract of Cyprus rotundus rhizomes reveals the presence of aromatic acids, triterpenoids, reducing sugars, alkaloids, phenolic compounds, saponins, tannins, flavonoids, phytosterols and absence of xanthoproteins. 102

11 Table 6.1 Phytochemical screening of Methanol extract fractions of Cyperus rotundus L. rhizomes S. NO Phytoconstituents H:EA 70:30 H:EA 60:40 Methanol extract fractions of Cyperus rotundus L. rhizomes Hexane(H) :Ethyl acetate (EA) ; Ethyl acetate (EA) : Methanol (M) EA 100% EA: M 98:02 EA: M 95:5 EA: M 90:10 EA: M 80:20 EA: M 60:40 EA: M 40:60 EA: M 20:80 1 Triterpenoids M 100% 2 Reducing sugar Alkaloids Phenolic compounds Saponins Xanthoprotein Tannins Aromatic acid Flavonoids Phytosterols (+) Presence (-) Absence 103

12 Table 6.2 Phytochemical screening of ethyl acetate extract fractions of Cyperus rotundus L. rhizomes. Ethyl acetate extract fractions of Cyperus rotundus L. rhizomes S.NO Phyto constituents DCM:M 80:20 DCM:M 70:30 DCM:M 60:40 Dichloro Methane (DCM) : Methanol (M) DCM:M 50:50 DCM:M 40:60 DCM:M 30:70 DCM:M 20:80 DCM:M 10:90 M 100% 1 Triterpenoids Reducing sugar Alkaloids Phenolic compounds Saponins Xanthoprotein Tannins Aromatic acid Flavanoids Phytosterols (+)Presence (-) Absence 104

13 Phytochemical screening of various fractions of methanol extract of Cyprus rotundus rhizomes reveals the presence of Triterpenoids, reducing sugars, alkaloids, phenolic compounds, Saponins, tannins, flavanoids, phytosterols and absence of xanthoprotein and aromatic acids. The total phenolics in methanol extract, ethyl acetate extract and fraction DCM: Methanol (30:70) of ethyl acetate extracts are 57.46±0.84, ± 0.51 and ± 0.88 respectively. The total flavanoid in methanol extract, ethyl acetate extract and fraction DCM: Methanol (30:70) of ethyl acetate extracts are 1.10 ± 0.07, 0.29 ± and 22 ± 0.05 respectively. The total tannin in methanol extract, ethyl acetate extract and fraction DCM: Methanol (30:70) of ethyl acetate extracts are ± 0.83, ± 0.19 and 5.32 ± 1.00 respectively. 105

14 Table 6.3 Estimation of Total phenolics, Total Flavanoids and total Tannins present in the Methanolic extract, Ethyl acetate extract and Ethyl acetate extract fraction DCM: S.No Sample Methanol (30:70) of Cyperus rotundus L. rhizomes. Total Phenolics mg TAE/g extract Total Flavanoid Mg RE/g extract Total Tannin mg TAE/g extract 1. Methanol crude ± ± ± Ethyl acetate crude Fraction DCM:M (30:70) ± ± ± ± ± ± 1.00 Values are means of three independent analyses of the extract + standard deviation (n=3). TAE Tannic acid equivalent RE Rutin equivalent 106

15 107

16 UV-SPECTRAL ANALYSIS Fig-6.1 UV- spectrum of the test fraction EA: Methanol (80:20) of Ethyl acetate extract gives prominent peak of 241.6, 208.0, 248.0, 257.6, with ,3.064, 3.020, 3.013, 0.053absorbence which supports the bioactive compound in the root extract. EA: M (80:20) WAVELENGTH ABS O WAVELENGTH ABS

17 UV spectrum of the test fraction DCM: Methanol (30:70) of ethyl acetate extract gives prominent peak of with absorbance which supports the bioactive compound in the root extract. Fig 6.2 DCM: M (30:70) WAVELENGTH ABS

18 Fig 6.3 In GC analysis of the test fraction DCM: Methanol (30:70) of ethyl acetate extract of C.rotundus 110

19 111

20 112

21 Fig 6.4 In GC analysis of the test fraction ethyl acetate: Methanol (80:20) of methanol extract of C.rotundus 113

22 114

23 115

24 116

25 In GC analysis of the test fraction EA: Methanol (80:20) of methanol extract of C.rotundus gives prominent peak. The most abundant peaks with retention time 8.98, 16.59, 19.13, 24.00, and were observed along with other peaks with smaller abundant out of the six more abundant peaks, the peak at and show the response for aromatic carboxylic acid and esters. Others are showing the response for aliphatic esters, acids and amides. In GC analysis of the test fraction DCM: Methanol (30:70) of Ethyl acetate extract of C.rotundus gives prominent peak. The most abundant peaks with retention time 12.89, 23.50, 23.90, and were observed along with other with little abundant. Out of five peaks the most abundant peaks with RT 23.5 and were quite interesting. The peak at is due to Bicyclo (4.1.0)heptanes, a terpene compound It belongs to bicyclic monoterpenoid class I (6+3 membered ring). It belongs to carane group. Car-3-ene car-2-ene 117

26 The car-3- ene and car-2-ene are found to be present in natural source is essential oils. Finar, (1973). p-cymene, p-cymenene has been reported earlier in Cyperus rotuntus (El-Gohary 2004). The carenes are obtained from m-cymene skeleton is p-cymene m-cymene Sylvesterene are also reported in natural source as essential oil. The most probable compound belonging to the peak at is justified by the above evidences. The DCM:Methanol (30:70) fraction of ethyl acetate extract of Cyperus rotuntus rhizomes is found to contain 3,3,3, trifluoro N-(-4-fluorophenyl)- bicyclo (4.1.0)heptane 118

27 This fraction shows excellent antimicrobial activities and antioxidant activities. Further phytochemical studies may lead to the exact bioactive compound principle. This most probable compound seems to be the first report in the rhizome extracts of Cyperus rotuntus Recent literature survey in this plant indicates the bioactive potential of this plant Cyperus rotundus L. The plant root extract is found to contain various phytochemicals. The selected phytochemicals such as phenolic compound, flavanoid and tannin, have been estimated. The GC-MS pattern of ethyl acetate extract fraction dichloromethane: methanol (30:70) indicates the presence of 3,3,3, trifluoro N-(-4-fluorophenyl)- bicyclo (4.1.0) heptane. 119