Antigiardial Activity Of Citrullus Lanatus Var Citroides Biology Essay

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The present study was conducted to investigate the antigiardial activities of Citrullus lanatus var. citroides (wild watermelon) fruits petroleum ether, ethyl acetate, butanol crude extracts as well as Cucurbitacin E and Cucurbitacin L 2-O-β-glucoside pure isolated compounds from Citrullus lanatus var. citroides. Cucurbitacin E and Cucurbitacin L 2-O-β-glucoside were revealed strong potent antigiardial activity against Giardia lamlia in vitro with IC50= 2 and 5 ng/ml after 5 days respectively. The ethyleacetate extract was the best among all examined extracts followed by petroleum ether and butanol with IC50 0.1, 0.2 and 0.5 µg/ml respectively. The results suggest that all the crude extracts and isolated compounds were active against Giardia lamblia, hence C. lanatus var. citroides may be recommended as new source for the treatment of giardiasis.

Introduction

Wild melon (Citrullus lanatus var. citroides Cucurbitaceae) is low climbing, hairy and annual plants. In Sudan found commonly on sandy or clay soils in savannah zone of central Sudan, Darfur, Kordofan, Red Sea and northwards to Khartoum (Loiy, 2009). The fruit, eaten when fully ripe or even when almost putrid, is used as a febrifuge (Grieve, 1984). The fruit is also diuretic, being effective in the treatment of dropsy and renal stones (Chiej, 1984). The rind of the fruit is prescribed in cases of alcoholic poisoning and diabetes (Duke and Ayensu, 1985).

Cucurbitaceae plants are known to contain bioactive compounds such as cucurbitacin, triterpenes, sterols and alkaloids. Plants containing cucurbitacin were early recognized in folk medicine to have biological values. Scientific studies mainly refer to Middle East and Asia where cucurbit plants were used actively as herbal remedies. Cucurbit plants demonstrated anti-inflammatory, antitumor, liver protective and immunoregulatory activities (Ram, 1999 and Hu et al., 1982). Cucurbitacins are a group of highly oxygenated steroidal triterpenes characterized by the cucurbitane skeleton. Aglycons isolated from various species have been given the general name of cucurbitacin. Various letters in consecutive order of isolation follow this name, although in certain cases other names have been given (Lavie and Glotter, 1971).

Metronidazole sometimes causes adverse effects, e.g., myoplasia, neuralgia, and allergic dermatitis (Upcrof et al, 2006); hence new antigiardiasis drugs are probably required. With the purpose of searching for new antigiardiasis agents, Citrullus lanatus which was used traditionally for treatment of clinical signs associated with giardiasis and the plants was selected to evaluate the activity of their ethyl acetate, butanol and petroleum ether crude extracts against Giardia lamblia trophozoites in vitro.

Material and Method

Plant materials and Extraction:

The plant used in this study was Citrullus latanus var. citriode collected from AL- Musawarat, Northern Sudan, collected on February 2006. The taxonomic identification of this plant was carried out at Medicinal & Aromatic Plants Research Institute, National Center for Research by W.E.A/Alla. A voucher specimen was deposited at the herbarium of the institute. Fruits were cut into thin slices and dried at room temperature; seeds were separated and ground into a coarse powder. Dried fruit powdered extracted with chloroform, the filtrates were collected together and the residue was brought to dryness and extracted with ethanol and was modified to aqueous extract, which was extracted successively with equal volumes of two organic solvents of increasing polarity (ethyl acetate and butanol).

Isolation of compounds from C. lanatus var. citroides fruits pulps:

Vacuum liquid chromatography (VLC):

Vacuum liquid chromatography was performed on column (25 x 15 cm) packed with silica gel of particle size (0.04-0.06 mm) (60-120 mesh) and compacted with vacuum and pressing ten times. The crude extract of ethyl acetate (12 g) was subjected to (VLC). The concentrated sample was applied on the wall of the column.

The elusion used to fractionate ethyl acetate crude extract (12 g) was chloroform: methanol mixture of increasing polarity. Portions of 100 ml were collected, combined on the basis of TLC analysis using solvent system chloroform: methanol (9.5: 0.5, 9: 1 and 8: 2). Eight fractions were obtained, fraction five (5 g) was semi-pure, which was subjected to column chromatography, on a glass column (36 x 3.5 cm) packed with silica gel (115 g) of particle size (0.04-0.063 mm), and crystallized to give compound (1) (Eth.c60) (300 mg). Fraction six (3 g) also was semi-pure, it was purified throw small column using the same eluent to give compound (2) (EA VLC 42-46) (400 mg).

Analytical Techniques:

Infra red (IR) spectroscopy:

The IR spectra were recorded on Perkin-Eelmer model 1650 FTIR spectrophotometer using 50 mg KBr and 1mg of the isolated compounds. The following abbreviations were reported: S (strong), M (medium) and W (week).

Nuclear magnetic resonance (NMR) spectra:

1H NMR spectra:

These were recorded on NMR: Bruker Avance 400 spectrometer apparatus. Samples were dissolved in deuteriochloroform (CDCl3) or deuterioacetone (AD3OD) using tetramethylsilane, as an internal standard (0.00 ppm). Data were presented in following order; chemical shift relative to tetramethylsilane, multiplicity and intensity as to the number of protons; coupling constant J; assignment (if appropriate). The following abbreviations were adopted; s (single); d (double); t (triplet); m (multiplet); dd (double of doublets) signal.

¹³C NMR Spectra:

These were reported at Ac 150 MHz instrument. Tetramethylsilane was used an internal standard (0.00ppm). Chemical shits were reported relative to tetramethlysilane, assignments were based on the multiplicities and chemical shifts. Multiplicities were determined from polarization transfer technique (DEPT) or from direct response to C-H couplings (APT).

Mass spectrometry:

Electron Impact Mass Spectra (EI- MS) were recorded on Finnigan MAT 31 mass spectrometer with a MATSPECO Data System. Peak matching and field desorption (FD-MS) experiments were performed.

Melting point:

Melting point was measured using electrothermal melting point apparatus model No. 1A6304.

Parasite isolate

G. lamblia used in all experiments were taken from patient Ibrahim Malik Hospital (Khartoum). All positive samples were examined by wet mount preparation. Then the positive sample was transported to MAPRI in nutrient broth medium. Trophozoites of G lamblia were maintained in RPMI 1640 medium containing 5% bovine serum at 37  1C. The trophozoites were maintained for the assays and were employed in the log phase of growth.

In vitro susceptibility assays

In vitro susceptibility assays used the sub- culture method of Cedilla et al., (2002). This is highly stringent and sensitive method for assessing the anti-protozoal effects (gold standard) particularly in Entamoba histolytica, Gairdia intestinalis and T. vaginalis (Arguello et al., 2004).

5 mg from each extract and compound was dissolved in 50 µl of dimethyl sulfoxzide (DMSO) at eppendorf tube containing 950 µl D.W in order to reach concentration of 5 mg/ml (5000ppm). The concentrates were stored at -20 °C for further analysis.

Sterile 96-well microtite plate was used for different plant extracts, positive control and negative control.

Twenty µl of complete RPMI medium were placed in the wells-except the first three wells C-1 (which 40 µl of an extract solution 5 mg/ml were added in the first three wells and the final concentrations were 1000 µg/ml). 20 µl of complete RPMI medium were placed in the wells in the following C-2 was 500µg/ml and C-3 which was 250 µg/ml. 80 µl of culture medium was complemented with parasite and added to all wells. The final volume in the wells was 100 µl.

Each test included metronidazole pure compound [(1-(2-hydroxyethl)-2-methyl-5 nitroimidazole], a trichomonocide was used as positive control in concentration 312.5 gml, whereas untreated cells used as a negative controls (culture medium plus trophozoites). Samples were taken for counting at 0, 24, 48, 72 and 96.

For counting the samples were mixed with Trypan blue in equal volume. The final number of parasites was determined with haemocytometer in triplicate.

The mortality % of parasite for each extracts activity was carried out according to the following formula:

Mortality of parasite (%) = (Control negative - tested sample withextract) Ã- 100%

Control negative

Only 100% inhibition of the parasite considered, when there was no motile parasite observed.

Statistical analysis

All data were presented as means ± S.D. Statistical analysis for all the assays results were done using Microsoft Excel program. Student t.test was used to determine significant difference between control and plant extracts at level of P < 0.05.

Results

Characterization of compound (1):

Compound (1) was obtained as an amorphous brown powder form Ethyl acetate extract. It has Rf value 0.47 in solvent system (Ch: MeOH (85:15)). It developed brown colour with vanillin reagent. M. P. 1002.66. IR spectrum showed absorption at: 3434 cm‾¹ (OH) b, 2929 cmˉ¹ (C-H), 1686 cmˉ¹ (C=O) v. s, 1637 (C=C) m. s, 1076 (C-O) m. s. (Table 1). The 1HNMR Spectrum of compound (1) displayed tow singlet each of three protons integrations at δ 1.48 ppm two at1.31 ppm, two at1.04ppm and one at 1.37 ppm indicated the presence of 7 methyl groups (Table 2)

1H-NMR (400 MHZ CDCD3), 1.04 s (3H), 1.87 d (1H) J= 8Hz. 2.05 (2H) J= 16 Hz, 5.75 s (1H), 4.08 d (1H) J= 12Hz, 2.6 s (3H). 1.85 d (2H) J= 8Hz, 2.18 d (1H) J=20 Hz, 1.3 s (3H), 1.87 d (2H) J= 8Hz, 1.6 d (2H) J=28 Hz, 3.23 m (1H) 3.57 s (1H), 1.90 s (1H), 1.37 s (3H), 3. 6 s (3H), 7.02 s(3H), 1.43 s (3H), 2.25 s (3H). ¹H- and ¹³C NMR was presented in (Table 2). El-MS m/z: 558.32 which was confirmed by HREI-MS to give a molecular formula C32H44O8. The NMR data of this compound were identical to those of Cucurbitacin E as previously reported; 1H NMR (Lavie et al., 1962) and13C NMR (Valde and Lavie 1983). Consequently, the structure of compound (1) was identified as cucurbitacin E. (Figure 1)

Cucurbitacins are obtained originally from Cucurbitaceae and are cytotoxic triterpenoid substances. Series of cucurbitacin cognates were identified and their pharmacological effects, such as anti-tumor, purgative, anti-inflammatory, and antifertility activities have also been reported (Chen et al. 2005). It has been reported that cucurbitacin E possesses anti-tumor activity and caused alterations in cell morphology by disrupting actin cytoskeleton (Duncan et al., 1996).

Table 1: IR spectral data of compound (1)

*Wave length (cm‾¹)

Assignments

Wave length of (34-35.60) peaks(cm‾¹)

480, 440

580, 550

610

820, 790

910, 870

970, 940

1045, 1015

1140, 1120, 1090

1180

1300

1260, 1240

1300

1360

1440

1660, 1580

2900

3350

C-C-C deform

C-C=O in-plane bend

C-OH out-of-plane bend

CH2 rock

C-H out-of-plane bend

Ring stretching

C-H out-of-plane bend

C-C-C in-plane bend

C-H in-plane bend

CH2 wag

C-H in-plane bend

O-H deform

CH 3 symmetric bend

CH 3 asymmetric bend

C=O stretching mode

C-H stretch

O-H stretch

-

-

614.26

-

-

-

1076,1028

-

-

-

1263.53

-

1371.89

1454.78

1637.40

2929.17

3434.82

*Nakanishi, K., P. H. Solomon (1977). Infrared Absorption Spectroscopy, Holden-Day, San Francisco.

Table 2: ¹³C and ¹H-NMR spectral data for compound (1)

Serial number

Carbon atom No.

Type

δc

ppm

δH

ppm

1

30

CH3

18.6

1.04

2

18

CH3

19.2

1.04

3

32

CH3

21.4

2.35

4

21

CH3

25.3

1.37

5

28

CH3

27.1

1.31

6

29

CH3

27.1

1.31

7

26

CH3

27.5

1.48

8

27

CH3

27.5

2.48

9

7

CH2

24.4

2.05,1.96

10

1

CH2

35.3

1.85,1.60

11

10

CH2

48.3

2.18

12

9

C

48.4

-

13

13

C

50.0

-

14

14

C

50.4

-

15

4

C

48.6

-

16

17

CH

59.6

1.9

17

16

CH2

71.0

3.23

18

2

CH

71.3

4.08

19

25

C

80.3

-

20

20

C

81.5

-

21

12

CH2

48.3

1.86,1.60

22

24

CH

121.6

7.02

23

25

C

80.3

-

24

6

CH

122.8

5.75

25

5

C

140.5

-

26

31

C

170.2

-

27

11

C

213.2

-

28

3

C

213.4

-

29

23

C

204.5

5.68

30

22

CH

155.4

7.01

31

16

C

200.6

-

32

20

C

204.5

-

Figure 1: Cucurbitacin E compound (1)

Characterization of compound (2):

Compound (2) (VLC42-46) was obtained as an amorphous powder from ethyl acetate extract of the fruit pulp. It has the following characteristics:

Rf value 0.47 in solvent system (CHCl3 : MeOH (85:15)), developed purple colour with vanillin reagents. It dissolved in acetone and MeoH.

IR spectrum showed absorption at: 3402 cm­¹ (OH), 2901 cm­¹(C-H), 1638.34 cm­¹ (C=C) (Table 3).

The 1H and 13C NMR spectra showed the presence of one sugar moiety, it was identified as a β-glucopyranosyl a terminal unit, with compound (2). Enzymatic hydrolysis of compound (2) with γ-amylase gave cucurbtacin L, confirming that the β-glucopyranosyl unit is a sugar connected to a ß-glucopyranosyl unit at C-2. Furthermore, the 13C NMR spectrum also revealed the attachment of a terminal sugar to C-2 of a β-glucopyranosyl unit due to the downfield shift of this atom (+9.4 ppm) and upfield shift of C-30 (_0.9 ppm) (table 4). El-MS m/z: 676.35. which was confirmed by HREI-MS to give a molecular formula of C36H52O12. Consequently, the structure of compound (2) was identified as cucurbitacin L 2-O-β-glucopyranosyl. (Figure 2)

Cucurbitacin L 2-O-β-glucopyranosyl previously was isolated with two other nor-cucurbitacin glycosides from the root of specimen of Wilbrandia sp(Cucurbitaceae) (Maria et al., 1993). From the fruits of Trichosanthes tricuspidata (Cucurbitacaeae), 14 cucurbitane glycosides were isolated along with cucurbitacin 2-O-b-glucopyranoside. Structural elucidations were based on chemical and spectroscopic analyses. (Tripetch et al., 2002).

Table 3 : IR spectral data of compound (2)

Wave length (cm‾¹)*

Assignments

Wave length of (VLC42-46) peaks(cm‾¹)

480, 440

580, 550

610

820, 790

910, 870

970, 940

1045, 1015

1140, 1120, 1090

1180

1300

1260, 1240

1300

1360

1440

1660, 1580

2900

3350

C-C-C deform

C-C=O in-plane bend

C-OH out-of-plane bend

CH2 rock

C-H out-of-plane bend

Ring stretching

C-H out-of-plane bend

C-C-C in-plane bend

C-H in-plane bend

CH2 wag

C-H in-plane bend

O-H deform

CH 3 symmetric bend

CH 3 asymmetric bend

C=O stretching mode

C-H stretch

O-H stretch

-

-

613.64

-

-

939.44

1040,1018

1076.61

1158.8

-

1263.86

-

1372.13

1460.29

1637.63

2976.73

3429.47

*Nakanishi, K., P. H. Solomon (1977). Infrared Absorption Spectroscopy, Holden-Day, San Francisco.

Table 4: ¹³C and ¹H-NMR spectral data for compound (5) (VLC42-46)

Serial number

Carbon atom No.

Type

δc

ppm

δH

ppm

1

25

CH3

17.6

1.30

2

18

CH3

19.2

1.04

3

30

CH3

25.3

1.36

4

31

CH3

25.3

1.36

5

26

CH3

25.3

1.38

6

23

CH3

29.6

1.28

7

32

CH3

29.6

1.28

8

13

CH

35.0

2.85

9

3

CH

41.6

1.79

10

9

CH2

46.3

1.71

11

6

CH2

48.4

2.27

12

4

C

48.8

-

13

1

C

50.0

-

14

2

CH2

50.5

1.04

15

17

C

51.2

-

16

7

CH

59.6

1.86

17

38

CH2

62.2

3.79

18

8

CH2

71.0

3.53

19

29

C

71.9

-

20

34

CH

75.1

3.71

21

36

CH

75.1

3.40

22

35

CH

76.9

3.49

23

37

CH

81.6

3.76

24

33

CH

109.3

5.68

25

21

CH

119.1

6.29

26

11

CH

120.7

5.51

27

14

CH

124.4

5.51

28

12

C

136.7

-

29

15

C

147.3

-

30

22

CH

155.4

7.01

31

16

C

200.6

-

32

20

C

204.5

-

33

5

C

213.1

-

Figure 2: compound (2) Cucurbitacin L 2-o-β-glucoside

The activity of petroleum ether extract of Citrullus lanatus gave mortality 90% after 120 hours in concentration 500 ppm, while in metrondizole shown 100% mortality at the same time Figure (3).

In contrast, no growth was observed after 96 hours in incubated at 500 ppm concentrations of Citrullus lanatus in ethyl acetate extract. However, at other concentrations of 250 ppm and 125 ppm, the cells were viable even after 120 h of incubation Figure (4).

Figure (5), shows the activity of butanol extract of Citrullus lanatus. The high dose of extract shows 90% mortality after 120 hours.

In Figure (6) shows the activity of Cucurbitacin-E extract of Citrullus lanatus. The high dose of extract gave 100% mortality after 96 hours, this was not the same result appeared with metrondizole.

As shown in Figure (7), the activity of Cucurbitacin-L extracts of Citrullus lanatus. All the cells were alive at all concentration after 120 hours, while in control positive gave 100 % mortality at the same time.

Discussion

Giardia lamblia is one of the most common intestinal pathogenic protozoan parasites (Newman et al. 2001). It is becoming increasingly important among HIV/AIDS patients. There are reports that some cases of acute and chronic diarrhea in AIDS patients may be associated with giardial infection (Merchant and Shroff 1996). However, Metronidazole, the common drug of choice, can cause mutagenicity in bacteria (Legator et al. 1975) and is carcinogenic in rodents (Rustia and Shubik 1972). It also possesses undesirable side effects and treatment failures have been reported (Llibre et al. 1989).

Our present in vitro investigation reveals promosing results for the use of the plant for cultivated parasites to formulate such ingredient of the plant extract as the drug. However, the IC50 of petroleum-ether, ethylacetate and butanol extracts are 0.18, 0.15 and 0.45 ppm and their IC90 were found to be 432, 122 and 406 ppm respectively. While the IC50 and IC90 of Cucurbitacin-E and Cucurbitacin L 2-o-β-glucoside are 0.002, 0.005 ppm and 15, 32 ppm respectively. The present study highlights the efficiency of C. lanatus extracts and isolated compounds acquisition alternative natural and chemical treatment for giardiasis.

In conclusion this result enhances the ethnobotanical uses of the plant as antidiarrheal in cases associated with giardiasis in central Sudan. Further investigations regarding the mode of action and other related pharmacological studies such as in vivo investigation, drug formulation and clinical trials are highly recommended.

Figure (3): In vitro activity of Citrullus lanatus petroleum ether extract against G. lamblia

Figure (4): In vitro activity of Citrullus lanatus ethyl acetate extract against G. lamblia

Figure (5): In vitro activity of Citrullus lanatus butanol extract against G. lamblia

Figure (6): In vitro activity of Citrullus lanatus Cucurbitacin-E against G. lamblia

Figure (7): In vitro activity of Citrullus lanatus Cucurbitacin L 2-o-β-glucoside against G. lamblia

Table (5) IC50 and IC90 of Citrullus lanatus extracts and isolated compounds against G. lamblia after 120 hours.

Sample tested

IC50/ppm

IC90/ppm

Citrullus lanatus Petrolium ether extract

0.182327

431.94

Citrullus lanatus ethylacetate extract

0.145179

122.25

Citrullus lanatus butanol extract

0.480735

406.65

Cucurbitacin-E

0.002381

15.06

Cucurbitacin L 2-o-β-glucoside

0.004910

32.43

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