Antigiardial Activity Of Citrullus Lanatus Var Citroides Biology Essay

The present survey was conducted to look into the antigiardial activities of Citrullus lanatus volt-ampere. citroides ( wild Citrullus vulgaris ) fruits crude oil quintessence, ethyl ethanoate, butanol petroleum infusions every bit good as Cucurbitacin E and Cucurbitacin L 2-O-I?-glucoside pure isolated compounds from Citrullus lanatus volt-ampere. citroides. Cucurbitacin E and Cucurbitacin L 2-O-I?-glucoside were revealed strong powerful antigiardial activity against Giardia lamlia in vitro with IC50= 2 and 5 ng/ml after 5 yearss severally. The ethyleacetate infusion was the best among all examined infusions followed by crude oil quintessence and butyl alcohol with IC50 0.1, 0.2 and 0.5 Aµg/ml severally. The consequences suggest that all the petroleum infusions and stray compounds were active against Giardia lamblia, therefore C. lanatus volt-ampere. citroides may be recommended as new beginning for the intervention of giardiasis.

Introduction

Wild melon ( Citrullus lanatus volt-ampere. citroides Cucurbitaceae ) is low mounting, hairy and one-year workss. In Sudan found normally on sandy or clay dirts in savannah zone of cardinal Sudan, Darfur, Kordofan, Red Sea and due norths to Khartoum ( Loiy, 2009 ) . The fruit, eaten when to the full ripe or even when about putrid, is used as a antipyretic ( Grieve, 1984 ) . The fruit is besides diuretic, being effectual in the intervention of edema and nephritic rocks ( Chiej, 1984 ) . The rind of the fruit is prescribed in instances of alcoholic toxic condition and diabetes ( Duke and Ayensu, 1985 ) .

Gourd family workss are known to incorporate bioactive compounds such as cucurbitacin, triterpenes, steroid alcohols and alkaloids. Plants incorporating cucurbitacin were early recognized in common people medical specialty to hold biological values. Scientific surveies chiefly refer to Middle East and Asia where cucurbit workss were used actively as herbal redresss. Cucurbit workss demonstrated anti-inflammatory, antitumor, liver protective and immunoregulatory activities ( Ram, 1999 and Hu et al. , 1982 ) . Cucurbitacins are a group of extremely oxygenated steroidal triterpenes characterized by the cucurbitane skeleton. Aglycons isolated from assorted species have been given the general name of cucurbitacin. Assorted letters in back-to-back order of isolation follow this name, although in certain instances other names have been given ( Lavie and Glotter, 1971 ) .

Metronidazole sometimes causes inauspicious effects, e.g. , myoplasia, neuralgy, and allergic dermatitis ( Upcrof et al, 2006 ) ; hence new antigiardiasis drugs are likely required. With the intent of seeking for new antigiardiasis agents, Citrullus lanatus which was used traditionally for intervention of clinical marks associated with giardiasis and the workss was selected to measure the activity of their ethyl ethanoate, butyl alcohol and crude oil ether rough infusions against Giardia lamblia trophozoites in vitro.

Material and Method

Plant stuffs and Extraction:

The works used in this survey was Citrullus latanus volt-ampere. citriode collected from AL- Musawarat, Northern Sudan, collected on February 2006. The systematic designation of this works was carried out at Medicinal & A ; 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 pieces and dried at room temperature ; seeds were separated and ground into a harsh pulverization. Dried fruit powdered extracted with trichloromethane, the filtrates were collected together and the residue was brought to dryness and extracted with ethyl alcohol and was modified to aqueous infusion, which was extracted in turn with equal volumes of two organic dissolvers of increasing mutual opposition ( ethyl ethanoate and butyl alcohol ) .

Isolation of compounds from C. lanatus volt-ampere. citroides fruits mushs:

Vacuum liquid chromatography ( VLC ) :

Vacuum liquid chromatography was performed on column ( 25 x 15 centimeter ) packed with silica gel of atom size ( 0.04-0.06 millimeter ) ( 60-120 mesh ) and compacted with vacuity and pressure 10 times. The rough infusion of ethyl ethanoate ( 12 g ) was subjected to ( VLC ) . The concentrated sample was applied on the wall of the column.

The slip used to fractionate ethyl acetate petroleum infusion ( 12 g ) was trichloromethane: methanol mixture of increasing mutual opposition. Parts of 100 milliliters were collected, combined on the footing of TLC analysis utilizing solvent system trichloromethane: methyl alcohol ( 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 centimeter ) packed with silica gel ( 115 g ) of atom size ( 0.04-0.063 millimeter ) , and crystallized to give compound ( 1 ) ( Eth.c60 ) ( 300 milligram ) . Fraction six ( 3 g ) besides was semi-pure, it was purified throw little column utilizing the same eluent to give compound ( 2 ) ( EA VLC 42-46 ) ( 400 milligram ) .

Analytic Techniques:

Infra ruddy ( IR ) spectrometry:

The IR spectra were recorded on Perkin-Eelmer theoretical account 1650 FTIR spectrophotometer utilizing 50 mg KBr and 1mg of the stray compounds. The undermentioned abbreviations were reported: S ( strong ) , M ( medium ) and W ( hebdomad ) .

Nuclear magnetic resonance ( NMR ) spectra:

1H NMR spectra:

These were recorded on Nuclear magnetic resonance: Bruker Avance 400 spectrometer setup. Samples were dissolved in deuteriochloroform ( CDCl3 ) or deuterioacetone ( AD3OD ) utilizing tetramethylsilane, as an internal criterion ( 0.00 ppm ) . Data were presented in following order ; chemical displacement relation to tetramethylsilane, multiplicity and strength as to the figure of protons ; matching changeless J ; assignment ( if appropriate ) . The undermentioned abbreviations were adopted ; s ( individual ) ; vitamin D ( dual ) ; T ( three ) ; m ( multiplet ) ; Doctor of Divinity ( dual of doublets ) signal.

A?A?C NMR Spectra:

These were reported at Ac 150 MHz instrument. Tetramethylsilane was used an internal criterion ( 0.00ppm ) . Chemical craps were reported comparative to tetramethlysilane, assignments were based on the multiplicities and chemical displacements. Multiplicities were determined from polarisation transportation technique ( DEPT ) or from direct response to C-H yokes ( APT ) .

Mass spectroscopy:

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

Melting point:

Melting point was measured utilizing electrothermal runing point setup theoretical account 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 saddle horse readying. Then the positive sample was transported to MAPRI in alimentary broth medium. Trophozoites of G lamblia were maintained in RPMI 1640 medium incorporating 5 % bovine serum at 37 i‚± 1i‚°C. The trophozoites were maintained for the checks and were employed in the log stage of growing.

In vitro susceptibleness checks

In vitro susceptibleness checks used the sub- civilization method of Cedilla et al. , ( 2002 ) . This is extremely rigorous and sensitive method for measuring the anti-protozoal effects ( gilded criterion ) peculiarly in Entamoba histolytica, Gairdia intestinalis and T. vaginalis ( Arguello et al. , 2004 ) .

5 milligram from each infusion and compound was dissolved in 50 Aµl of dimethyl sulfoxzide ( DMSO ) at eppendorf tubing incorporating 950 Aµl D.W in order to make concentration of 5 mg/ml ( 5000ppm ) . The dressed ores were stored at -20 A°C for farther analysis.

Sterile 96-well microtite home base was used for different works infusions, positive control and negative control.

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

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

For numbering the samples were assorted with Trypan blue in equal volume. The concluding figure of parasites was determined with haemocytometer in triplicate.

The mortality % of parasite for each infusions activity was carried out harmonizing to the undermentioned expression:

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

Control negative

Merely 100 % suppression of the parasite considered, when there was no motile parasite observed.

Statistical analysis

All informations were presented as agencies A± S.D. Statistical analysis for all the checks consequences were done utilizing Microsoft Excel plan. Student t.test was used to find important difference between control and works infusions at degree of P & lt ; 0.05.

Consequences

Word picture of compound ( 1 ) :

Compound ( 1 ) was obtained as an formless brown pulverization signifier Ethyl ethanoate infusion. It has Rf value 0.47 in dissolver system ( Ch: MeOH ( 85:15 ) ) . It developed brown coloring material with vanillin reagent. M. P. 1002.66. IR spectrum showed soaking up at: 3434 cmaˆ?A? ( OH ) B, 2929 cmE‰A? ( C-H ) , 1686 cmE‰A? ( C=O ) v. s, 1637 ( C=C ) m. s, 1076 ( C-O ) m. s. ( Table 1 ) . The 1HNMR Spectrum of compound ( 1 ) displayed tow vest each of three protons integratings at I? 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 vitamin D ( 1H ) J= 8Hz. 2.05 ( 2H ) J= 16 Hz, 5.75 s ( 1H ) , 4.08 vitamin D ( 1H ) J= 12Hz, 2.6 s ( 3H ) . 1.85 vitamin D ( 2H ) J= 8Hz, 2.18 vitamin D ( 1H ) J=20 Hz, 1.3 s ( 3H ) , 1.87 vitamin D ( 2H ) J= 8Hz, 1.6 vitamin 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 ) . A?H- and A?A?C NMR was presented in ( Table 2 ) . El-MS m/z: 558.32 which was confirmed by HREI-MS to give a molecular expression C32H44O8. The NMR information of this compound were indistinguishable to those of Cucurbitacin E as antecedently reported ; 1H NMR ( Lavie et al. , 1962 ) and13C NMR ( Valde and Lavie 1983 ) . Consequently, the construction of compound ( 1 ) was identified as cucurbitacin E. ( Figure 1 )

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

Table 1: IR spectral informations of compound ( 1 )

*Wave length ( cmaˆ?A? )

Assignments

Wave length of ( 34-35.60 ) extremums ( cmaˆ?A? )

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 crook

C-OH out-of-plane crook

CH2 stone

C-H out-of-plane crook

Ringing stretching

C-H out-of-plane crook

C-C-C in-plane crook

C-H in-plane crook

CH2 wit

C-H in-plane crook

O-H deform

CH 3 symmetric crook

CH 3 asymmetric crook

C=O stretching manner

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: A?A?C and A?H-NMR spectral informations for compound ( 1 )

Consecutive figure

Carbon atom No.

Type

I?c

ppm

I?H

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 )

Word picture of compound ( 2 ) :

Compound ( 2 ) ( VLC42-46 ) was obtained as an formless pulverization from ethyl acetate infusion of the fruit mush. It has the undermentioned features:

Rf value 0.47 in dissolver system ( CHCl3: MeOH ( 85:15 ) ) , developed purple coloring material with vanillin reagents. It dissolved in propanone and MeoH.

IR spectrum showed soaking up at: 3402 cmA­A? ( OH ) , 2901 cmA­A? ( C-H ) , 1638.34 cmA­A? ( C=C ) ( Table 3 ) .

The 1H and 13C NMR spectra showed the presence of one sugar mediety, it was identified as a I?-glucopyranosyl a terminal unit, with compound ( 2 ) . Enzymatic hydrolysis of compound ( 2 ) with I?-amylase gave cucurbtacin L, corroborating that the I?-glucopyranosyl unit is a sugar connected to a ?-glucopyranosyl unit at C-2. Furthermore, the 13C NMR spectrum besides revealed the fond regard of a terminal sugar to C-2 of a I?-glucopyranosyl unit due to the downfield displacement of this atom ( +9.4 ppm ) and upfield displacement of C-30 ( _0.9 ppm ) ( table 4 ) . El-MS m/z: 676.35. which was confirmed by HREI-MS to give a molecular expression of C36H52O12. Consequently, the construction of compound ( 2 ) was identified as cucurbitacin L 2-O-I?-glucopyranosyl. ( Figure 2 )

Cucurbitacin L 2-O-I?-glucopyranosyl antecedently 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 informations of compound ( 2 )

Wave length ( cmaˆ?A? ) *

Assignments

Wave length of ( VLC42-46 ) extremums ( cmaˆ?A? )

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 crook

C-OH out-of-plane crook

CH2 stone

C-H out-of-plane crook

Ringing stretching

C-H out-of-plane crook

C-C-C in-plane crook

C-H in-plane crook

CH2 wit

C-H in-plane crook

O-H deform

CH 3 symmetric crook

CH 3 asymmetric crook

C=O stretching manner

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: A?A?C and A?H-NMR spectral informations for compound ( 5 ) ( VLC42-46 )

Consecutive figure

Carbon atom No.

Type

I?c

ppm

I?H

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-I?-glucoside

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

In contrast, no growing was observed after 96 hours in incubated at 500 ppm concentrations of Citrullus lanatus in ethyl ethanoate infusion. However, at other concentrations of 250 ppm and 125 ppm, the cells were feasible even after 120 H of incubation Figure ( 4 ) .

Figure ( 5 ) , shows the activity of butyl alcohol infusion of Citrullus lanatus. The high dosage of infusion shows 90 % mortality after 120 hours.

In Figure ( 6 ) shows the activity of Cucurbitacin-E infusion of Citrullus lanatus. The high dosage of extract gave 100 % mortality after 96 hours, this was non the same consequence appeared with metrondizole.

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

Discussion

Giardia lamblia is one of the most common enteric infective protozoon parasites ( Newman et al. 2001 ) . It is going progressively of import among HIV/AIDS patients. There are studies that some instances of ague and chronic diarrhoea in AIDS patients may be associated with giardial infection ( Merchant and Shroff 1996 ) . However, Metronidazole, the common drug of pick, can do mutagenicity in bacteriums ( Legator et al. 1975 ) and is carcinogenic in gnawers ( Rustia and Shubik 1972 ) . It besides possesses unwanted side effects and intervention failures have been reported ( Llibre et al. 1989 ) .

Our present in vitro probe reveals promosing consequences for the usage of the works for cultivated parasites to explicate such ingredient of the works infusion as the drug. However, the IC50 of petroleum-ether, ethylacetate and butyl alcohol infusions are 0.18, 0.15 and 0.45 ppm and their IC90 were found to be 432, 122 and 406 ppm severally. While the IC50 and IC90 of Cucurbitacin-E and Cucurbitacin L 2-o-I?-glucoside are 0.002, 0.005 ppm and 15, 32 ppm severally. The present survey highlights the efficiency of C. lanatus infusions and stray compounds acquisition alternate natural and chemical intervention for giardiasis.

In decision this consequence enhances the ethnobotanical utilizations of the works as antidiarrheal in instances associated with giardiasis in cardinal Sudan. Further probes sing the manner of action and other related pharmacological surveies such as in vivo probe, drug preparation and clinical tests are extremely recommended.

Figure ( 3 ) : In vitro activity of Citrullus lanatus crude oil quintessence infusion against G. lamblia

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

Figure ( 5 ) : In vitro activity of Citrullus lanatus butyl alcohol infusion 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-I?-glucoside against G. lamblia

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

Sample tested

IC50/ppm

IC90/ppm

Citrullus lanatus Petrolium ether infusion

0.182327

431.94

Citrullus lanatus ethylacetate infusion

0.145179

122.25

Citrullus lanatus butyl alcohol infusion

0.480735

406.65

Cucurbitacin-E

0.002381

15.06

Cucurbitacin L 2-o-I?-glucoside

0.004910

32.43