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RESEARCH ARTICLE

Separating and determining the effective compounds percentage in Ecballium elaterium L. Cucurbitacins Syria

Manal Darwish*, Mohamed Isam Hasan Agha², Soufi Barkil³, Oussama Mansour⁴

¹Department of Pharmacognosy, Faculty of Pharmacy, Al-Andalus University for Medical Science,

Al-Qdmous, Syria.

²Department of Pharmacognosy, Faculty of Pharmacy, Damascus University

³Department of Toxicology and Pharmacology-Faculty of Pharmacy-Damascus University

⁴Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Al-Alndalus University for Medical Science, Al-Qdmous, Syria.

*Corresponding Author E-mail: mansouroussama@yahoo.fr

ABSTRACT:

The Ecballium elaterium L. which grows in the Mediterranean region is considered one of the well known plants in folk medicine in treating liver diseases, especially jaundice. Recent studies have indicated its effectiveness in this area in addition to its valid use in the treatment of Enfluenza, Analgesic and antipyretic and Anti-in flammation. Its fruit is usually used due to the high percentage of the active ingredients and the ease of extraction without ruining or damaging these active ingredients. This extraction contains a set of tri-terpene compounds responsible for the pharmaceutical effectiveness of the plant. These compounds are called Cucurbitacins and they have several types. The study aims at conducting a series of tests and chemical assay in order to determine the juice content of chemical components and calibrate Cucurbitacins existed in the plant using the available ways (HPLC). The plant fruit was collected from the coastal region and identified by morphological characteristics of sex and gender. Afterwards, it was manually squeezed , purified, and properly tested to detect and calculate the concentrations of Cucurbitacins especially Cuc I and Cuc E. HPLC Examination of the intense methanolic juice, conducted on the waves (230 nm, 254 nm), has confirmed the presence of Cuc I and Cuc E (concentration of Cuc E was greater than that of Cuc I ).

KEY WORDS: Ecballium elaterium L., Cucurbitacin (Cuc), HPLC.

INTRODUCTION:

Ecballium elaterium L, which is from the Cucurbitaceae family^(1,2,7) widespread in Syria (the coastal and the southern region)⁽⁸⁾, with its active components of the Cucurbitacins has been considered extremely affective in curing many diseases^(3,4,5). It is widely used in treating liver diseases particularly (Hepatitis⁽⁹⁾). Moreover, it is used in the treatment of rheumatism⁽⁶⁾ Viral disease^{(9) ,}sinusitis⁽¹⁰⁾herpes zoster^(11,12).

Received on 29.03.2015 Modified on 08.04.2015

Research J. Pharm. and Tech. 8(7): July, 2015; Page 829-835

DOI: 10.5958/0974-360X.2015.00135.3

The plant includes several elements in its liquid juice, and the intense extract of the ripe fruit consist of proteins, which range in molecular weight between 13 → 103 KDa, lipids, sugars, minerals and Cucurbitacins⁽²⁾ (Figure1) which are classified as a group of active compounds ( triterpens, tetracyclic) that are different from each other where a set of hydroxyl is in positions C2, C3, C19 and C24, the cationic function is in position C3, the double bond is between C1 and C2, and between C23 and C24, acetalation of the hydroxyl set is in position C26. The taste of the juice is also bitter, and it contains cucurbitacins B, D, E, I, R, and L which lots of biological activities are attributed to. Ecballium elaterium L. is a highly toxic plant and not appropriate for internal use; it causes severe irritation to the digestive tract⁽²⁾ although it is used to treat liver toxicity⁽¹³⁾. The plant extract, especially its fruit, is rich in Elatrium which in large doses may cause nausea and vomiting; moreover, and in case of repeated use, it may cause severe inflammation in the stomach and in testiness that leads to death⁽¹⁴⁾. Ocular exposure can cause conjunctivitis and corneal edema erosion. Dermatitis occurs within minutes of exposure due to the irritation of the mucous membrane with varying degrees of severity (intensity). Intranasal administration can lead to irritation of the mucous membranes, and it is manifested in pharyns, dyspnea, drooling, dysphagia, and upper airway obstruction^(15,16).

Figure 1: The general structure of Cucurbitacins

MATERIALS AND METHODS:

Cucurbitacins I, Cucurbitacins E were obtained from Genay France company, Acetonitrile ACN, Methanol MeOH ,Water H₂O for HPLC (Shamlab Company).

· HPLC type JASCO equipped with the followings:

· (PU-980 Intelligent HP, UV-detector pump 970 Intelligent UV / VIS).

· ColumnC18 (250 mmx4.6mm), (Kromasil).

· Ultrasonic Model 310 T (Germany).

· Filters measure 0.45μM (Whatman).

· Rotavpor model 111 BUCHI RE Labconco.

· Centrifuge Heraeuslobofuge 200.

· Stirrer model Snijders 34532.

Preparation of the Juice:

Ripe fruits were collected from costal region and squeezed to get the crude fruit, filtered in order to remove seeds and the remaining tissues, and then they were centrifuged at 5,000 r / min for 40 minutes. Afterwards, they were filtered twice through membrane filters with the dimensions of 0.45 μM. Clear juice (500 ml) as pale watery liquid was obtained, and it was kept in sterile tubes at a temperature (-20C˚)⁽²⁾

Analysis of Cucurbitacins of the Juice:⁽²⁾

The extraction of the active substances from the fruits was carried by adding the clear juice, obtained from the former stage, to pure methanol, where 100 ml of the juice was mixed with an equal volume of methanol (100 ml) for two hours at room temperature and using a magnetic motor to ensure homogeneous stirring and distribution in methanol in order to extract the active ingredients⁽²⁾properly. It was observed separation of some white parts of the juice which were fibers and proteins. The centrifugation of the solution was carried at speed 10,000 r / min for 15 minutes/ ˚4C in special covered tubes. Afterwards, the floating solution was collected, weighed, and placed in a flask of the rotary evaporator. The temperature was set on ˚40C, and the pressure inside the flask (65 ± 2 bar). This pressure was suitable to vaporize the methanol from the liquid. This lasted for two hours until full vaporization of the liquid. Eventually, the active substances were obtained and preserved in sterile tubes at temperature -20 ˚C. It is worth mentioning that we have kept juice samples from the various stages of the work (pure water juice, methanolic juice before evaporation).

Preparation of Standards:

Cuc E: solution was obtained with concentration of 1 mg / ml (equivalent to 1000 μg / ml). Then, a standardized series was conducted, and the first standardized solution Cuc E was obtained with concentration of 0.1 mg / ml equivalent to 100 μg / ml, and the second standardized solution Cuc E with concentration of 0.01 mg/ml equivalent to10μg/ml.

Cuc I : was prepared in the same way and the basic solution was obtained with concentration of 1 mg / ml equivalent to 1000 μg / ml. The first standardized solution Cuc I was obtained with concentration of 0.1 mg / ml equivalent to 100 μg / ml and the second Cuc I with concentration of 0.01 mg / ml equivalent to 10mg / ml. Solutions have been prepared and placed in a sealed airtight glass tubes with labels reading each concentration with the standard name, and they were left untouched and preserved at a temperature of -20 C^o until use.

High Performance Liquid Chromatography (HPLC):

The work has been done on waves length of (230- 254 nm). A comparison between the two waves length was conducted having in mind that the main work was done on 230 nm wave after testing the results. Since Cucurbitacins are polar materials, they carry at least one hydroxyl group, so C18 non-polar material was chosen separation within a column that has the dimensions (250 × 4.6 mm). The aqueous mobile phase (including water) was selected provided with organic corrigents of methanol and acetonitrile in which the active substances dissolve in at the rate of methanol / acetonitrile / water 30: 30:40. The used standards Cuc E and CucIwere solved well in methanol, acetonitrile and water especially that these solvents constitute the mobile phase. Pumping system is Isocratic chromatography flow rate was 1 ml / min and the size of the sample injected was 20μl. We mixed the standards with the extract or the experimented material with the same concentration that was injected for each standard alone, and the extract was injected alone in a process called internal standard injection. (0.1ml) of the second standard solution for Cuc I, (0.1 ml) of the first standard solution for Cuc E, and (0.25ml) from the intense methanolic extract of plant were taken, and then the mixture was extended to (1ml) in the mobile phase. After wards, the obtained extract was injected at the wavelength 230 nm (the principal wave length). This was done after injecting the mixture of the tow standards together alone, and the extract alone in same concentration, that was found in, in the internal standard. Then, the peaks correspondences and their areas were compared to make sure that the expected peak in extract of Cuc E and Cuc I is the same injected and standardized material.

RESULTS AND DISCUSSION:

Appropriacyof the device at the standard Cuc I: The device was appropriate through injecting five consecutive samples from the standard using the wave length 230 nm and a concentration of 10 μg / ml (Figure 2), and the value of the relative standard deviation RSD was less than 2%

Calculating the concentration of the plant intense extract: Comparing the results of the injection of the extended methanolic extract with standars Cuc I and Cuc E , it is possible the concentration of the plant extract as the following:

1-Measuring at 230 nm wave length - concentration of Cuc E in the extended extract = (concentration of standard Cuc E × area of standard Cuc E in extract) / (area of standard Cuc E) = (100 × 674741.218) /1047715.061 = 64.4 µg/ml Thus, the concentration of Cuc E in intense methanolic extract of plant is: (Concentration × volume) before extension = (concentration × volume) after extension Concentration = (64.4 × 1) /0.25=257.6 μg / ml = 0.26mg / ml so Concentration in the crude juice 100 ml: Concentration = (0.26 × 3) /100=0.008 mg / ml - concentration of Cuc I in the extended extract = (concentration of standard Cuc I × area of standard Cuc E in extract) / (area of standard CucI) = (10 × 660177.860) /302250.596 = 21.8 µg/ml

Thus, the concentration of CucI in intense methanolic extract of plant is: Concentration = (21.8 × 1) /0.25=87.2 μg / ml = 0.09 mg / ml so concentration in the crude juice 100 ml: Concentration = (0.09 × 3) /100=0.003 mg / ml

2- Measuring at 254 nm wave length - concentration of Cuc E in the extended extract = (concentration of standard Cuc E × area of standard Cuc E in extract) / (area of standard Cuc E) = 62.14 µg/ml - concentration of Cuc I in the extended extract = (concentration of standard Cuc I × area of standard Cuc E in extract) / (area of standard Cuc I)21.03= µg/ml All the previous calculations can be summarized in table (1): Table (1): areas and concentrations of mixed standards together and their concentrations in extract when use wave lengths 230 nm, 254 nm

Peak Area		Concentration µg/ml		Sample
230 nm Wave Length	254 nm Wave Length	230 nm Wave Length	254 nm Wave Length	Sample
543201.504	460949.554	10.00	10.00	Cuc I Standard
2510867.410	2345239.584	100.00	100.00	CucE Standard
302250.596	252338.376	10.00	10.00	Cuc I Mixed Standard (CucI+Cuc E)
1047715.061	876887.589	100.00	100.00	CucE Mixed Standard (CucI+Cuc E)
660177.860	530669.373	21.80	21.03	Cuc I Extract
674741.218	544918.975	64.40	62.14	CucE Extract

Figure 4: chromatography diagram of standards mixed on 230 nm wave length

Figure 5: chromatography diagram of extract mixed on 230 nm wave length

Figure 6: chromatography diagram of standards mixed on 254 nm wave lenght

Figure 7: chromatography diagram of extract mixed on 254 nm wave length

Table 2 : concentrations and areas of Cuc I, Cuc E in the standards , extract, and the internal standard

Cuc I Standard

Cuc I Extract

Cuc I Extract)+Standard)

Cuc E Standard

Cuc E Extract

Cuc E Extract)+(Standard

Samples

329366.031

703416.108

1031843.718

1532809.048

577260.700

2084620.305

Peak Area

10.0000

21.3566

31.3281

100.0000

37.6600

137.0000

Concentration

µg/ml

Figure 8 : chromatography diagram to standards mixture

Figure 9: chromatography diagram for extract mixture

Figure 10 : chromatography diagram for internal standard mixture)

CONCLUSION:

The extract content of active substances in this study has been determined by conducting a series of particular chemical tests. Cucurbitacins, particularly compounds Cuc I and Cuc E in juice, has been detected, and their concentrations calculated. The fact that Cucurbitacinsis are responsible for the biological activity of the plant, their existence have been confirmed by the high performance liquid chromatography (HPLC) examination, which was conducted on the wave length (230 nm, 254 nm) to the intense methanolic extract. Concentration of Cuc E compound is greater than the concentration of Cuc I compound. This result matches international studies and research^(2). There is no statistical difference in the concentrations values on the two waves lengths; however, the absorption is bigger and better on the wavelength (230 nm).

ACKNOWLEDGEMENT:

I would like to thank Mr. Ayham Aljghami, Instructor at The Higher Institute of Languages-Tishreen University-Syria, for the language assistance provided during the writing process of this article.

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