PHYTOCHEMICAL AND BIOLOGICAL INVESTIGATIONS OF ATRIPLEX SEMIBACATA R . BR . GROWING IN EGYPT

The lipid content of Atriplex semibacata growing in Egypt was studied. The unsaponifiable fraction was identified by GLC. A series of hydrocarbons ranging from C14C28 in addition to cholesterol, stigmasterol and the triterpenoids  and  amyrin were identified. GLC analysis of fatty alcohols fraction revealed the presence of six fatty alcohols in which dotriacontanol (C32H66O) was the major (14.68%). Six compounds (five coumarins and one phenolic acid) were isolated for the first time from A. semibacata. The coumarin constituents isolated from the chloroform and the ethyl acetate fractions of the aqueous alcoholic extract of A. semibacata were identified as scopoletin, umbelliferorne, coumarin, scopolin, 7-methoxy coumarin in addition to a phenolic acid P-coumaric acid. Also, the flavonoidal compounds isolated from the n-butanol fraction of the plant revealed the presence of kaempferol 3-O glucoside and acacetin. Their identity was proved by m.p., TLC, PC, UV and MS analysis. The alcohol extract showed significant antimicrobial activity against G-ve bacteria, moderate activity against G+ve bacteria. On the other hand, the pet. Ether extract showed marked activity against G+ve bacteria and fungi, also the G-ve bacteria was greatly inhibited by the chloroform extract. The different extracts of the plant exhibited no cytotoxic activity against Erlich-ascites carcinoma cells line at the tested concentrations, also showed a strong antioxidant activity using DPPH.


Introduction
The family chenopodiaceae is one of the largest families of the flowering plants. It is represented in Egypt by 25 genera and 300 species. The most important genera are Ambrosia, Atriplex, Beta, suaeda and Halocnemum (Tackholm, 1974). The occurrence of flavonoids in several chenopodiaceae species have been reported (Rizk, 1986). Also coumarins, alkaloids and terpenes were detected in several species (Rizk, 1982).
The genus Atriplex is represented in Egypt by 15 species (Tackholm, 1974) and little information was represented about the constituents of these genera. Atriplex are characterized by their high content of sodium chloride, they are said to be useful as fodder plants (Watt and Breyer, 1962). The study of the chemical composition of several species shows that the plants have a relatively high nutritive value for ruminants and many are useful as a feed supplement (Rizk, 1986), also it was found from the literature that the ethyl alcohol extract of A. leucolata and A. lampa showed respectively a molluscidal and antimicrobial activities (Shoeb, et al., 1987;Arellano, et al., 1988;Rizk, et al., 1986). Phytochemical screening of several species revealed the presence of coumarins, saponines, alkaloids and few species contain flavonoids. Alkaloids were detected in A. littoralis and A. nitens and phenolic acids in A.astata (Rizk, 1986). Four flavonol glycosides were isolated from A. littoralis (Wiestawa, 2004) and a new flavone glycoside, identified as kaempferol-3-O-3acetyl--L-arabinopyranosyl-(1-6) --D-glucopyranoside has been isolated from the aerial parts of Thalictrum Atriplex (Guangyao, et al., 2000). Previous studies revealed the isolation of protocatechuic acid, caffiec acid, P-coumaric acid, kaempferol and sitosterol from the same plant (Gao, et al., 1999). It was found from literature that four triterpenoid saponines were isolated from A. semibacata (Shaker and Seifert, 2003).
From the medical point, it was found that A. hortensis and A. patula were used as a folk remedy for plethora, lung ailments, diuretic and emetic. Also it was found that A. halimu can be used for hypoglycemic treatments (Yaniv, et al., 1987).
Nothing was reported about the lipid and coumarin constituents of A. semibacata, therefore the present work deals with the study of lipid, coumarin and non-identified flavonoidal constituents of A. semibacata growing in Egypt and the study of the antimicrobial and antitumor activities activity of total extracts and isolated compounds, as well as studying their antioxidant activity.

Plant material
Atriplex semibacata R.Br. was collected from Borg El-Arab near Alexandria during June 2009. The plant was identified by Dr. M. El-Gebaly and Dr. S. El-Kawashty, taxonomists, National Research Centre, Cairo, Egypt, to whom the authors are deeply indebted. The aerial parts of the plant were air dried and ground into fine powder. A voucher specimen was kept in the herbarium of NRC.
Antimicrobial activity: The tested organisms were obtained from Medical Microbia and Immunity Dept , Faculty of Medicine , Cairo University..

Antitumor activity:
The antitumor activity of the different extracts were tested against Ehrlich-ascites of carcinoma in vitro in the National Institute of Cancer, Cairo.

Isolation of Lipids:
About 2 Kg of the air dried powdered plant (aerial parts) of A. semibacata was extracted with pet. ether (40-60). The pet. ether extract was evaporated and the residue (28g) was dissolved in boiling acetone (250ml), cooled and the formed amorphous precipitate (3.1g) was separated out. The acetone soluble fraction was saponified (N/2 alc. KOH) and the unsaponifiable matter (1.68) was separated (Radwan and Abdel-Shafeek, 2006). Results are shown in table (1,2).
Isolation of coumarins and phenolic acids: About 1.8 Kg of defatted dried powdered of A. semibacata was macerated with 80% ethanol. The combined alcoholic extracts were evaporated in vacuo at 40 0 C. The residue (48g) was dissolved in hot distilled water and left over night. The aqueous filtrate was portioned with successive portions of chloroform (3 X 200 ml), followed by ethyl acetate (3 X 200 ml) and finally with n-butanol (3 x 200 ml). The extracts were dehydrated separately over anhydrous sodium sulfate and evaporated in vacuo at 45 0 C. The chloroform, ethyl acetate and n-butanol free residues amounted to 1.9 g, 2.2 g and 2.45 g respectively.
The chloroform fraction was subjected to preparative paper chromatography (3MM, 15% acetic acid). Four main zones (R f 0.74, 0.69, 0.61 and 0.41) were cut and eluted separately by 90% methanol. The eluted fractions were further purified on sephadex LH-20 column using 90% methanol as eluent. The ethyl acetate fraction was also subjected to preparative paper chromatography (3MM, 15% acetic acid) and two main zones (R f 0.49 and 0.36) were cut and eluted separately by 80% methanol. The n-butanol fraction was subjected to preparative PC (3 MM, 15% acetic acid) and two main zones (F f 0.54 and 0.09) were cut and eluted separately with 90% methanol. The eluted fractions were further purified on sephadex LH-20 column using 90% methanol.
Antimicrobial and Antifungal activities: Seven representative strains of bacteria and fungi isolates were selected viz Staphylococcus aureus, Sarcina lutea, Bacillus subtilis, Pseudomonas aeruginosa, Proteus mirabilis, Candida albicans and Saccharamyces cerviceae. The different extracts and fractions of the plant in dimethyl formamide (DMF) were tested in vitro against the selected strains of bacteria and fungi isolates. The Antimicrobial activities were carried out by using agar diffusion disc method (Lorian, 1980). Results of antimicrobial and antifungal activities are shown in table (3).

Antitumor activity:
The plant extracts were screened in vitro using a single tumor (Ehrlich-ascites carcinoma cells) using a dose of 100 g/ml, 50 g/ml and 25 g/ml of each extract (table 4). This screening was carried out employing the method described earlier (

Determination of scavenging effect on DPPH radicals:
The effect of the plant extracts on DPPH was studying employing the modified method described by (Yamaguchi et al., 1998). The decrease of the absorbance at 516 nm of the DPPH solution after addition of the sample (plant materials) was measured in a glass cuvette. An aliquot of 0.1 ml M. methanol solution of DPPH was mixed with the methanolic solution of the sample, so that the relative concentration of plant materials versus the stable radical in the cuvette was 0.13, then the solution with tested sample was shaken vigorously. The absorbance was mentioned at the start and at 30 min. after being kept in the dark against a blank of methanol without DPPH. All tests were run in duplicate and averages were calculated (Nicolaos and Maria 2002). The antioxidative of these samples were compared with Trolox. Where % RSA= 100% X Abs of blank 516 nm -Abs of sample 516 nm Abs of blank 516 nm The results are expressed as radical scavenging activity (%RSA) as shown in Table 5.

Unsaponifiable Fraction
GLC analysis for the unsaponifiable fraction proved to be a mixture of triterpenes, sterols and a series of hydrocarbons. Identification of the compounds was carried out by comparison of their retention times with the available reference compounds Table 1.

Fatty alcohol Fraction
GLC analysis for the acetone precipitate fraction of A. semibacata revealed the presence of six fatty alcohols in which dotriacontanol (C 32 H 66 O, 14.68%) was the major component as illustrated in Table 2.

P-coumaric acid (compound A)
The residue obtained from the eluted band (R f 0.74) isolated from The chloroform fraction by preparative paper chromatography (3MM, 15% acetic acid) and eluted by 90% methanol, gave after purification on sephadex LH-20 column using 90% methanol, a single compound which gave a blue color under UV light. The UV spectrum in methanol showed peaks at (317, 268 and 216 nm) that found to be identical to those of P-coumaric acid (Gao, et al., 1999 ;Yang, et al., 2002).
Also the R f of the compound was identical to that of P-coumaric acid in different solvents and adsorbents. Further confirmation was performed by carrying out EI-Mass spectrum which showed a molecular ion peak M +  164 which corresponding to the molecular formula C 9 H 8 O 3 and fragments at m/z 147, 119 that are identical to that reported for Pcoumaric acid (Gao, et al., 1999 ;Foaad, et al., 1997).

Scopoletin (compound B)
The residue obtained from the eluted band (R f 0.69) isolated from The chloroform fraction (21 mg) by preparative paper chromatography (3MM, 15% acetic acid) and eluted by 90% methanol, gave after purification on sephadex LH-20 column using 90% methanol, a single compound which gave a blue color under UV light and no color with p-anizaldehyde. Upon crystallization from methanol, it gave needles (m.p. 203 o C). The UV spectrum in methanol showed peaks at (342, 293 sh , 260 sh and 226 nm) these data are identical to those of scopoletin (Eyre and Spottis, 1965 ;Murray, et al., 1982). Further confirmation was performed by carrying out EI-Mass spectrum which showed a molecular ion peak M +  at m/z 192 which corresponds to the molecular formula C 8 H 8 O 4 and fragments at M/z 177 (M + -CH 3 ) , 164 (M + -CO), 149, 121 that are identical to that reported for scopoletin (Murray, et al., 1982).

Umbelliferone (compound C)
The residue obtained from the eluted band (R f 0.61) isolated from The chloroform fraction (16 mg) by preparative paper chromatography (3MM, 15% acetic acid) and eluted by 90% methanol, gave after purification on sephadex LH-20 column using 90% methanol, a single compound which gave a blue fluorescence under UV light and no color with panizaldehyde. The UV spectrum in methanol showed peaks at (320, 257 and 222 nm). Upon crystallization from methanol, it gave colorless crystals (m.p. 226-227 o C). These data are identical to those of Umbelliferorne (Yang, et al., 2002 ;Foaad, et al., 1997). The EI-Mass spectrum showed a molecular ion peak M +  at m/z 162 which corresponds to the molecular formula C 9 H 6 O 3 and fragments at M/z 134 (M + -CO), 106 that are identical to that reported for Umbelliferorne (Murray, et al., 1982).

Coumarin (compound D)
The residue obtained from the eluted band (R f 0.41) isolated from The chloroform fraction (24 mg) by preparative paper chromatography (3MM, 15% acetic acid) and eluted by 90% methanol, gave after purification on sephadex LH-20 column using 90% methanol, a single compound which gave a blue color under UV light and no color with p-anizaldehyde. Upon crystallization from methanol, it gave faint yellow crystals (m.p. 71-72 o C), also the UV spectrum in methanol showed peaks at (310, 274 and 215 nm). These data are the same of that reported for coumarin (Yang, et al., 2002). The EI-Mass spectrum showed a molecular ion peak M +  at m/z 146 which corresponds to the molecular formula C 9 H 6 O 2 and fragments ions at m/z 118, 90 that are identical to that reported for coumarin (Murray, et al., 1982).

Scopolin (compound E)
The residue obtained from the eluted band (R f 0.49) isolated from The ethyl acetate fraction (18 mg) by preparative paper chromatography (3MM, 15% acetic acid) and eluted by 90% methanol, gave after purification on sephadex LH-20 column using 90% methanol, a single compound which posses the same R f values as authentic scopolin using different solvents and adsorbents. Crystallization was carried out from methanol to yield yellowish crystals (m.p. 217-218 o C) (Yang,  et al., 2002). Acid hydrolysis (2N HCl) of the compound gave scopoletin which was identified by co-chromatography using TLC, PC, UV and MS M +  at m/z 192 and fragments at M/z 177, 164, 149, 121 that are identical to that reported for scopoletin (Murray, et al., 1982). The sugar moiety identified as glucose by PC. Whatman No.1, ethyl acetate -pyridinewater (12: 5: 4) and n-butanol-benzene-pyridine (5: 13: 3).

7-Methoxy coumarin (compound F)
The residue obtained from the eluted band (R f 0.36) isolated from the ethyl acetate fraction (12 mg) by preparative paper chromatography (3MM, 15% acetic acid) and eluted by 95% methanol, gave after purification on sephadex LH-20 column using 90% methanol, a single compound which gave blue fluorescence under UV light and no color with p-anizaldehyde. The UV spectrum in methanol showed peaks at (349, 322, 250, 216 and 205 nm), these data are identical to those of 7-methoxy coumarin (herniarin). The EI-Mass spectrum showed a molecular ion peak M +  at m/

Discussion
Fractionation of the constituents of pet. ether extract of Atriplex semibacata was carried out and the components of various fractions were identified by GLC. The unsaponifiable fraction was found to contain a series of hydrocarbons ranging from C 14 -C 28 , in which C 28 (32.94%) was the main component. Cholesterol and stigmasterol were represented by 7.21% and 4.9% respectively and the triterpenoids  and  -amyrin were represented by 11.23% and 2.72%. GLC analysis of fatty alcohols fraction revealed the presence of a mixture of six fatty alcohols in which dotriacontanol (C 32 H 66 O) represented the major alcohol (14.68%). The study of the coumarin fraction revealed the isolation and identification of scopoletin, umbelliferorne, coumarin, scopolin, 7-methoxy coumarin in addition to a phenolic acid P-coumaric acid. Also, the study of the flavonpidal compounds isolated from the n-butanol fraction of the plant revealed the isolation and identification of kaempferol 3-O glucoside and acacetin. Their identity were proved by m.p., TLC, PC, UV and MS analysis, in addition to acid hydrolysis of the glycoside compounds. This the first record of the coumarins in Atriplex semibacata. The results of antimicrobial activities Table 3 showed that the alcohol extract exhibited higher antimicrobial activity against G-ve bacteria, Pseudomonas aeruginosa and Proteus mirabilis,moderate antimicrobial activity against G+ve bacteria, Staphylococcus aureus, Bacillus subtilis. On the other hand the pet.ether extract showed a significant antimicrobial activity against G+ve bacteria, Staphylococcus aureus and fungi, Candida albicans, also it is clear from table (3) that G-ve bacteria was greatly inhibited by the chloroform extract. The results of the antitumor activity Table 4 of the different extracts of the plant showed no cytotoxic activity against Erlich-ascites carcinoma cells line at the tested concentrations (100 g/ml, 50 g/ml and 25 g/ml). Also, the radical scavenging effect of the tested extracts and isolated compounds using DPPH free radical were represented in Table 5. The chloroform, ethyl acetate and n-butanol extracts showed a strong antioxidant activity due to the presence of the main coumarin and flavonoidal compounds. Also, unsaponifiable fraction, posses a moderate antioxidant activity followed by fatty alcohol fraction compared to Trolox (standard antioxidant compound).

Conclusion
The significant antimicrobial and antifungi activities of the petroleum ether and chloroform extracts may be attributed to the presence of the coumarin compound and unsaponifiable fraction. The biological activities of different extracts were available, but the antimicrobial and antifungi activities of the different isolated compounds were not available due to their small quantities. Therefore, we will complete the research in a large scale to isolate enough quantities to measure the biological activity of the isolated compound in the near future in another research.