Investigation of lipoidal matter, antimicrobial and diuretic activities of leaves and fruits of Juniperus phoenicea L growing in Egypt.
GC/MS analysis of unsaponifiable matter of petroleum ether extracts
of the dried needles (leaves) and berries (fruits) of Juniperus
phoenicea L growing in Egypt resulted in the identification of 76 and 72
compounds constituting 99.52% and 99% of the total fractions
respectively. The major compound of leaf unsaponifiable matter was
[gamma]-cadinene representing 22.75%, while transpinocarveol was the
major compound of fruit unsaponifiable matter representing 20.49%. Fatty
acid contents of both extracts were also investigated resulting in the
identification of 12 and 15 fatty acids in the saponifiable matter of
leaves and fruits respectively. Antimicrobial activity and diuretic
action of the total and successive extracts of leaves and fruits were
Key words: Juniperus phoenicea, Cupressaceae, needles, berries, fatty acids, GC/MS analysis, antimicrobial, diuretic activity.
Fatty acids (Analysis)
Fatty acids (Investigations)
Salma, El-Sawi A.
Hemaia, Montawae M.
Mohamed, Sleem A.
Abdel-Rahman, El-Shabrawy E.
Amani, Sleem A.
Amal, Ali M.
Maii, Ismail A.
|Publication:||Name: Australian Journal of Medical Herbalism Publisher: National Herbalists Association of Australia Audience: Academic Format: Magazine/Journal Subject: Health Copyright: COPYRIGHT 2011 National Herbalists Association of Australia ISSN: 1033-8330|
|Issue:||Date: Winter, 2011 Source Volume: 23 Source Issue: 4|
|Topic:||Event Code: 980 Legal issues & crime Computer Subject: Company legal issue|
|Product:||Product Code: 2834570 Renal Diuretic Preparations; 2899200 Fatty Acids & Derivatives NAICS Code: 325412 Pharmaceutical Preparation Manufacturing; 325998 All Other Miscellaneous Chemical Product and Preparation Manufacturing SIC Code: 2834 Pharmaceutical preparations; 2899 Chemical preparations, not elsewhere classified|
The genus Juniperus belongs to family Cupressaceae which is a conifer family with worldwide distribution. It includes 27 to 30 genera with about 130-140 species (Silba 1986, Van Royen 1979). Juniperus phoenicea is the juniper growing in Egypt in Sinai near the Red Sea (Yelleg, Halal and Maghara mountains) in rocky ridges, Mediterranean region, extending to Central Arabia (Boulos 1999).
Whereas a number of papers have been devoted to GC/MS analysis of the essential oils from J. phoenicea leaves and fruits (Banthorpe 1973, Admas 1996, Rezzi 2001, Cosentino 2003, Marongiu 2004, Ibrahim 2005, El-Sawi 2007), there is very little known about the composition of lipoidal matter of the petroleum ether extracts of them with the exception of the GC/MS analysis of the petroleum ether extracts of needles and berries of J. phoenicea growing in Libya (Aboul-Ela 2005).
Several pharmacological activities of J. phoenicea have been investigated, including cytotoxic (El-Sawi 2007), antioxidant (Ibrahim 2005), anti-inflammatory (Comte 1995), hypoglycemic (Aboul-Ela 2005), and hepatoprotective as well as the antimicrobial activity of the essential oil (Ibrahim 2005).
This study of the unsaponifiable and saponifiable fractions of the petroleum ether extracts of leaves and fruits of J. phoenicea L. growing in Egypt was carried out to identify the sterols and terpenes, as well as fatty acid content using GC/MS analysis. Identification of the constituents was carried out by comparison of their recorded mass spectra and fragmentation patterns with those available in the literature together with the database libraries available at National Research Center, Cairo, Egypt, Wiley (Wiley Int.) USA, National Institute of Technology USA and/or published data (Adams 1995, Jennings 1981.
The present study investigated the antimicrobial activities of the total 80% ethanol extract as well as the diuretic activities of total 80% ethanol and successive extracts of leaves and fruits J. phoenicea growing in Egypt.
Fresh aerial parts of J. phoenicea L family Cupressaceae were collected from north Sinai, Magara Mountain, Egypt, and were kindly identified by Prof Dr Nabeel El- Hadidi, Botany Department, Faculty of Science, Cairo University. The plant was dried, ground and a voucher specimen was kept at Pharmacognosy Department, National Research Centre, Cairo, Egypt.
Preparation of total extracts
Five hundred grams of air dried powdered leaves and fruits of J. phoenicea were exhaustively extracted by reflux with 80% ethanol. The combined extract was evaporated under reduced pressure.
Preparation of successive extracts
Five hundred grams of air dried powdered leaves and fruits of J. phoenicea were exhaustively and successively extracted in Soxhlet apparatus using petroleum ether, chloroform, ethyl acetate and methanol. These extracts were evaporated to dryness under vacuum at 40[degrees]C. All extracts were kept for chemical and biological investigation.
Investigation of the petroleum ether extracts of leaves and fruits of J. phoenicea (Tsuda 1960)
Saponification of the petroleum ether extracts
Half a gram of the residue of the petroleum ether extracts of leaves and fruits off J. phoenicea was separately refluxed for 6 h with 100 mL of 0.5 N alcoholic KOH in a boiling water bath. The saponified extracts were concentrated to one third their volumes. The cooled reaction mixtures were diluted with equal volumes of distilled water and exhaustively extracted with ether (negative test for sterols).
The combined ethereal extracts for both extracts were washed several times with water until free of alkalinity and dehydrated over anhydrous sodium sulphate. After evaporation of ether to dryness the residues of both extracts were separately used in studying the unsaponifiable matter.
The alkaline aqueous solutions remaining after extraction of the unsaponifiable matter were acidified with hydrochloric acid to liberate the fatty acids which were extracted several times with ether. The combined ethereal extracts were washed several times with distilled water till free of acidity, filtered over anhydrous sodium sulphate and then the filtrates were evaporated to dryness. The residues of both extracts were separately kept for studying the fatty acid contents.
Preparation of fatty acid methyl esters
The residues of fatty acids of leaves and fruits of J. phoenicea obtained from both extracts were separately dissolved in 50 mL absolute methanol mixed with 0.25 mL sulphuric acid, refluxed for about three hours, cooled, diluted with about 100 mL distilled water then transferred to a separating funnel. The resulting fatty acid methyl esters of both extracts were separately extracted several times with ether. The combined ethereal extracts were washed several times with water until free of acidity and dehydrated over anhydrous sodium sulphate. The solvents were evaporated and the residues were subjected to GC/MS analysis (Finar 1967).
GC/MS for unsaponifiable matter and fatty acid methyl esters were performed using GC/MS Finnigan Mat SSQ 7000, Digital DEC EL, 70 eV.
Bioactivity study: antimicrobial activity
Test organisms used were obtained from the faculty of Agriculture, Cairo University.
A. Bacteria: Three gram positive bacterial strains
Staphylococcus aureus, Bacillus subitilis and Bacillus cereus were used.
B. Fungi: Aspergillus niger, Aspergillus flavus, Maccrophomina phasioli and Botrytis allii.
C. Yeast: Saccharomyces cereviseae and Candida pseudotropicalis.
Nutrient agar media (0.3 g/L beef extract, 5.0 g/L peptone, 3.0 g/L sodium chloride and 20 g/L agar). All the chemicals used in preparation of media were of the analytical grade, distilled water was used.
The antimicrobial activity was carried out using the inverted Petri plate method (Dubey 2005). The test organisms were grown on the nutrient agar media. Routine sterilisation was carried out by autoclaving for 20 mins at 121[degrees]C adjusting pH at 7. Presterilised filter paper discs (5 mm in diameter) were impregnated with specific weight of the test extract (100 ng/disc) and fixed to the bottom of inverted Petri plate previously inoculated with the test organisms. Diameter of inhibition zones were measured in mm. Results were compared with those of control drugs; ampicillin for bacteria and clotrimazole for fungi and yeasts.
Bioactivity study: diuretic activity
Adult albino rats of Sprauge Dawely Strain weighing 130-150 gm were obtained from the animal house colony of National Research Centre, Dokki, Egypt. They were kept under the same hygienic conditions and well balanced diet and water.
Diet consisted of vitamin mixture (1%), mineral mixture (4%), corn oil (10%), sucrose (20%), cellulose (0.2%), casein 95% (10.5%) and starch (54.3%).
Dosages of the drugs were calculated according to Paget and Berne (1964) and were administered orally by gastric tube.
In vivo study
Seventy two adult male albino rats were divided into twelve groups each of six animals and were held in the metabolic cages, fasted for 18 hours, given water only.
Group 1 received 1 mL saline and kept as normal control
Groups 2-11 received oral 100 mg/kg b/wt of total 80% ethanol extract, petroleum ether extract, chloroform extract, ethyl acetate extract, ethanol extract of needles and berries.
Group 12 received 5 mg/kg b/wt of the positive control, moduretic drug (amiloride HCl + hydrochlorothiazide). (Goldstein 1964).
The volume of urine was measured after 2, 4 and 24 hours. Serum urea and creatinine levels were assessed.
Results and discussion
Unsaponifiable matter investigation
Analysis of the unsaponifiable matter of the petroleum ether extracts of J. phoenicea leaves and fruits by GC/MS resulted in the identification of 76 and 72 compounds constituting 99.52% and 99% of the total fractions respectively. The major compound of the leaf unsaponifable matter was [gamma]-cadinene representing 22.75%, followed by trans-caryophyllene (14.6%) and [alpha]-muurolene (12.49%), while trans-pinocarveol was the major compound of the fruit unsaponifiable matter representing 20.49%, followed by [alpha]-terpineol (10.01%) and 2-ethyl hexanol (9.03%).
The results showed that the percentage of the non oxygenated compounds was more than the percentage of the oxygenated compounds in leaf unsaponifiable matter where they constituted 90.85% and 8.67% respectively. The major constituent of the non-oxygenated compounds was [gamma]-cadinene constituting 22.75%, the non-oxygenated compounds were divided into terpenoids (90.5%) and hydrocarbons (0.35%), while the major constituent of the oxygenated compounds was a hydroxylated compound phenyl-tert-butanol constituting 2.01%, followed by carotol (0.94%). The oxygenated compounds were divided into hydroxylated compounds constituting 7.28%, ethers (0.02%), esters (0.04%), ketones (0.22%), aldehydes (0.65%) and oxides (0.46%).
Conversley the percentage of the oxygenated compounds was more than the percentage of the non oxygenated compounds in fruit unsaponifiable matter where they constituted 72.08% and 26.92% respectively. The major constituent of the non oxygenated compounds was the terpenoid verbenene constituting 7.26% followed by p-mentha-1,3,8-triene (4.07%). The non oxygenated compounds were divided into a hydrocarbon constituting 0.42% and terpenoids (26.5%), while the major constituent of the oxygenated compounds was the hydroxylated compound trans-pinocarveol constituting 20.49%, the oxygenated compounds were divided into hydroxylated compounds constituting 53.56%, ethers (4.44%), esters (1.03%), ketones (3.49%), aldehydes (4.47%) and oxides (3.49%).
The results indicated that 21 compounds were present in both leaves and fruits but in different concentrations. Full tabled results are available in tables 1 and 2 from www.nhaa.org.au/Publications/AJMH/downloads.
Analysis of the saponifiable matter of the petroleum ether extracts of J. phoenicea leaves and fruits by GC/MS resulted in the identification of 12 and 15 fatty acids constituting 70.45% and 96.96% of the total fractions respectively. The major compound of the fatty acids in leaves was octadecanedioic acid dimethyl ester constituting 20.78% followed by 12-hydroxy- methyl dodecanoate (14.6) then 12,13-epoxy stearic acid methyl ester (13.7%), while the major compound of the fatty acids in fruits was methyl-16-acetyl hydroxyl palmitate constituting 26.16% followed by methyl palmitate (20.41%) then 12-hydroxy methyl dodecanoate (19.69%). The results showed that saturated and unsaturated fatty acids in the leaves were 63.3% and 7.15% respectively, while in fruits 95.57% and 1.39% respectively. These results revealed that leaves and fruits contained high percentages of saturated fatty acids, but leaves contained a higher percentage of unsaturated fatty acids than fruits. The results also indicated that 7 fatty acids were present in both leaves and fruits but in different concentrations. Full tabled results are available in table 3 from www. nhaa.org.au/Publications/AJMH/downloads.
To our knowledge this is the first study of the petroleum ether fraction to identify the sterols, terpenes and fatty acid contents by GC\MS technique in the Egyptian species. The compounds detected were found to be different from those detected from the Libyan species (Aboul-Ela 2005).
Reviewing current literature, nothing has been reported concerning the antimicrobial activity of different extracts of J. phoenicea leaves and fruits growing in Egypt. Here we tested the antimicrobial activity of the total 80% ethanol extracts of leaves and fruits against some bacterial strains, fungi and yeasts. Results are shown in table 4.
Total extracts of leaves and fruits showed notable activities against the tested bacteria. Total leaf extract showed higher activity against S. aureus and B. cerus while total fruit extract showed higher activity against B. subtilis. Both extracts showed higher activity against B. cereus relative to the standard antibiotic ampicillin.
Total leaf extract showed notable activity against M. phasioli and B. allii and was inactive against A. niger and A. flavus, while total fruit extract showed medium activity against M. phasioli relative to the standard antifungal drug clotrimazole, and was inactive against A niger, A. flavus and B. allii. Total leaf and fruit extracts showed notable activities against the tested yeasts relative to clotrimazole. Total leaf extract showed higher activity against S. scereviseae, while total fruit extract showed higher activity against C. pseudotropicalis.
The effects of total and successive extracts of leaves and fruits of J. phoenicea on the kidney of male albino rats were assessed by measuring the volume of urine as well as serum urea and creatinine levels in comparison to moduretic drug. Results are shown in Table 5.
Total alcoholic extracts of leaves and fruits showed diuretic effect as they increased the volume of urine at 2, 4 and 24 h time interval relative to control group and moduretic drug. Total leaf extract showed a slightly more diuretic effect (15.4 mL) than total fruit extract (15.1 mL) at 24 h. All successive extracts of leaves showed diuretic effect as they increased the volume of urine at 2, 4 and 24 hour time intervals relative to control group and to the moduretic drug. Ethanol extract showed the most diuretic effect (13.6 mL) followed by chloroform extract (12.4 mL), then petroleum ether (12.2 mL) and ethyl acetate (12.1 mL) at 24 h relative to control group (6.2 mL) and to the moduretic drug (16.7 mL). Similarly all successive extracts of fruit showed diuretic effect as they increased the volume of urine at 2, 4 and 24 h time interval relative to control group and to moduretic. Ethanol extract showed the most diuretic effect (13.1 mL) followed by chloroform extract (12.9 mL), then petroleum ether (12.8 mL) and ethyl acetate (12.7 mL) at 24 h relative to control group (6.2 mL) and to moduretic (16.7 mL).
Total alcoholic extracts of leaves and fruits more or less kept the urea levels, total leaf extract had the better effect than total fruit extract (19.4 and 17.2 mg/dL respectively) relative to control group (18.3 mg/dL) and to the moduretic drug. All successive extracts of leaves and fruits had conservative effect on urea levels relative to control group and to the moduretic.
All extracts of leaves and fruits kept the serum creatinine levels relative to control group and to the moduretic drug.
Chemical and biological investigation of J. phoenicea L growing in Egypt revealed that:
* The compounds detected in the petroleum ether extract unsaponifiable matter were largely similar to those detected in the essential oil of J. phoenicea growing in Egypt (Ibrahim 2005). Among these compounds were [alpha]-pinene, [gamma]- terpinene, trans-caryophyllene, [gamma]-cadinene, dehydroabietane, longifolene and aromadendrene.
* The total alcoholic extracts of leaves and fruits exhibited good antimicrobial activities against gram positive bacteria, yeasts and fungi. These results were in agreement with the effect exhibited by the leaf essential oil of the same plant (Ibrahim 2005) where the antimicrobial activity of the leaf essential oil of J. phoenicea inhibited the growth of those microorganisms at a dilution of 1: 50 with liquid paraffin (v\v).
* The total and successive extracts of leaves and fruit possessed high diuretic action with more or less constant serum urea and creatinine levels.
Juniper berries and extracts were found in some over the counter diuretic preparations. Juniper species were known in folk medicine to be used as a diuretic and urinary antiseptic for the treatment of infected urethra, bladder and ureters and for the treatment of kidney and bladder stones (Hoffmann 1990, Mills 1994). The volatile oil of juniper berries was used for the treatment of urinary tract inflammations (McGuffin 1997).
The antimicrobial and diuretic action of the plant may be due to the presence of some terpenoidal compounds. Its high concentration of volatile oils (especially terpeniol) act directly on the kidneys stimulating the flow of urine by raising the rate of glomerulus filtration; the process by which blood is purified and wastes filtered out. In acute nephritic illness, juniper returns normal secretory action to the renal epithelium and normalises blood pressure. While overdoses can irritate the kidneys, the risk of toxicity is low if the berry is used properly. Juniper possesses some antibiotic and disinfectant properties (Blumenthal 1998). Janku et al (1957) mention that the activity is most likely due to the action of terpinen-4-ol, which is known to increase renal glomerular filtration rate. Terpineol and terpenic alcohol also contribute to the diruretic properties of juniper oil. Isoborneol, borneol and cadinene demonstrated antiseptic properties, combined with the ability of juniper oil to stimulate kidney activity contributes to its use as a mild diuretic and as a hypotensive agent (Brandies 1996, Jean Valnet 1982, and Marcel 1990).
Thus the presence of [alpha]-pinene, [alpha]-terpinene, [gamma]-terpinene, terpinolene, [beta]-terpineol, a-terpineol (one the major compounds in fruit extract), 4-terpineol, isoborneol, [gamma]-cadinene (the major compound in leaf extract), [alpha]-cadinene, cadina-1, 4-diene and [sigma]-cadinene may contribute to the diuretic and antiseptic action of the leaf and fruit extracts of the Egyptian J. phoenicea.
Conversley results showed that the plant did not alter serum urea and creatinine levels. Urea is a waste product formed from the breakdown of proteins and healthy kidneys filter urea and other waste products from blood. A high blood level of urea (uremia) indicates that the kidneys may not be working properly or that the body is dehydrated. If blood urea levels show lower than normal, it could indicate liver disease, damage or malnutrition (Nicoll 2008). Creatinine is a break down product of creatine phosphate in muscle and is chiefly filtered out of the blood by the kidneys. If the filtering of the kidney is deficient, creatinine blood levels rise. A rise in blood creatinine level is observed only with marked damage to functioning nephrons (Delanghe 1989, Gross 2005).
From these results it may be concluded that the leaves and fruits of J. phoenicea L growing in Egypt possess antimicrobial activity and high diuretic action while maintaining renal function, urea and creatinine levels. This action suggests the use of the plant as a topical disinfectant, urinary tract antiseptic and as a safe diuretic antihypertensive drug.
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El-Sawi A Salma (1), Montawae M Hemaia (1), Sleem A Mohamed (2), El-Shabrawy E Abdel-Rahman (2), Sleem A Amani (3), Ali M Amal (4), Ismail A Maii (1) *
(1) Pharmacognosy Department, National Research Centre, Dokki, Cairo, Egypt
(2) Pharmacognosy Department, Faculty of Pharmacy, Cairo University
(3) Pharmacology Department, National Research Centre, Dokki, Cairo, Egypt
(4) Microbial Chemistry Department, National research Centre, Dokki, Cairo, Egypt
* Corresponding author email: firstname.lastname@example.org
Table 4: Antimicrobial activities of total alcoholic extracts of leaves and fruits of J. phoenicia Inhibition zone in mm [+ or -] SE Total leaf Total fruit Test organism extract extract Control * (100 [micro] (100 [micro] (100 [micro] \disc) \disc) \disc) Bacteria Staphylococcus aureus 13 [+ or -] 0.1 12 [+ or -] 0.2 22 [+ or -] 0.8 Bacillus subtilis 18 [+ or -] 0.3 20 [+ or -] 0.6 24 [+ or -] 0.5 Bacillus cerus 15 [+ or -] 0.3 14 [+ or -] 0.3 10 [+ or -] 0 Fungi Aspergillus niger 0 0 9 [+ or -] 0.3 Aspergillus flavus 0 0 20 [+ or -] 0.5 Maccrophomina phasioli 10 [+ or -] 0.4 12 [+ or -] 0.1 21 [+ or -] 0.7 Botrytis allii 18 [+ or -] 0.4 0 NT Yeast Saccharomyces cereviseae 13 [+ or -] 0.2 15 [+ or -] 0.4 11 [+ or -] 0.2 Candida pseudo- tropicalis 22 [+ or -] 0.5 18 [+ or -] 0.6 NT Control * : ampicillin for bacteria, clotrimazole for fungi and yeasts. NT: not tested Table 5: Effects of total and successive extracts of leaves and fruits of J. phoenicia on the kidney of male albino rats Group Inhibition zone in mm [+ or -] SE 2 hours 4 hours Control (1ml saline) 0.9 [+ or -] 0.01 1.8 [+ or -] 0.03 Total leaves extract 2.9 [+ or -] 0.3 * 5.9 [+ or -] 0.1 * Pet ether leaves ext 2.8 [+ or -] 0.3 * 5.2 [+ or -] 0.2 * Chloroform leaves ext 2.6 [+ or -] 0.1 * 5.1 [+ or -] 0.1 * Ethyl acetate leaves ext. 2.5 [+ or -] 0.1 * 4.8 [+ or -] 0.1 * Ethanol leaves ext. 2.4 [+ or -] 0.1 * 5.6 [+ or -] 0.2 * Total fruits ext. 3.1 [+ or -] 0.2 * 6.2 [+ or -] 0.3 * Pet. Ether fruits ext. 2.7 [+ or -] 0.2 * 4.9 [+ or -] 0.3 * Chloroform fruits ext. 2.3 [+ or -] 0.1 * 5.2 [+ or -] 0.4 * Ethyl acetate fruits ext. 2.1 [+ or -] 0.3 * 5.3 [+ or -] 0.2 * Ethanol fruits ext. 2.2 [+ or -] 0.2 * 5.8 [+ or -] 0.2 * Moduretic 3.5 [+ or -] 0.2 * 7.6 [+ or -] 0.5 * Group Inhibition zone Serum in mm [+ or -] SE urea 24 hours (mg/dL) Control (1ml saline) 6.2 [+ or -] 0.3 18.3 [+ or -] 0.4 Total leaves extract 15.4 [+ or -] 2.7 * 19.4 [+ or -] 0.2 Pet ether leaves ext 12.2 [+ or -] 0.2 * 16.4 [+ or -] 0.2 Chloroform leaves ext 12.4 [+ or -] 0.3 * 16.3 [+ or -] 0.4 Ethyl acetate leaves ext. 12.1 [+ or -] 0.2 * 16.5 [+ or -] 0.3 Ethanol leaves ext. 13.6 [+ or -] 0.2 * 15.9 [+ or -] 0.7 Total fruits ext. 15.1 [+ or -] 2.3 * 17.2 [+ or -] 0.3 Pet. Ether fruits ext. 12.8 [+ or -] 0.4 * 16.7 [+ or -] 0.6 Chloroform fruits ext. 12.9 [+ or -] 0.3 * 15.3 [+ or -] 0.3 Ethyl acetate fruits ext. 12.7 [+ or -] 0.5 * 17.1 [+ or -] 0.4 Ethanol fruits ext. 13.1 [+ or -] 0.1 * 16.8 [+ or -] 0.5 Moduretic 16.7 [+ or -] 0.8 * 19.1 [+ or -] 0.3 Group Serum creatinin (mg/dL) Control (1ml saline) 1.2 [+ or -] 0.03 Total leaves extract 1.2 [+ or -] 0.03 Pet ether leaves ext 1.4 [+ or -] 0.01 Chloroform leaves ext 1.2 [+ or -] 0.02 Ethyl acetate leaves ext. 1.3 [+ or -] 0.03 Ethanol leaves ext. 1.1 [+ or -] 0.01 Total fruits ext. 1.1 [+ or -] 0.02 Pet. Ether fruits ext. 1.4 [+ or -] 0.03 Chloroform fruits ext. 1.3 [+ or -] 0.01 Ethyl acetate fruits ext. 1.2 [+ or -] 0.01 Ethanol fruits ext. 1.1 [+ or -] 0.02 Moduretic 1.3 [+ or -] 0.02 * Significantly different from control group at p < 0.01. Values are means of 6 observations hydrochlorothiazide).
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