- Corresponding Author:
- S. Chanda
Phytochemical, Pharmacological and Microbiological Laboratory, Department of Biosciences, Saurashtra University, Rajkot-360 005, India
|Date of Submission||11 November 2008|
|Date of Revision||20 July 2009|
|Date of Acceptance||22 July 2009|
|Indian J Pharm Sci, 2009, 71 (4): 406-412|
Many plants used in Saurashtra folk medicine have been reported to exhibit high antibacterial and antioxidant activities. In the present study, some parts of five plants, Guazuma ulmifolia L., Manilkara zapota L., Melia azedarach L., Syzygium cumini L. and Wrightia tomentosa R.& S., were evaluated for their antibacterial activity, total phenol content, flavonoid content, 2,2-diphenyl-1-picrylhydrazyl free radical scavenging activity and phytochemical analysis, using successive extraction by cold percolation method with petroleum ether, ethyl acetate, methanol and water. In vitro antibacterial activity was evaluated against five bacterial strains viz. Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella typhimurium and Enterobacter aerogenes by agar well diffusion method. Among the plants screened, W. tomentosa leaf and fruit showed the best antibacterial activity. The Gram-positive bacteria were more susceptible than Gram-negative bacteria. Methanol extract of M. zapota showed the best 2,2-diphenyl-1-picrylhydrazyl free radical scavenging activity. Highest total phenol content was shown by M. zapota and S. cumini in methanol extract, while highest flavonoid content was shown by W. tomentosa stem in petroleum ether extract and ethyl acetate extract. In all the plants, cardiac glycosides and triterpenes were more as compared to other phytoconstituents.
Antibacterial activity, antioxidant activity, Guazuma ulmifolia L., Manilkara zapota L., Melia azedarach L.
The frequency of life-threatening infections caused by pathogenic microorganisms has increased worldwide and is becoming an important cause of morbidity and mortality in immuno compromised patients in developed countries . There is an urgent need to control antimicrobial resistance by improved antibiotic usage and reduction of hospital cross infection ; however the development of new antibiotics should be continued as they are of primary importance to maintain the effectiveness of antimicrobial treatment . In developing countries, the World Health Organization  estimates that about three quarters of the population relies on plant based preparations used in their traditional medicinal system and as the basic needs for human primary health care. Therefore, several medicinal plants have been evaluated for possible antimicrobial activity and to get remedy for a variety of ailments of microbial origin [5,6].
Oxidative stress is an important contributor to the pathophysiology of a variety of pathological conditions including cardiovascular dysfunction, atherosclerosis, inflammation, carcinogenesis, drug toxicity, reperfusion injury and neurodegenerative diseases . Plants (fruits, vegetables, medicinal herbs) contain a wide variety of free radical scavenging molecules, such as phenolic compounds, nitrogen compounds, vitamins, terpenoids and some other endogenous metabolites, that are rich in antioxidant activity [8-11]. Scavenging of free radicals by antioxidants could reduce the fibrosis process in the tissue . Antioxidants are considered as possible protective agents reducing oxidative damage to the human body . Antioxidants are naturally abundant in fruits and are able to neutralize free radicals donating an electron and converting them to harmless molecules .
Natural antioxidants have a wide range of biochemical activities, including inhibition of ROS generation, direct or indirect scavenging of free radicals, and alteration of intra cellular redox potential . Antioxidants provide protection to living organisms from damage caused by uncontrolled production of reactive oxygen species and the concomitant lipid peroxidation, protein damage and DNA strand breakage . An antioxidant, which can quench reactive free radicals, can prevent the oxidation of other molecules and may, therefore, have health promoting effects in the prevention of degenerative diseases . In addition, it has been reported that there is an inverse relationship between dietary intake of antioxidant rich food and the incidence of human diseases .
Hence, in this article, antibacterial and antioxidant activities of the extracts obtained from five plants from their different parts (leaf, stem and fruit) that have been traditionally used as general health supplements are examined. The plants studied were Guazuma ulmifolia L. (Sterculiaceae), Manilkara zapota L. (Sapotaceae), Melia azedarach L. (Meliaceae), Syzygium cumini L. (Myrtaceae) and Wrightia tomentosa R. & S. (Apocynaceae). The aim of this current work was to evaluate the antibacterial and antioxidant potential of extracts in different solvents of all these plants, as well as their total phenol and flavonoid contents.
Materials and Methods
Collection of plant material
The plants were collected from Rajkot, Gujarat, India in September 2007 and identified at the Department of Biosciences, Saurashtra University, Rajkot, Gujarat, India. The ethnobotanical information of the screened plants is given in Table 1 . The plant parts were thoroughly washed with tap water, air dried, homogenized to fine powder and stored in air tight bottles.
|Plant Species||Vernacular Name||Family||Parts used||Therapeutic uses|
|GuazumaulmifoliaL.||Rudrakshi||Sterculiaceae||Leaf||Bark tonic used as demulcent and elephantiasis.|
|ManilkarazapotaL.||Chiku||Sapotaceae||Leaf||Bark is an antibiotic, astringent and febrifuge.|
|Chicle from bark is used in dental surgery.|
|MeliaazedarachL.||BakanLimdo||Meliaceae||Leaf,||The leaves are useful in hysteria, leprosy, cough,|
|Stem||bronchitis and scabies.|
|SyzygiumcuminiL.||Jambu||Myrtaceae||Leaf||Bark, leaf, fruit used in diabetes, leucorrhoea,|
|stomachalgia and gastropathy.|
|Wrightiatomentosa||Dudhlo||Apocynaceae||Leaf,||Root, bark, seed, used in piles, fever colic,|
|R. & S.||Stem,||diarrhea and round worm|
Table 1: The Ethnobotanical Information of the Screened Plants.
The dried powder of plants was extracted successively in petroleum ether, ethyl acetate, methanol and distilled water by cold percolation method . Ten grams of dried powder was taken in 100 ml of solvent in a conical flask, plugged with cotton wool and then kept on a rotary shaker at 190–220 rpm for 24 h. After 24 h, the extract was filtered with eight layers of muslin cloth; the residue was dried and used for extraction in another solvent while the filtrate was centrifuged at 5000 g for 10 min, the supernatant was collected and the solvent was evaporated. The dried extract of each solvent, was stored at 4° in airtight bottles. The extraction was done at least three times and the mean values are presented.
The bacterial strains used are identified strains and were obtained from National Chemical Laboratory, Pune, India. Two Gram-positive bacteria, Bacillus subtilis ATCC6633 and Staphylococcus aureus ATCC29737, and three Gram-negative bacteria, Pseudomonas aeruginosa ATCC27853, Salmonella typhimurium ATCC23564 and Enterobacter aerogenes ATCC13048 were studied.
A loop full of the strain was inoculated in 25 ml of nutrient broth in a conical flask and incubated at room temperature on a rotary shaker for 24 h to activate the test bacteria. The inoculum size was 1×108 cells. Muller Hinton agar No. 2 (Himedia) was used for the antibacterial susceptibility study. The bacterial assay was performed by agar well diffusion method [21,22]. The media and the test bacterial cultures were poured into Petri dishes (Hi-Media). The test strain (200 μl) was inoculated into the media when the temperature reached 40-42°. Care was taken to ensure proper homogenization. After the media was solidified; a well was made in the plates with the help of a cup-borer (8.5 mm). The well was filled with 100 μl of the extract (500 μg/well) and the plates were incubated over night at 37°. The bacterial growth was determined by measuring the diameter of the zone of inhibition. The experiment was done three times and the mean values are presented. For each bacterial strain, controls were maintained where pure solvent (DMSO) was used instead of the extract.
The preliminary phytochemical screening of different extracts was done to ascertain the presence of bioactive components. The presence of alkaloids (dragendroff, Mayer, Wagner), flavonoids, tannins, phlobatannins, triterpenes, steroids, saponins and cardiac glycosides was determined [23,24].
Total phenol determination
Total phenolic content of the extracts was determined by Folin Ciocalteu reagent method  with some modifications. Plant extract (1 ml) was mixed with Folin Ciocalteu reagent (0.1 ml, 1 N), and allowed to stand for 15 min. Then 5 ml of saturated Na2CO3 was added. The mixtures were allowed to stand for 30 min at room temperature and the total phenols were determined spectrophotometrically at 760 nm. The calibration curve was prepared by preparing gallic acid (10-100 μg/ml) solution in distilled water. Total phenol values are expressed in terms of gallic acid equivalent (mg/g of extracted compound).
Aluminum chloride colorimetric method  with some modifications was used to determine flavonoid content. Plant extract (1 ml) in methanol was mixed with 1 ml of methanol, 0.5 ml aluminum chloride (1.2%) and 0.5 ml potassium acetate (120 mM). The mixture was allowed to stand for 30 min at room temperature; the absorbance of the reaction mixture was measured at 415 nm. The calibration curve was prepared by preparing quercetin (5-60 μg/ml) solution in methanol. Flavonoid content is expressed in terms of quercetin equivalent (mg/g of extracted compound).
2,2-Diphenyl-1-picrylhydrazyl (DPPH) free radicals scavenging activity
The free radical scavenging activity of the extracts of various plant species was measured using the modified method of McCune and Johns . The extracts were dissolved in methanol. Various concentrations of the extracts (100-1000 μg) were added to DPPH solution in methanol (0.1 mM). The mixture was shaken vigorously and allowed to stand for 10 min at room temperature. The absorbance of the resulting solution was measured at 517 nm. The radical scavenger activity is expressed in terms of the amount of antioxidants necessary to decrease the initial DPPH absorbance by 50% (IC50).
Results and Discussion
The extractive yield of different plant parts is given in Table 2. The extractive yield varied among the different plants and also among the solvents used. Petroleum ether and ethyl acetate extract showed less extractive yield as compared to aqueous and methanol extracts. Among the plants screened maximum yield was in methanolic extract of G. ulmifolia (11.28%) and minimum extractive yield was in petroleum ether extract of S. cumini (0.36%).
|Botanical Name||Parts used||Extracts||% Yield (w/w)|
|G. ulmifolia||Leaf||Petroleum ether||0.64|
|M. zapota||Leaf||Petroleum ether||4.21|
|M. azedarach||Leaf||Petroleum ether||0.79|
|M. azedarach||Stem||Petroleum ether||0.48|
|S. cumini||Leaf||Petroleum ether||0.36|
|W. tomentosa||Leaf||Petroleum ether||0.69|
|W. tomentosa||Stem||Petroleum ether||1.33|
|W. tomentosa||Fruit||Petroleum ether||3.32|
Table 2: Extractive Yield (%) of Different Plants in Different Solvents with Increasing Polarity.
The crude powder of all the eight plant parts were subjected to preliminary phytochemical analysis and the results are shown in Table 3. All the eight plant parts showed the presence of triterpenes and cardiac glycosides in higher amount while other phytoconstituents were present in trace amount or absent. It is possible that these secondary metabolites might be responsible for the bioactivity of the plant extracts .
|Plant name||Parts used||Alk||Fla||Tan||Phl||Tri||Ste||Sap||Car|
-: No presence; +: Less presence; ++: Moderate presence; +++: High presence; Alk: Alkaloids; Dra: Dragendroff; May: Mayer; Wag: Wagner; Fla: Flavonoids; Tan: Tannins; Phl: Phlobatannins; Tri: Triterpenes; Ste: Steroids; Sap: Saponins; Car: Cardiac glycosides
Table 3: Preliminary Phytochemical Analyses of Medicinal plants Screened.
The antibacterial activity of eight parts of five plants belonging to five different families was assayed in vitro by agar well diffusion method against five different bacterial strains. The parts used for the study were leaf, stem and fruit, while the solvents used were petroleum ether, ethyl acetate, methanol and water. Therefore, in all, 32 extracts were evaluated for antibacterial activity as shown in Table 4. Aqueous extract did not show any antibacterial activity against almost all the bacteria studied. Similar results have been reported in the literature [29,30]. Among the plants screened, W. tomentosa leaf and fruit showed best antibacterial activity. Petroleum ether extracts of almost all plants showed antibacterial activity against both Gram-positive bacteria while they were inactive against Gram-negative bacteria. Highest antibacterial activity was shown by ethyl acetate extract of W. tomentosa leaf against S. aureus and by petroleum extract of S. cumini against B. subtilis. The most susceptible bacterium was S. aureus, while the most resistant bacteria were S. typhimurium and E. aerogenes.
|Plant Name (Used Parts)||Extracts||Bacteria|
BS = Bacillus subtilis; SA = Staphylococcus aureus; PA = Pseudomonas aeruginosa; ST = Salmonella typhimurium; EA = Enterobacter aerogenes; PE = Petroleum ether; EA = Ethyl acetate; ME = Methanol; AQ = Aqueous; - means no activity; Values are Mean ± SEM, n=3
Table 4: Antimicrobial Activities of Medicinal Plants Screened
From the above results, it can be concluded that Gram-positive bacteria are susceptible to plant extracts more as compared to Gram-negative bacteria. Various workers have already reported similar results [31-33]. The difference in sensitivity might be ascribed to the difference in morphological constitutions between Gram-positive and Gram-negative organisms. Many plant species present inhibition zones of differing diameters; however, size difference of the inhibition zone depends primarily upon many factors for e.g. diffusion capacity of substances (present in the extracts) in the agar medium, antimicrobial activity of diffused substances, growth and metabolic activity of microorganisms in the medium. Inhibition zone diameter can further be associated with polarities of substances which make up the tested extracts and also with cell wall composition of test organisms since Gram-positive bacteria present cell walls with lower lipid levels than do Gram-negative bacteria .
Plant phenolics constitute one of the major groups of compounds acting as primary antioxidant free radical terminators. These compounds possess a wide spectrum of chemical and biological activities including radical scavenging properties. Several studies have described the antioxidant properties of medicinal plants, foods and beverages which are rich in phenolic compounds [35-37].
The total phenol content of eight plant parts is shown in Table 5. Amongst the four extracts, methanol extracts showed higher amount of phenolic content followed by aqueous extracts, while petroleum ether extracts showed trace amount of phenolic content. Highest amount of total phenol content was in methanol extracts of M. zapota (194.06) and S. cumini (179.89). The flavonoid content of eight plant parts is shown in Table 6. Amongst the four extracts, petroleum ether extracts showed higher amount of flavonoid content, while aqueous extracts showed trace amount of flavonoid content.
|Plant Name||Parts Used||Extracts|
|Petroleum ether||Ethyl acetate||Methanol||Aqueous|
Values are Mean ± SEM, n=3
Table 5: Total Phenol Content (mg/g) of Medicinal Plants Screened.
|Plant Name||Parts Used||Extracts|
|Petroleum ether||Ethyl acetate||Methanol||Aqueous|
Values are Mean ± SEM, n=3
Table 6: Flavonoid Content (mg/g) of Medicinal Plants Screened.
Various assays are used to test for antioxidant activity but the mostly widely used methods are those that involve generation of free radical species which are then neutralized by antioxidant compounds [38,39]. DPPH radical is commonly used as a substrate to evaluate antioxidant activity. In the present work, eight parts of five plant species, in various solvents were evaluated for their free radical scavenging activity. Out of 32 extracts investigated, 19 extracts showed IC50 value more than 1000 μg/ml, while the remaining 13 plant extracts showed a varied level of DPPH scavenging activity. IC50 values ranged from 24.5 μg/ml to 800 μg/ml (Table 7). Ascorbic acid was used as standard and its IC50 value was 11.4 μg/ml. The lowest IC50 value was of leaf of M. zapota in methanol extract (IC50 = 24.5 μg/ml) and the highest IC50 value was of leaf of G. ulmifolia (IC50 = 655 μg/ml). Amongst all the four extracts, methanol extract showed better antioxidant activity.
|Plant Name||Parts Used||IC50 Value in µg/ml|
|Petroleum ether||Ethyl acetate||Methanol||Aqueous|
Table 7: Dpph Free Radical Scavenging Activity in Various Solvent Extracts of Different Plants.
Natural extracts with proven antioxidant activity are usually composed with their phenolic moiety, for example flavonoids, coumarins and tocopherols. Organic acids, carotenoids and tannins can also be present and act as antioxidants or have a synergistic effect with phenolic compounds . However in present work, methanol extract of M. zapota showed promising result in total phenol content and antioxidant activity, while in other plant extracts there was weak correlation between phenolic content and antioxidant potency. This suggests that non-phenolic compounds may also be responsible for the observed antioxidant activity as also suggested by Yam et al .
In the present study, antimicrobial and antioxidant activity of eight plant parts were screened. Five bacterial strains were used for the antimicrobial activity. Amongst all the plants screened, W. tomentosa leaf and fruit showed best antibacterial activity; while M. zapota showed best DPPH free radical scavenging activity in methanol extract. The Gram-positive bacteria were more susceptible than Gram-negative bacteria. Amongst the four extracts used, methanol extracts followed by ethyl acetate extracts showed better antimicrobial activity. Highest total phenol content was shown by M. zapota and S. cumini in methanol extract, while highest flavonoid content was shown by W. tomentosa stem in petroleum ether extract and ethyl acetate extract. In all the plants, cardiac glycosides and triterpenes were more as compared to other phytoconstituents.
Based on these results, it can be concluded that plant extracts have great potential as antimicrobial compounds against microorganisms and they can be used in the treatment of infectious diseases caused by resistant microorganisms. They can also be a source of natural antioxidants. Due to their antibacterial and antioxidant activities W. tomentosa and M. zapota extracts have promising potential as a source of natural antioxidant and antimicrobial agents. Such screening of various natural organic compounds and identification of active agents is the need of the hour because successful prediction of lead molecule and drug-like properties at the onset of drug discovery will pay off later in drug development.
- Al-Bari MAA, Sayeed MA, Rahman MS. Characterization and antimicrobial activities of a phenolic acid derivative produced by Streptomyces bangladeshiensis: A novel species collected in Bangladesh. Res J Med Sci 2006;1:77-81.
- French GL. Clinical impact and relevance of antibiotic resistance. Adv Drug Deliv Rev 2005;57:1514-27.
- van der Waaij D, Nord CE. Development and persistence of multi-resistance to antibiotics in bacteria: An analysis and a new approach to this urgent problem. Int J Antimicrob Agents 2000;16:191-7.
- World Health Organization. Traditional medicine-growing needs and potential. WHO policy perspectives on medicine, No. 2. WHO/ EBM/2002. Geneva: WHO; 2002.
- KameshwaraRao C. Database of medicinal plants. Government of Karnataka, India 2000. p. 1-23.
- Subramani SP, Goraya GS. Some folklore medicinal plants of Kolli hills: Record of a WattivaidyasSammelan. J Econ Taxon Bot 2003;27:665-78.
- Aruoma OI. Free, radicals, oxidative trace and antioxidants in human health and diseases. J Am Oil Chemist’s Soc 1998;75:199-212.
- Cotelle N, Bernier JL, Catteau JP, Pommery J, Wallet JC, Gaydou EM. Antioxidant properties of hydroxyl flavones. Free RadiatBiol Med 1996;20:35-43.
- Velioglu YS, Mazza G, Gao L, Oomah BD. Antioxidant activity and total phenolics in selected fruits, vegetables and grain products. J AgricFood Chem 1998;46:4113-7.
- Zheng W, Wang SY. Antioxidant activity and phenolic compounds in selected herbs. J Agric Food Chem 2001;49:5165-70.
- Cai YZ, Sun M, Corke H. Antioxidant activity of betalains from plants of the Amaranthaceae. J Agric Food Chem 2003;51:2288-94.
- Thresiamma KC, Kuttan R. Inhibition of liver fibrosis by ellagic acid. Indian J PhysiolPharmacol 1996;40:363-6.
- Yam MF, Basir R, Asmawi MZ, Rosidah Ahmad M, Akowuah GA. Antioxidant and hepatoprotective activities of Elephantopustomentosusethanol extract. PharmaBiol 2008;46:199-206.
- Leonard SS, Cutler D, Ding M, Vallyathan V, Castranova V, Shi X. Antioxidant properties of fruit and vegetable juices: More to the story than ascorbic acid. Ann Clin Lab Sci 2002;32:193-200.
- Abdollahi M, Larijani B, Rahimi R, Salari P. Role of oxidative stress in osteoporosis. Therapy 2005;2:787-96.
- Ghosal S, Tripathi VK, Chauhan S. Active constituents of Emblicaofficinalis: Part 1: The chemistry and antioxidant effects of two newhydrolysable tannins, emblicanin A and B. Indian J Chem 1996;35:941-8.
- Shahidi F. Natural antioxidants: Chemistry, health effects and applications, Urbana, IL: AOCS Press; 1997.
- Rich-Evan CA, Sampson J, Bramely PM, Hollwa DE. Why we do except carotenoids to be antioxidants in vivo. Free Rad Res1997;26:381-98.
- Anjaria J, Parabia M, Dwivedi S. Ethnovet heritage Indian ethnoveterinary medicine: An overview, Ahmadabad, India: Pathik Enterprise; 2002.
- Vaghasiya Y, Chanda SV. Screening of methanol and acetone extracts of fourteen Indian medicinal plants for antimicrobial activity. Turk J Biol 2007;31:243-8.
- Perez C, Paul M, Bazerque P. An antibiotic assay by the agar well diffusion method. Acta Bio Med Exp 1990;15:113-5.
- Parekh J, Chanda S. Screening of aqueous and alcoholic extracts of some Indian medicinal plants for antibacterial activity. Indian J Pharm Sci 2006;68:835-8.
- Harborne JB. Phytochemical methods, London: Chapman and Hall Ltd; 1973. p. 49-188.
- Parekh J, Chanda S. Antibacterial and phytochemical studies on twelve species of Indian medicinal plants. Afr J Biomed Res 2007;10:175-81.
- Mc Donald S, Prenzler PD, Autolovich M, Robards K. Phenolic content and antioxidant activity of olive extracts. Food Chem 2001;73:73-84.
- Chang C, Yang M, Wen H, Chern J. Estimation of total flavonoid content in Propolis by two complementary colorimetric methods. J Food Drug Anal 2002;10:178-82.
- McCune LM, Johns T. Antioxidant activity in medicinal plants associated with the symptoms of diabetes mellitus used by the Indigenous people of the North American boreal forest. J Ethnopharmacol 2002;82:197-205.
- Nino J, Narvaez DM, Mosquera OM, Correa YM. Antibacterial, antifungal and cytotoxic activities of eight Asteraceae and two Rubiaceae plants from Colombian biodiversity. Braz J Microbiol2006;37:566-70.
- Idu M, Oronsaye FE, Igeleke CL, Omonigho SE, Omogbeme OE, Ayinde BA. Preliminary investigation on the phytochemistry and antimicrobial activity of SennaalataL. leaves. J ApplSci 2006;6:2481- 5.
- Owolabi OJ, Omogbai EKI, Obasuyi O. Antifungal and antibacterial activities of the ethanolic and aqueous extract of Kigeliaafricana (Bignoniaceae) stem bark.Afr J Biotechnol 2007;6:1677-80.
- Lin J, Opoku AR, Geheeb-Keller M, Hutchings AD, Terblanche SE, Jager AK, et al. Preliminary screening of some traditional Zulu medicinal plants for anti-inflammatory and antimicrobial activities. J Ethnopharmacol 1999;68:267-74.
- Moshi MJ, Mbwambo ZH. Some pharmacological properties of extracts of Terminaliasericea roots. J Ethnopharmacol 2005;97:43-7.
- Parekh J, Chanda S. In vitro antimicrobial activities of extract of LaunaeaprocumbensRoxb. (Labiateae),Vitisvinifera(Vitaceae) and Cyperusrotundus(Cyperaceae).Afr J Biomed Res 2006b;9:89-93.
- Bandeira MF, Teixeira MF, Abinader CD, Parente RC, Lima PS. Avaliaçãoin vitro da sensibilidade da Candida albicansaohidróxido de cálcioassociadoaoóleo de copaíba. Rev Dentística 2006;6:12-22.
- Brown JE, Rice-Evans CA. Luteolin rich artichoke extract protects low-density lipoprotein from oxidation in vitro. Free Radiat Res1998;29:247-55.
- Krings U, Berger RG. Antioxidant activity of some roasted foods. Food Chem 2001;72:223-29.
- Wang H, Gao XT, Guo CZ, Cai L, Yao WB. In vitro and in vivo antioxidant activity of aqueous extract from Choerospondias axillariesfruit. Food Chem 2008;106:888-95.
- Arnao MB, Cano A, Acosta M. The hydrophilic and lipophilic contribution to total antioxidant activity. Food Chem 2001;73:239-44.
- Masoko A, Eloff JN. Screening of twenty four South African Combretum and six Terminalia species (Combretaceae) for antioxidant activities.Afr J Trad CAM 2007;4:231-9.
- Dapkevicius A, Venskutonis R, Beek TA, Linssen PH. Antioxidant activity of extracts obtained by different isolation procedure from some aromatic herbs grown in Lithuania. J Sci Food Agric 1998;77:140-6.