Chemical and biological studies on the pigments of tomato (lycopersicon esculentum Mill.) and litchi (litchi Chinensis Sonn.) of Bangladesh

Abstract

Tomato (Lycopersicon esculentum Mill.) is one of the most important widely consumed vegetables of the Solanaceae family, of Bangladesh whereas Litchi (Litchi chinensis Sonn.) is one of the most beautiful as well as uncommon sub-tropical evergreen tree belonging to the Sapindaceae family and sub-family Nepheleae, native to Southeast Asia, is fast becoming popular throughout the world because of its attractive appearance and delicious taste.

Tomatoes and their products such as tomato pulp, ketchup, juice and sauce are important source of micronutrients; these contain certain minerals (notably potassium) and carboxylic acids, including ascorbic, citric, malic, fumaric and oxalic acids. All of these are essential for health because of their antioxidant and antimicrobial activities in plants as well as in animals and humans.

Among vegetables, Lycopersicon esculentum are rich sources of carotenoids – principally lycopene, followed by β-carotene, lutein, phytofluene, phytoene and tocophrols are also present. Lycopene the most abundant pigment (60-64 %) is mainly responsible for the characteristic deep-red colour of ripe tomato fruits and tomato products. Because of the presence of long-chain conjugated double bonds, lycopene has been reported to possess antioxidant and antimicrobial activity and is superior to lutein or β - carotene.

Numerous epidemiological studies have suggested that a large consumption of raw tomato and tomato based products may experience a decreased risk for developing certain cancers such as prostate cancer, lung and stomach cancer, and cardiovascular diseases. The antioxidant and antimicrobial capacity, together with provitaminic properties typical of other tomato carotenoids has drawn attention towards widely consumed fruits over years. The second most important carotenoid is β-carotene, which represents about 7% of the total carotenoid content.

Preliminary trials were carried out before to proceed for Soxhlet extraction these involve use of ethyl acetate (EtOAc) as solvent for extraction. Moreover different solvents i.e. ethyl acetate (100%), di-mixture (acetone: pet ether, 1:1, v/v) and tri-mixture (n-hexane: acetone: ethanol, 2:1:1, v/v/v) were employed. Ethyl acetate was the best solvent for extracting of trans- and cis-isomers of lycopene and β-carotene. The consumption of tomato would exert nutritional, biological and health benefits by virtue of their antioxidant, antimicrobial and anti-inflammatory activity. Attempts were taken into account to explain the scientific basis for the medicinal and nutritional benefits of these fruits. The phytochemical contents, antioxidant and antimicrobial activity were also assessed. The in-vitro antimicrobial activity of the test microorganism was performed by agar disc diffusion method by measuring the diameter of zone of inhibition in terms of millimeter with a calibrated scale.

The isolation, purification and identification of chemical structure of the isolated lycopene and β - carotene involve the use of thin layer chromatography (TLC), vacuum liquid chromatography (VLC), column chromatography and spectroscopic methods. The tomato extract described in this application is the ethyl acetate (100%), acetone: pet ether (1:1, v/v) and n-hexane: acetone: ethanol (2:1:1, v/v/v) extracts of ripe tomato fruits with lycopene content ranging from 17 to 29 mg/100 g. The lycopene content in tomato differs with the variety, geographic location, technique of cultivation, soil condition and degree of ripeness of tomato fruits.

The significant antimicrobial activity of active extracts was evaluated using bacterial cultures of Staphylococcus aureus (S. aureus), Pseudomonas aeruginosa (P. aeruginosa), Escherichia coli (E. coli), Streptococcus pneumonia (S. pneumonia), and fungal cultures of Aspergillus niger (A. niger), Aspergillus flavus (A. flavus), Candida albicans (C. albicans) was compared with the Ciprofloxacin and Ketoconazole as standard antimicrobes. Chloroform extract showed more antibacterial and antifungal activities and most of the extracts executed moderate to good antimicrobial activity against the tested micro-organisms.

The various extracts of tomato were active against the entire tested microorganisms for anti-bacterial activity with the range of Minimum Inhibitory Concentration (MIC) values for S. aureus (MIC: 17-33 μg /ml), E. coli (MIC: 16-32 μg /ml) S. pneumonia (MIC: 1733

μg /ml) and P.aeruginosa (MIC: 15-39 μg /ml) and for anti-fungal activity with the range of MIC values for A. niger (MIC: 17-34 μg/ml), A. flavus (18-37 μg/ml) and C. albicans (16-35 μg/ml).

Fruits and vegetables are known to contain a variety of different antioxidant compounds such as ascorbic acid, tocopherol, glutathione and carotenoids, which may all contribute to protection against oxidative damage. General phytochemical screening of the Litchi chinensis revealed the presence of alkaloids, steroids, phenolic compounds, tannins, saponins and fatty acids. These phenolic compounds belong to the class of anthocyanins, flavanol or flavonoids and are antioxidant compounds remains in the pericarp, seeds and flowers of litchi fruits. In general, phenolic acids and flavonoids exhibit powerful antioxidant activities that inhibit key enzymes in mitochondrial respiration, offer protection against coronary heart diseases, and have anti-inflammatory, antitumor and antimicrobial activities. That’s why Litchi chinensis has wide folk medicinal uses.

The extraction was carried out using dried powder of litchi fruits pericarp (LFP) with distilled methanol (MeOH) in a soxhlet apparatus. The MeOH extract was concentrated in vacuo. These concentrated mass was treated with pet ether. The pet ether fraction was examined by TLC (silica gel: pet ether, 90:10) showed the presence of five spots of which two were violet, two pink and one green. The presence of pink colour spot was thought to be an indication of the presence of either steroid or fatty acid material or both. However its Salkowski and Liberman-Burchard reaction gave positive results confirming the presence of steroids. Upon VLC and crystallization of the fraction T-7 and purified it to mini column chromatography and the light yellowish fraction TS-2 was left undisturbed at room temperature gave white sharp needle crystals. The crystals were dissolved in chloroform and transferred to a vial marked SS-I. Its TLC study showed a single spot with the R value of 0.38. After recrystallization isolated compound was characterized by UV, IR, f 1 H‐NMR, 13 C‐NMR, DEPT-135 spectrum and it was identified Stigmasterol. This is the first report so far of occurrence and details spectroscopic description of this compound from litchi fruit pericarp (LFP). The antibacterial activities of litchi fruit parts both pulp and waste (peel and seeds) extracted using two different solvents (ethanol and acetone), were evaluated against medically important bacteria i.e. E. coli, P. aeruginosa, S. aureus, and B. subtilis. The antibacterial activity of litchi pulp and waste were compared with Bacitracin as standard antibiotic. The results revealed that litchi waste ethanolic extract exerted the strongest inhibition against all tested bacterial strains compared to other extracts while pulp acetonic extract showed no effect against most of tested bacteria.

Extracted Stigmasterol was further used to determine its antimicrobial activity. The antimicrobial activity ranged from 12 mm to 23.5 mm for the Stigmasterol and 19.5 mm to 27.5 for Gentamicin against E. coli, P.aeruginosa, S. aureus and K. pneumoniae. In comparison with standard antibacterial compound Gentamicin (20 μg /mL) it was found that purified Stigmasterol (20 μg /mL) has antimicrobial activity almost equivalent to the standard.

The antioxidant activities were found in the Litchi Fruits Pericarp (LFP) approximately 15% of total fruits. Possibly these are due to the presence of phenolics, flavonoids and ascorbic acids. To achieve the antioxidant activity LFP was treated with preheated Charcoal to make them free from oily and colouring materials. This cleaned mixture contains powerful antioxidant compounds, such as ascorbic acid, phenolic acids and flavonoids which are capable of blocking the harmful effects of free radicals in the body. Antioxidant activities of phenolic and flavonoid compounds of LFP of litchi extracts were evaluated using Ferric (Fe 3+ ) reducing antioxidant power (FRAP) assay activity.