Hossain et al. Bioethanol fuel production from rotten banana as an environmental waste management and sustainable energy. Afr. J. Microbiol. Res. 2011

Hot and cold solvent and aqueous extract of leaves, barks, fruits and green husks of J. regia from different countries revealed broad spectrum antibacterial activity against gram-positive and gram-negative bacteria viz. Bacillus cereus, Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, Staphylococcus epidermidis,Micrococcus luteus, Salmonella typhimurium, Enterococcus faecalis, Bacillus thuringiensis, Protomonas extroquens, and Proteus sp. using agar streak method, disc diffusion method and microplate alalmar blue assay (Deshpande et al., 2011; Poyrazolu et al., 2010; Pereira et al., 2008; Oliveira et al., 2008; Pereira et al., 2007; Qa’dan et al., 2005a b; Citoglu and Altanlar, 2003; Upadhyay et al., 2010b). The antimicrobial activity against gram-negative bacteria were selective since not all the fruit extract of J. regia cultivator inhibited the growth of Pseudomonas aeruginosa and E. coli. cv. Lara inhibited the growth of K. pneumoniae (MIC of 100 mg/mL), cv. Mayette inhibited the development of P. aeruginosa and E. coli with minimum inhibitory concentrations (MICs) of 50 and 10 mg/mL, respectively, and cv. Mellanaise inhibited the growth of E. coli and K. pneumoniae at concentration of 100 mg/mL (Ali-Shtayah et al., 1999). Mexican aqueous bark and leaves extract exhibited no antimycobacterial activity. Only the hexane and methanol extract showed antimycobacterial activity with MIC of 100 and 125 mg/ml, respectively using Soxhlet extractor (Cruz-Vega et al., 2008). Over 45% of Iranian clinical isolates of Helicobacter pylori strain were inhibited by J. regia aqueous and equal mixture of methanol, diethyl ether and petroleum benzene extract (Nariman et al., 2004). In a recent study, juglone was shown to potently inhibit the three key enzymes from Helicobacter pylori, cystathionine γ-synthase (HpCGS), malonyl-CoAacyl carrier protein transacylase (HpFabD), and β-hydroxyacyl-ACP dehydratase (HpFabZ) with the half maximal inhibitory concentration (IC₅₀) values of 7.0±0.7, 20±1, and 30±4 μmol/L, respectively. Therefore, HpCGS, HpFabD, and HpFabZ are considered to be the potential targets of juglone (Kong et al., 2008). The antibacterial activity of Jordanian J. regia leaves extract to acne developing organism revealed that 12.5% S. epidermidis isolates were resistant to the leaf extract where as all Propionibacterium acnes isolates were sensitive even to 10% of the extract (Qa'dan et al., 2005b).

Antifungal activity

J. regia fruits, leaves and bark aqueous and solvents extract exhibited antifungal activity against wide range of fungi using disc diffusion method, agar dilution method, agar streak dilution and Raddish method. Pereira et al. (2008) reported that all the walnut varieties exhibited antifungal activity against Candida albicans and Cryptococcus neoformans when soxhleted with light petroleum ether (b.p. 40-60°C). The higher inhibition was observed with cv. Lara extract (MIC of 1 mg/mL). However, C. albicans and C. neoformans were only resistant to cv Mallanaise extract. Cold extraction of fruit, leaves and bark inhibited the growth of Microsporum canis, Trichophyton mentagrophytes, and Trichophyton violaceum (Ali-Shtayah et al., 1999). On the other hand, the aqueous extract of green husks showed no antifungal activity against C. albicans and C. neoformans (Oliveira et al., 2008). Methanol, acetone, chloroform and ethyl acetate bark extract revealed antifungal activity against A. niger, Alternaria alternata, Trihoderma viresn, fusarium solani, Pichia guiliermondii, Pichia jadinii and all Candida speices tested (Upadhyay et al., 2010c ; Ahmad et al., 1973).

Antiviral activity

Mei-zhi et al. (2007) reported that 95% ethanol and ethyl acetate leaves extract of J. regia, inhibited tobacco mosaic virus (TMV). The methanol extract of J. regia inhibited Sindbis virus at a minimum concentration of 1.5 µg/ml (Mouhajir et al., 2001).

Antioxidant activity

The antioxidant potential of ethyl acetate, butanol, meta-nol, ether and aqueous methanol extract of walnut kernels, husks and leaves were measured by different methods such as reducing power, scavenging activity on 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals and lipid oxidation inhibition by β-carotene linoleate system. All the extracts showed strong antioxidant activity (Qamar and Sultana, 2011; Carvalho et al., 2010; Abbasi et al., 2010; Rahimipanah et al., 2010; Zhang et al., 2009b; Almeida et al., 2008; Oliveira et al., 2008; Pereira et al., 2008; Pereira et al., 2007; Fukuda et al., 2003). Bullo et al. (2010) reported a decrease in the antioxidant burden observed in enzymatic and non-enzymatic antioxidant systems after the consumption of a whole-walnut or a walnut-skin diet in C57BL/6 mice. The same author also reported that consumption of walnuts and walnut skins have no deleterious effect on low-density lipoprotein (LDL) oxidizing capability, despite their higher contents of omega-6 PUFAs. Several phenolic compounds isolated from J. regia such as pyrogallol, p-hydroxybenzoic acid, vanillic acid, ethyl gallate, protocatechuic acid, gallic acid, 3,4,8,9,10-pentahydroxydibenzo pyran-6-one, tannins, glansrins, adenosine, adenine, etc, could provide a chemical basis for some of the health benefits claimed for J. regia in foods and folk medicine (Zhang et al., 2009a; Fukuda et al., 2003).

Antidiabetic activity

Fukuda et al. (2004) demonstrated a strong inhibitory activity of walnut polyphenols and the polyphenolic components like Casuarictin, tellimagradin II and Tellimagradin I on different enzymes like glycosidase, sucrose, maltase and amylase. In addition to the above findings, researchers also noticed that walnut polyphenol-rich fraction has triglyceride lowering effect and urine peroxide lowering effect in genetically inherited Type II diabetes mellitus (db/db) mice at the dose of 200mg/kg/day. The consumption of walnut leaf pellets in alloxan induced diabetic rats at the dose of 185 mg/kg reduced fasting blood sugar significantly and the histomorphometric study of pancreas showed a sign of regeneration of β-cells in the treated group (Jelodar et al., 2007). J. regia leaves methanolic extract at dose of 250 mg/kg decreases the postprandinal plasma blood glucose levels in both short and long term models. The plant extract significantly inhibited α-glucosidase activity in vitro for both maltase and sucrase enzymes and showed no changes in the insulin and glut-4 genes expression. The author attributed the inhibitory action of the plant extract to gallic acid and caffeoylquinic acid in the leaves (Teimori et al., 2010).

Anthelmintic activity

Kale et al. (2011) reported that stem park of J. regia acetone extract exhibited significant activity at all dilution tested when compared to the Albendazole standard against Eicinia feotida. The benzene, methanol and ethanol soxhlet extracts of J. regia stem bark on adult Indian earthworm, Pheretima posthuma exhibited significant anthelmintic activity as comparable to that of standard drug Piperazine citrate (Upadhyay et al., 2010a). The 95% ethanol, petroleum ether and ethyl acetate extract of green walnut hull have obvious anti-feeding effect on armyworm and the small vegetable-moth. The research group indicated that anti-feeding rate, death rates as well as growth inhibition rate of armyworm

have correspondingly changed in dose dependant manner (Me-zhi et al., 2006).

Anti-inflammatory activity

The ethanolic extracts of J. regia leaves exhibited potent anti-inflammatory activity as potent as indomethacin against carrageenan-induced hind paw edema model in mice without inducing any gastric damage (Erdemoglu et al., 2003). Mokhtari et al. (2008) stated that the alcohol extract of walnut leavesin dose of 1.5 mg/kg caused a significant nociception decrease in acute phase of formalin test where as the aqueous (2.87 and 1.64 g/kg) and ethanolic (2.044 and 1.17 g/kg) extracts of leaves showed antinociceptive activity in hotplate test suggesting a promising analgesic and anti-inflammatory agents against diseases such as rheumatoid arthritis. On the basis of Qamar and Sultana (2011) result, a protective role of methanolic J. regia extract against CSE-induced acute lung toxicity in Wistar rats was suggested. The extract significantly decreased the levels of Lactate dehydrogenase (LDH), total cell count, total protein and increased the glutathione (GSH) level in bronchoalveolar lavage fluid. It also significantly restored the levels of Glutathione reductase (GR), catalase and reduced the xanthine oxidase (XO) activity in lung tissue.

Antidepressant activity

The macerated hexane extract of J. regia fruit produced significant antidepressant activity at both doses of 100 and 150 mg/kg body weight when compared with standard drug fluoxetine on male Wistar rats. The antidepressant activity was evaluated by forced swimming and tail suspension test (Rath and Pradhan, 2009).

Antityrosinase activity

Ozer et al. (2007) suggested that gel formulation containing ellagic acid and plant leaves extract of J. regia is effective in treating uneven skin pigmentation. The ethanolic leaves extract could be suggested as new sources of skin-whitening agents. Aitani and Shimoda (2005) reported that melanin formation was inhibited at concentration 1 to 30 μg/ml in Pre-cultured B16 melanoma cells incubated with medium containing walnut polyphenols and their result indicated that walnut polyphenols is more superior to the popular skin-lightening agent, ascorbic acid and arbutin upon data comparison.

Hepatoprotective activity

Orally fed Walnut polyphenols prepared from the kernelpellicle demonstrated a dose dependent lowering effect in glutamyl oxaloacetic transaminase (GOT) and glutamyl pyruvic transaminase (GPT) in carbon tetrachloride (CCl₄) induced liver damage in mice model after a single oral administration (200 g/kg). Result indicated that walnut polyphenols is more superior to Curcumin, a commonly used hepatoprotective agent. The effect of each active component of in vitro evaluation of walnut polyphenols on CCl₄-induced cytotoxicity in primary cultured rat hepatocytes showed that tellimagrandin I, casuarictin, tellimagrandin II, and rugosin C (Figure 1) are inhibitory on CCl₄-induced cytotoxicity in primary cultured rat hepatocytes however, tellimagrandin I of walnut polyphenols is believed to be the most important active compound responsible for hepatoprotective effect (Hiroshi et al., 2008). The same author, Hiroshi et al. (2006) reported that 50% EtOH extract from endocarps of walnuts on mice liver injury models induced by carbon tetrachloride at the dose of 100 and 200 mg/kg significantly suppressed GOT and GPT deviations. Polyphenolic constituents, tellimagrandins I and II, rugosin C and casuarictin were found to be principal constituents with hepatoprotective activity against oxidative damage.

Hypotriglyceridemic activity

Oral administration of a polyphenol-rich extract (WP) from walnuts (100 and 200 mg/kg) in high fat diet fed mice significantly reduced liver weight and serum triglycerides (TG) where as hepatic β-oxidation in cytosol, including peroxisome, was enhanced by WP (50-200 mg/kg). A polyphenol-rich extract was found to possess hypotriglyceridemic activity via enhancement of peroxisomal fatty acid β-oxidation in the liver. These results suggest that tellimagrandin I is involved in the hypotriglyceridemic mechanism (Shimoda et al., 2009).

Anticancer activity

Juglone has been reported to inhibit intestinal carcinogenesis induced by azoxymethane in rats and might be a promising chemopreventive agent in human intestinal neoplasia (Sugie et al., 1998). Juglone was also proven to be a potent cytotoxic agent in vitro in human tumor cell lines, including human colon carcinoma (HCT-15) cells, human leukemia (HL-60) cells and doxorubicin-resistant human leukemia (HL-60R) cells (Kamei et al., 1998; Segura-Aguilaretal, 1992). In a recent study, Juglone inhibited the growth and induce apoptosis of sarcoma and 180 SGC-7901 cells in vivo. The mechanism is mediated by the activation of the mitochondrial death pathway, which requires the generation of reactive oxygen species (ROS), down-regulation of Bcl-2 protein expression and up-regulation of Bax protein expression (Ji et al., 2011). Walnut methanolic extracts obtained from J. regia seed, green husk and leaf showed concentration dependent growth inhibition against human renal cancer cell lines A-498, 769-P and the colon cancer cell line Caco-2. Concerning A-498 renal cancer cells, all extracts exhibited similar growth inhibition activity (IC₅₀ values between 0.226 and 0.291 mg/mL), while 769-P renal and Caco-2 colon cancer cells, walnut leaf extract showed a higher antiproliferative efficiency (IC₅₀ values of 0.352 and 0.229 mg/mL, respectively) than green husk or seed extracts (Carvalho et al., 2010). The tested dried fine powder of J. regia light petroleum seed extract in cancer induced in Swiss albino mice with the help of 7,12-Dimethylbenz(a)anthracene (DMBA) and croton oil showed the petroleum extract was significant in reducing the cancer cells (Kumudhavalli et al., 2010).

Other medicinal uses

Willis et al. (2009) examined the effects of walnut diet on motor and cognitive ability in aged rats for 8 weeks. The three treated groups (2, 6 and 9%) revealed that the 2% walnut diet improved performance on rod walking, while the 6% walnut diet improved performance on the medium plank walk; the higher dose of the 9% walnut diet impaired reference memory, however the researcher attributed this to the number of polyphenolic compounds that could be negatively effecting reference memory at a higher dose. A 2004 study by the NYS Institute for Basic Research in Developmental Disabilities (OMRDD) revealed that methanolic extract of walnut was able to inhibit and defibrillize fibrillar amyloid β- protein (the principal component of amyloid plaques in the brains of patients with Alzheimer's). It is proposed that polyphenolic compounds present in walnuts may be responsible for its anti-amyloidogenic activity (Chauhan et al., 2004). Similarly, it was found that two of its major components in walnuts, gallic and ellagic acid, act as "dual-inhibitors" of the enzyme acetylcholinesterase which, in association with amyloid inhibits protein aggregation, and inhibit the site of acetylcholinesterase responsible for the breakdown of acetylcholine. These results suggest that walnuts may reduce the risk or delay the onset of Alzheimer's disease by maintaining amyloid-protein in the soluble form and prevent the break down of acetylcholine (Society for Neuroscience, 2007).