Histochemical evaluation of catechins in PEG stressed transgenic tea plants using catechin-specific-diazotized sulfanilamide reagent.

We investigated the applicability of catechin-specific-reagent (CSR) for histochemical evaluation of catechins. The diazotized arylamine moiety in CSR reacts specifically with the A-ring of catechins to yield a golden yellow complex. This makes it highly specific for spectrophotometric quantification of catechins. Therefore, microtome cut sections of untransformed and osmotin-expressing transgenic leaves and stem of tea were stained with CSR. We found catechins in the form of golden yellow globules. The catechin globules increased in the structurally intact and highly turgid cells of osmotin expressing transgenic tea plants after stress treatment with 20% PEG; by contrast, the cells in non-transgenic plants accumulated fewer catechin globules. Spectrophotometric quantification of catechins also confirmed higher levels in transgenics compared to untransformed plants. We found elevated accumulation of catechins in stress tolerant cells of tea leaves.

Determination of antibacterial and antioxidant potential of some medicinal plants from saurashtra region, India.

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.

Assessment of anti-inflammatory and hepatoprotective potency of Polyalthia longifolia var. pendula leaf in Wistar albino rats.

The objective of the present study was to screen various solvent extracts of Polyalthia longifolia var. pendula (Annonaceae) leaf for anti-inflammatory activity and to evaluate the anti-inflammatory and hepatoprotective potency of the potent solvent extract. Successive extraction was performed with six different solvents, viz. petroleum ether, hexane, toluene, chloroform, acetone and methanol. Toluene, chloroform, acetone and methanol were used in acute inflammatory studies; the results revealed methanol as most potent extract. Hence three concentrations of methanolic extract (300, 600, 900 mg/kg) were used to evaluate its potential as an anti-inflammatory and hepatoprotective agent. Diclofenac sodium was used as the toxicant in hepatoprotective studies, in which various serum biochemical parameters and liver glycogen were assessed. The three concentrations of methanol showed anti-inflammatory activity comparable to that of the control (Diclofenac sodium). All the serum biochemical parameters studied revealed the hepatoprotective nature of the methanol extract, but a concentration effect was not observed. The results indicated that the methanolic extract of Polyalthia longifolia var. pendula leaf possesses significant anti-inflammatory and hepatoprotective activity. However, the appropriate effective concentration needs to be determined.

Effect of Hippophae rhamnoides leaf extract against Dengue virus infection in human blood-derived macrophages.

Dengue virus occurs as four distinct serotypes, called Dengue 1, 2, 3, and 4. Symptomatic dengue virus infection ranges from a self limited febrile illness, dengue fever (DF), to a more severe disease, dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). The anti-Dengue treatment is severely hampered as no specific therapeutic agents are available. Even present treatment strategies for Dengue are more supportive than curative. In the present study anti-dengue activity of Hippophae rhamnoides (Seabuckthorn, SBT) leaf extract was evaluated in Dengue virus type-2 infected blood-derived human macrophages as macrophages are the primary target of Dengue virus infection. Infected cells were treated with SBT leaf extract and compared with commercially available anti-viral drug, Ribavirin. The extract was able to maintain the cell viability of Dengue-infected cells at par with Ribavirin along with the decrease and increase in TNF-alpha and IFN-gamma respectively. Anti-dengue activity of SBT extract was further determined by the traditional plaque assay. These observations suggest that the SBT leaf extract has a significant anti-dengue activity and has the potential for the treatment of Dengue.

Antibacterial and preliminary phytochemical and physico-chemical analysis of Eucalyptus citriodora Hk leaf.

The present communication deals with some studies on the antibacterial, physico-chemical and phytochemical parameters of different extracts of Eucalyptus citriodora leaf. The antibacterial study was performed using the agar ditch method on some clinically important bacteria, namely Pseudomonas pseudoalcaligenes, Proteus vulgaris, Citrobacter freundii, Staphylococcus subflava, Bacillus megaterium, and Enterobacter aerogenes. Physico-chemical parameters namely water, methanol, 1,4-dioxane, DMF, acetone soluble extractives, total ash, melting point, and pH were determined according to pharmacopoeial procedures. Methanol gave the maximum extract while it was minimum in water. Phytochemical parameters were screened for alkaloids, tannins, cardiac glycosides, saponins, steroids and flavonoids. Tannins and flavonoids gave positive results, while steroids and glycosides were absent. The most susceptible bacteria was C. freundii, while the most resistant was P. vulgaris.

Effect of Seabuckthorn (Hippophae rhamnoides) flavone on immune system: an in-vitro approach.

There are several reports, which suggest that the consumption of foods rich in flavonoids is associated with a lower incidence of certain degenerative diseases, including cardiovascular disease. Flavones, of Seabuckthorn (SBT) (Hippophae rhamnoides L.) fruit berry can modulate the production and level of several signaling molecules associated with immune function and inflammation in vitro, including several cytokines. We have evaluated the immunomodulatory activity of ethanolic solution of SBT flavone (FLV) in human peripheral blood mononuclear cells (PBMCs). The SBT flavone was found to stimulate production of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) in PBMCs. However, increased expressions of p-IkappaB, NF-kappaB, and p-p38 were found in flavone-treated human PBMCs with significantly suppressed expression of CD25 (IL-2R). There was no alteration found in the nitric oxide (NO) production in mouse macrophage cell line RAW 264.7. These observations suggest that stimulation of IL-6 and TNF-alpha secretion may contribute to the putative beneficial effects of dietary flavone against microbial infection.

Phoenix sylvestris Roxb pollen allergy: a 2-year randomized controlled trial and follow-up study of immunotherapy in patients with seasonal allergy in an agricultural area of West Bengal, India.

BACKGROUND: Although the efficacy of allergen immunotherapy has been demonstrated in seasonal pollen allergy, there is no report of a double-blind placebo-controlled trial with standardized pollen extract in seasonal respiratory allergy from India. In the agricultural area of eastern India, Phoenix sylvestris Roxb or date sugar palm is grown or cultivated and seasonal allergic rhinitis is common during the pollen season. OBJECTIVE: The objective of the present study was to observe the clinical and immunological changes during a 2-year double-blind placebo-controlled trial of immunotherapy with standardized P sylvestris pollen extract in respiratory patients sensitive to pollen from this wild date palm. Thirty-five subjects with typical seasonal allergic rhinitis with or without bronchial asthma were selected. A symptom-medication score (based on a questionnaire and diary) was correlated with pollen counts as recorded in a Burkard sampler. Eighteen subjects were randomized to a specific immunotherapy (SIT) group receiving regular injections containing standardized allergen extract and 17 to a placebo control group. Changes in the level of specific immunoglobulin (Ig) E, IgG1, and IgG4 were recorded at 3-month intervals. Measurement of wheal diameter, total IgE level and forced expiratory volume in 1 second (FEV1) were performed before starting and a month after finishing therapy. RESULTS: The SIT group showed decreases of 33.5% and 57% from the baseline symptom-medication scores during the first and second treatment season, respectively. This group showed significant decreases in skin-reactivity to P sylvestris pollen extract and in specific IgE levels, and significant increases in FEV,, specific IgGI (1.95-3.2 times higher) and IgG4 (21.24-30.83 times higher). There were no significant changes in total IgE levels. The control group showed no significant changes for any parameter except the development of new sensitization in 2 cases (to Saccharum officinarum pollen grain and Alternaria species spores). The rate of local adverse reactions was 0.024%. CONCLUSION: After a 2-year study, allergen immunotherapy with standardized P sylvestris pollen extract was found to be effective in seasonal respiratory allergic subjects susceptible to P sylvestris pollen with a narrow range of sensitization.

Aerobiological and immunochemical studies on Carica papaya L. pollen: an aeroallergen from India.

BACKGROUND: Carica papaya L. is a fruit yielding tree, wildly grown or cultivated in the tropics and subtropics. Its pollen grain has been reported to be airborne and cause immunoglobulin E (IgE)-mediated hypersensitivity. OBJECTIVE: To conduct long-term aerobiological study on Carica pollen, along with aeroallergenic particles originating from it and to identify vis-a-vis characterize an important IgE-reactive component present in this pollen. METHODS: The seasonal and diurnal periodicities of airborne C. papaya pollen were recorded in a 5-year survey using a Burkard volumetric sampler. The allergenic potential was studied by skin prick tests, IgE-enzyme-linked immunosorbent assay (ELISA) and also by aeroallergen immunoblotting. The total pollen extract was fractionated by Sephacryl S-200 column, and out of the eluted five fractions, the maximum IgE-reactive fraction (as found in ELISA inhibition) was resolved into five major subfractions in reverse-phase high-performance liquid chromatography (RP-HPLC). The subfraction with optimum IgE reactivity was studied by activity gel, native and nonreducing sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The homogeneity of the isolated protein fraction was checked by crossed immunoelectrophoresis with rabbit antisera and IgE reactivity was confirmed by ELISA inhibition and immunoblotting using individual patient sera. RESULTS: The Carica pollen occurred in the air round the year with peaks during January and September-October. Among a patient population of 1000, skin-test results showed 27.8% +1 level and 5.6% +2/+3 level reactions. In aeroallergen immunoblotting of exposed Burkard tape segments, the detected allergen spots showed a significant correlation with airborne pollen count recorded. The pollen extract elicited loss of IgE reactivity when treated with reducing agent-like beta-mercaptoethanol and heat, but showed six IgE-reactive components in nonreducing IgE-immunoblot. The fraction 1 eluted from Sephacryl S-200 column showed highest IgE reactivity and resolved into five major components in RP-HPLC. Out of these, the fraction showing optimum IgE reactivity in IgE-ELISA inhibition and immunoblotting with patient antisera, elicited esterase activity and found to be a homogenous protein of 100 kDa. CONCLUSION: Carica papaya tree contributes significantly to the aeropollen and aeroallergen load of the suburban outskirts of Calcutta metropolis, India. The pollen extract contains an important IgE-reactive protein component of 100 kDa molecular weight with esterase activity.

Differences in concentrations of allergenic pollens and spores at different heights on an agricultural farm in West Bengal, India.

The aim of the study was to assess the vertical profile of the major airborne pollen and spore concentration in the lower heights (up to six meters) and to check their allergenic potential causing respiratory allergy in agricultural workers. The study was conducted using rotorod samplers mounted at different heights at weekly intervals for two consecutive years (November 1997-October 1999). The major pollen grains and fungal spores (from mass culture) were collected in bulk and studied by skin-prick tests to detect allergenicity. Of the recorded pollen, 10 major and perennial types (e.g., Poaceae, Cheno-Amaranthaceae, Cyperaceae, Areca, etc.) were considered for comparative analyses. The tree pollen count showed more or less good correlation with increasing heights, whereas herb/shrub members are dominant at lower heights during all the three seasons (winter, summer and rains). The 10 major and perennial fungal spore types included Aspergilli group, Cladosporium, Nigrospora, etc. The smaller spores were dominant at greater heights and larger spores and conidia were more prevalent at lower levels. The total spore count was higher just after the rainy season during winter. In terms of allergenicity, Saccharum officinarum (sugar cane) of Poaceae, showed highest reactivity (70.58%) in skin test carried out in 189 adult agricultural field workers with respiratory disorders living inside the study area. Among fungal spores, Aspergillus japonicus was the strongest allergen, evoking 74.07% positive reactions. Drechslera oryzae, the pathogen causing brown spot of rice was also found to be a potent allergen.

Clinicoimmunologic studies on Phoenix sylvestris Roxb. pollen: an aeroallergen from Calcutta, India.

BACKGROUND: This study highlights the allergenicity and allergenic components of the pollen of Phoenix sylvestris Roxb. (PS), or date sugar palm, which is predominantly airborne in the air of Greater Calcutta. METHODS: A 2-year aerobiologic survey was performed by Burkard sampler. PS pollen extract was used in skin tests of allergic patients, fractionated by (NH4)2SO4 and the Sephacryl S-200 column. The allergenicity of each fraction was checked by skin test and IgE ELISA inhibition. The principal allergenic fraction, Fr.lla, was separated in 11% SDS-PAGE, and its allergenicity was confirmed by IgE ELISA inhibition and immunoblotting. RESULTS: PS pollen grains were found to be prevalent in the air of the suburban zone of Calcutta from January to March with a peak in February. The pollen extract showed high (44.07%) positive skin reaction on 540 respiratory allergic patients. Among the (NH4)2SO4 cut fractions, Fr.II was the most active one, and it was resolved into four subfractions in the Sephacryl S-200 column. Fr.lla was the principal allergenic fraction, showing the presence of two components of 33 and 66 kDa in SDS-PAGE. In IgE immunoblotting, both of the components were found to be allergenic. CONCLUSIONS: The PS pollen grain is an important aeroallergen from Calcutta, India. The 33- and 66-kDa components are the major allergens present in the relevant pollen extract.

Antigenic relationship between four airborne palm pollen grains from Calcutta, India.

The pollen grains of Areca catechu, Borassus flabellifer, Cocos nucifera and Phoenix sylvestris, all belonging to the family Aracaceae (Palmae), are airborne and found to be potent in causing human respiratory allergy. The present study was undertaken to discover the antigenic relationship, if any, in the four relevant palm pollen grains. The study was conducted by using Borassus and Phoenix antisera raised in rabbit. These antisera were used in rabbit IgG specific ELISA-inhibition and rocket immunoelectro-phoresis (RIE) assays for all four palm pollen extracts. In ELISA-inhibition, a distinct inhibition was obtained with comparable amount of soluble pollen protein. The RIE precipitin bands also revealed the presence of common antigenic components in the palm pollen. After isolation and purification, such common antigens may be useful in allergen immunotherapy in asthmatics.

Effects of a high intake of unsaturated and saturated oils on intestinal transference of calcium and calcium mobilization from bone in an ovariectomized rat model of osteoporosis.

Intestinal transference of calcium and rate of bone turnover were evaluated in ovariectomized rats fed for 15 days with a high amount (30%) of lipid enriched with monounsaturated (groundnut oil), polyunsaturated (sunflower oil) and saturated (coconut oil) fatty acids. The results were compared with those for sham-operated control and ovariectomized groups fed a normal diet (7% groundnut oil). Irrespective of the saturation and unsaturation characteristics, all lipids (edible oils) used in our study considerably decreased the rate of in situ intestinal transference of calcium. Likewise, the activities of intestinal mucosal enzymes, alkaline phosphatase (AP) and calcium ATPase (Ca2+-ATPase) were decreased significantly in all the segments of the small intestine in a descending gradient. Significant changes in bone turnover and bone calcium (Ca) mobilization were confirmed in these animals by marked alterations in plasma AP activity, urinary calcium and phosphate excretion and calcium to creatinine (Ca:creatinine) ratio. Lipid supplementation (30%) in such ovariectomized rats using groundnut oil (monounsaturated), sunflower oil (polyunsaturated) or coconut oil (saturated) for 15 days further enhanced all of the above observed parameters. These results suggest that the intake of high amounts of lipids with different unsaturation and saturation characteristics may be an important factor in determining bone loss in ovariectomized rats.

Allergenic relationship among four common and dominant airborne palm pollen grains from Eastern India.

BACKGROUND: Palm pollen grains are predominant aeroallergens in the tropics including India. Evidence of allergenic crossreactivity had been reported from various parts of the world on different families, e.g. Poaceae, Asteraceae, etc. No such information is available about the palm pollen of tropical countries. OBJECTIVES: The present study was undertaken to find out the allergenic relationship, if any, in four common and important palm pollen in India. METHODS: A 2-year aerobiological survey was carried out at Madhyamgram situated at the suburban fringe of Calcutta Metropolis using Burkard volumetric sampler to know the seasonal variation of Areca catechu, Borassus flabellifer, Cocos nucifera and Phoenix sylvestris among others. Skin-prick tests (SPT) were performed with the relevant pollen extracts on the respiratory allergic patients. Sera from the subjects were tested directly by ELISA for estimating the allergen specific IgE. ELISA inhibitions and dot blotting were performed with pooled patients sera and four palm pollen extracts to detect the cross-reactivity. RESULTS: Among 70 patients, Areca catechu exhibited the maximum percentage (48.5%) of positive responses followed by Cocos nucifera (45.7%), Phoenix sylvestris (42.85%) and Borassus flabellifer (38.5%). On an average, 30-50% of the patients showed positive skin reactions and enhanced specific IgE level to more than one palm pollen extract. Further evidence of allergenic crossreactivity among the four palm pollen grains were provided by dot blotting and ELISA inhibition studies. In ELISA inhibition, a distinct inhibition was obtained with comparable amounts of the pollen extracts. CONCLUSION: The suburban aerobiological survey of Calcutta and SPT results confirmed that the relevant pollen types are significant contributors of aeroallergen load of the study area. ELISA inhibition studies with the pooled patients sera using antihuman IgE probe revealed the presence of shared allergenic components among the four palm pollen grains.

Aerobiologic and immunochemical studies on Borassus flabellifer pollen: evidence for a 90-kD allergen.

BACKGROUND: The pollen grains of Arecaceae have been proved to be a very common aeroallergen in India. Except for the pollen grains of Cocos nucifera, detailed information about the allergenicity of other dominant Indian palm pollen is not available. OBJECTIVES: To explore the aerobiologic and allergenic significance of the pollen of palmyra palm (Borassus flabellifer Linn.), one of the important and common palm trees in India, with a view to isolate and purify its major allergenic components. METHODS: The aerobiologic survey was performed continuously using Burkard volumetric and Rotorod samplers from July, 1994 to June, 1996 in a northern suburb of greater Calcutta. The allergenic potential of the pollen extract and different fractions were studied by skin prick tests (SPT). ELISA and ELISA inhibition experiments were performed with individual and pooled patient sera to detect the specific IgE level. By 2-step ammonium sulphate fractionation and gel filtration study, the allergic fraction Fr.IIA1 was isolated. Its molecular weight was determined by SDS-PAGE and homogeneity was confirmed by rocket and crossed immunoelectrophoresis using rabbit antisera. RESULTS: Pollen grains of B. flabellifer were found to be present in air from February to May and June contributing more than 7% of the total airborne pollen load of the study area. The pollen elicited maximum concentration at 19 to 20 hours in the evening and at a height of 4 m from ground level. Skin prick testing of 455 respiratory allergic patients with whole pollen extract exhibited 31.64% positive response. After 2-step ammonium sulphate fraction and gel filtration, a fraction (Fr.IIA1) having remarkable allergenicity was isolated. In native and SDS-PAGE, it was found as a single protein component of 90 kD and as homogeneous in RIE and CIE. The soluble protein-carbohydrate analysis indicated the probability of the component to be a glycoprotein. CONCLUSION: The aerobiologic, clinical, and immunochemical, studies demonstrated that the pollen of B. flabellifer is a dominant aeroallergen to cause respiratory trouble in the area where these trees grow. The isolated 90-kD component is one of the major allergens present in the pollen extract.

High-lipid diet intake is a possible predisposing factor in the development of hypogonadal osteoporosis.

The effects of a high-lipid diet on the intestinal transference of Ca2+ and the activities of intestinal enzymes were studied in ovariectomized rats. The plasma Ca2+ level and urinary loss of Ca2+ were also observed. Ovariectomy decreased both alkaline phosphatase (AP) and calcium ATPase (Ca(2+)-ATPase) activity, and also reduced the mucosal transference of Ca2+ in all parts of the small intestine. Although the plasma Ca2+ level did not change significantly, the urinary loss of Ca2+ was enhanced. A high-lipid diet supplement enhanced all these parameters in ovariectomized rats. It is therefore believed that a high-lipid diet may promote osteoporosis in ovariectomized rats as the result of Ca2+ transfer and enhanced urinary Ca2+ loss.

Role of nutrition in the survival after hepatotoxic injury.

Nutritional status is an important factor in determining susceptibility to toxic chemicals. While macro and micronutrients may affect many aspects of Stage I and Stage II of toxicity, in this paper, the influence of macronutrients as sources of energy required for cell division and tissue repair mechanisms on the outcome of hepatic injury is discussed. Male Sprague-Dawley rats maintained on normal rodent chow and 15% glucose (as a source of energy for the centrilobular hepatocytes) in drinking water for 7 days experienced an increased lethality from structurally and mechanistically different centrilobular hepatotoxicants (acetaminophen, thioacetamide, chloroform and carbon tetrachloride), while male Sprague-Dawley (S-D) rats fed rat chow containing palmitic acid (PA, 8% w/w, as a source of energy for the periportal hepatocytes) and L-carnitine (LC, 2 mg/ml, as a mitochondrial carrier for the supplemented fatty acids) in drinking water for 7 days were protected from a LD100 dose (600 mg/kg, i.p.) of thioacetamide (TA). Indices of cell division revealed that cell cycle progression in the liver played a very critical role in determining the final outcome of hepatotoxic injury. These results confirmed our hypothesis that cell division and tissue repair play a critical role in survival after life-threatening hepatotoxic injury. Any manipulation directed towards altering a prompt and exacting compensatory cell division and tissue repair responses after hepatotoxic injury would also alter the final outcome of the toxicity. These studies indicate that the source of cellular energy can decisively influence the compensatory response of the target tissue to alter the outcome of hepatotoxic injury. Since nutritional status is known to vary widely among human populations, these could contribute enormously to susceptibility of human populations to toxic chemicals.

Role of nutritional fatty acid and L-carnitine in the final outcome of thioacetamide hepatotoxicity.

Male Sprague-Dawley rats (n = 10/group) were fully protected from a lethal dose (600 mg/kg, i.p.) of thioacetamide by adding 8% (w/w) palmitic acid to the diet and L-carnitine (2 mg/ml) to drinking water for the previous 7 days. Supplements of palmitic acid or L-carnitine alone did not confer protection. Liver injury induced by thioacetamide peaked between 36 and 48 h in both control and supplemented rats. Liver damage regressed thereafter in supplemented rats but progressed in control rats. Immunohistochemical and histopathological observations confirmed biochemical indicators of liver damage. Thus, hepatic tissue repair after thioacetamide-induced tissue injury seems to be stimulated by supplements of fatty acids together with L-carnitine, a mitochondrial transfer agent. The extent to which nutritional supplements may aid in inducing the recovery of liver from injury caused by other hepatotoxic agents remains to be explored.