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Adaptogenic activity of glyco-peptido-lipid fraction from the alcoholic extract of Trichopus zeylanicus Gaerten (part II).

Abstract

Anti-stress activity was carried out on glyco-peptido-lipid (AF) fraction from the alcoholic extract of Trichopus zeylanicus Gaerten and demonstrated against a battery of tests in rats and mice. AF exhibited significant anti-stress activity in dose-related manners in all the parameters studied against different models used to induce non-specific stress viz physical and chemically. The major parameters studied were immobilization induced gastric ulceration, adjuvant-induced trauma (Stress); humoral antibody synthesis in normal and immuno-suppressed mice and delayed type of hypersensitivity (DTH) reaction, chemically stress-induced alteration in hepatic function and anti-oxidant activity. The extract of Withania somnifera root (a commercial preparation available locally, Dabur India ltd.) was used to compare the results. In the safety evaluation study the maximum tolerance dose (MTD) and oral L[D.sub.50] were found to be more than 3000 mg/kg, with no signs of abnormalities or any mortality observed for 15 days period under observation after single dose of drug administration. Feeding behaviour and fecal output were normal.

[c] 2005 Elsevier GmbH. All rights reserved.

Keywords: Trichopus zeylanicus; Adaptogens; Anti-stress; SNIR (non-specific increase of resistance of the organism); Immuno-stimulant; Withania somnifera; Galactosamine (GalN); Prophylactic; Inflammation; Sheep erythrocytes; Hem agglutination

Introduction

Stress is a phenomenon that manifests itself in our bodies in many different ways. Some of the common symptoms of stress include problems with sleep, depression, anxiety, irritability, fatigue and lethargy. Perhaps the single most important property of an adaptogen is its proven ability to combat stress in all forms. The response to stress is non-specific and independent of the nature of the stress as the stress-induced state produced by diverse stresses is indistinguishable (Selye, 1973).

Herbal drugs in the recent years have gained sufficient importance because of their efficacy and cost effectiveness (Subramoniam and Pushpangadan, 1999). Rasaayana drugs in the Ayurvedic literature are used for remedies in various ailments in the folklore and indigenous system of medicines since times immemorial. Adaptogens are pharmacological agents that induce a state of non-specific increase of resistance of the organism (SNIR) to aversive stimuli that threaten to perturb internal homeostasis (Brekhman, 1965; Brekhman and Dardymov, 1969; Voskresnsky et al., 1986). The corroborative and tonic plants generally known as Rasaayana drugs, known to prevent ageing, increase longevity and offer resistance to diseases by augmenting the immune system (Rege et al., 1999; Singh et al., 2001).

Trichopus zeylanicus Gaerten Trichopodaceae, is a wild plant, a rare genus, small glabrous herb growing in the Agasthyar hilly forests of Kerala, India. The tribal inhabitants (Kani tribe) of this area call this plant "Arogyappacha" meaning the greener of health, and use this plant as a health tonic and rejuvenator. This information is based on ethno-medico-botanical investigations (Pushpangadan et al., 1988; Sharma et al., 1989; Evans et al., 2002). The aim of the present study is to explore and identify an active fraction(s)/constituent(s) of Trichopus zeylanicus Gaerten that increases the non-specific resistance of the body to combat harmful influence of the stress.

Materials and methods

Plant material

The Trichopus zeylanicus Gaerten was collected from Agasthyar hills of Kerala (India) in the month of October-November and authenticated by taxonomists at Janki Ammal Herbarium of the Institute (Regional research Laboratory, Jammu, India) where a voucher specimen has been deposited vide accession No. 18385 for future reference.

Preparation of extract and fractionation

Shade dried, coarsely powdered whole plant (2.5 kg) of Trichopus zeylanicus Gaerten was extracted with ethyl alcohol and water (70:30) by percolation (10 times) to obtain 70% alcoholic extract (285 g, yield 11.40%). The extract so obtained (200 g) was dissolved in 10% methanol in water and extracted with hexane (3 X 500 ml), chloroform (3 X 500 ml) and n-butanol (4 X 500 ml) to give 16.6, 10.0 and 4.8 g fractions in 8.3%, 5.0% and 2.4% yield, respectively. The aqueous fraction left was lyophilized to give a dark brown semisolid material (169 g, 84% yield). Among fractions evaluated for biological activity, the aqueous fraction showed a significant effect (Singh et al., 2001).

Small portions of aqueous fraction AF were hydrolyzed with 6N HCl and methanolic-sulphuric acid (Lowry et al., 1951) individually. The hydrolysates were analyzed for the presence of amino acids and estimated to be about 20% by Lowry's method (Lowry et al., 1951). Hydrolysates were also analyzed for the carbo-hydrates and estimated by phenol-sulphuric acid and was found to be 40%. The presence of fatty acids (5%) was determined (Kates, 1972) by saponification of a small portion of aqueous fraction. Drastic hydrolysis (Puhlmann et al., 1992) of AF with 2 M TFA at 121 [degrees]C, a mixture of three distinct compounds were visible on TLC in butanol-pyridine-water (6:4:3) solvent system. The non-polar part of hydrolysate ([R.sub.f] 0.53) developed with iodine; the moderately polar hydrolysate ([R.sub.f] 0.23) gave yellow colouration with bromo-cresol green and the more polar hydrolysate ([R.sub.f] 0.06) charred with cerric ammonium sulphate.

The aqueous sub-fraction was passed through Sephadex G-100 gel repeatedly. This resulted into homogeneity of the bioactive constituent which was crystallized as white rhombic crystals (yield 40%) from deionized water m.p.>360[degrees]C, [[alpha]][.sub.D.sup.25] + 9[degrees]. On analysis of a small portion of crystalline material by flame photometry, sodium and potassium cations were detected in 1.5% and 50%, respectively. Amberlite (IR 120 plus) was used for exchanging of sodium and potassium ions. Desalted pure organic compound having [[alpha]][.sub.D.sup.25] + 23[degrees] (C 0.1, [H.sub.2]O), i.r. [[lambda].sub.max] 3450 (OH), 2850 (weak CH St), 1725 (acidic C = O), 1626 (CO-NH), 1404, 1194, 1040 and 620 (CO-NH) indicating thereby AF to be composed of carbohydrate, amino acid and fatty acids.

However, the fatty acid was separated from the mixture by preparing its methyl ester (Christie, 1982). The fatty acid present in AF hydrolysate was identified as myristic acid, [C.sub.14][H.sub.28][O.sub.2] by comparing the retention time with authentic sample on GLC (Gehrke and Leimer, 1971). The fraction AF on acid hydrolysis with 6N HCl afforded alanine and glutamic acid. These amino acids were identified by comparison with authentic samples by paper chromatography and gas liquid chromatography of their silyl derivatives (Jones and Albersheim, 1972). The polar hydrolysate of AF obtained with 2 M TFA was found to be a mixture of deoxy ribose and mannose. These sugars were identified by comparative paper chromatography with authentic samples and by gas liquid chromatography using their silylated derivatives (Jones and Albersheim, 1972). The above degradative study suggested that AF is a glycopeptido-lipid.

Animals

Charles Foster rats (150-180 g) and Swiss albino mice (25-30 g) of either sex, bred in the Institute's Animal House, were used in the present study. Animals were housed under standard conditions (23 [+ or -] 2[degrees]C, 60-70% relative humidity and 12h photo-period) and were maintained on standard rodent pellet diet (Lipton India Ltd., Bombay) and water ad libitum. The grouping of animals and treatment schedule is described in Table 1.

Treatment

Different doses of test drug, standard and vehicle were given once daily at the interval of 24 h for 15 days. Freshly prepared solution of AF (1% w/v) in normal saline was used in all the experiments except for toxicity studies where 10% solution was used. The control animals were given proportionate volume of vehicle. The extract of Withania somnifera roots (a commercial preparation available locally) was used to compare the results and the same has been referred as standard (Std). A 1.25% solution in 0.2% gum acacia was prepared for the treatment of animals (Table 1).

Anti-inflammatory evaluation

Adjuvant-induced polyarthritis in rats

Adjuvant arthritis (Newbould, 1963) in one of the hind paws of the male rat was induced by the subplantar injection of 0.05 ml freshly prepared suspension (5.0 mg/ml) of steam-killed dried Mycobacterium tuberculosis (Difco, USA) in liquid paraffin (I.P. S.D. fine Chemicals). The volume of the injected (primary response, Arrigoni-Martelli and Brahm, 1975) and un-injected "secondary" or immune-mediated responses (Arrigoni-Martelli et al., 1976), hinds paws were quantitated using plethysmographic recordings of paw volume (Volume differential meter, Model 7101, Ugo Basile, Italy) before and on alternate days from day 2 to day 14 and lastly on day 21 after the Adjuvant injection. Different doses of the test drug dissolved in normal saline and the vehicle (normal saline) given orally (p.o.) 1h before the injection and then once daily at an interval of 24h for 21 days (Table 2).

Immune-stimulant activity in normal and immunosuppressed mice

Immuno-stimulant activity in normal mice

Seven groups of mice were used (Table 3). The mice were immunized (Sharma et al., 1996) by an intraperitoneal injection of fresh sheep erythrocytes (SRBC) one hour after the drug treatment on day 0 and challenged by injecting the same amount of SRBC i.p. on day + 7 after taking a small amount of blood from orbital sinus from all the animals and serum separated for primary antibody titer. On day + 14 again blood was collected 1 h after the last dose administered from all the animals for secondary antibody titer (Sharma et al., 1992) by the hemagglutination technique (Nelson and Mildenhall, 1967) using serial two fold dilution in 'V' bottomed 96-well microtiter plates. The highest dilution showing visible agglutination was taken as the antibody titer (Table 3).

Immuno-stimulant activity in immuno-suppressed mice

Seven groups of mice were used. "Cyclophosphamide" (CY) 250 mg/kg, p.o. was given for two days at the interval of 24 h except groups 1 and 3 (Nouza et al., 1990). On day 3 all the animals were immunized (Sharma et al., 1996) with SRBC. Different doses of the test drug dissolved in normal saline and the vehicle (normal saline) given orally (p.o.) once daily at the interval of 24h from day 3 to 7. On day + 7 blood was collected 1 h after the last dose had been administered from all the animals for antibody titer (Sharma et al., 1992) by the hemagglutination technique (Nelson and Mildenhall, 1967). The highest dilution showing visible agglutination was taken as the antibody titer (Table 3).

Delayed type of hypersensitivity (DTH) reaction in mice

Mice were sensitized by injecting 20[micro]l of 5 X [10.sup.9] SRBC/ml subcutaneously (s.c.) into the right hind foot paw on day 0 and challenged by injecting the same dose of SRBC (i.d.) into the left hind foot paw on day + 7 (Doherty, 1981). The thickness of the left hind foot pad of each mouse was measured using a spheromicrometer (reading to 0.1 mm) 24h after the challenge, it was expressed as the mean [+ or -] SE for each group (Table 3).

Immobilization (physical)-induced stress in rats

Eight groups of rats were used. All the animals except control without stress and AF per se groups were immobilized on a wooden plank with the help of adhesive plaster (Fregley, 1953, Bhattacharya et al., 1987) individually for 5h on day 15, 1 h after the treatment. Blood from all the animals was collected for the estimation of serum biochemical parameters (Tables 4-6).

Immobilization (physical)-induced biochemical parameters in rats

The rats of all the groups were killed by decapitation, their adrenal glands were decapsulated, removed and weighed (Selye, 1936b). The corticosterone (Zenker and Bernstein, 1958), ascorbic acid (Roe and Kuether, 1949) and cholesterol (Zlatkis et al., 1953) in adrenal glands were estimated after homogenization in sterile cold normal saline (Table 4).

Immobilization (physical)-induced stress hepatic function (biochemical parameters) in rats

The livers of all the animals were quickly excised, cleaned of adhering tissue weight and homogenized in chilled 'Tris buffer (pH 7.4) in a potter-S-homogenizer (B. Braun, Germany) consisting of a Teflon pestle and glass homogenizer. The homogenization, centrifugation, etc. was carried out at 0-4[degrees]C. The hepatic lipid peroxidation was measured by estimating malondialdehyde (MDA) as described by Buege and Aust (1978). The results were calculated based on the molar extinction coefficient of 1.56 X [10.sup.5] [m.sup.-1] [cm.sup.-1] (Wills, 1969).

The hepatic glutathione was determined by the methods of Ellman (1959), David et al. (1987). An equal volume of chilled TCA (10%, w/v) was added to the chilled liver homogenate (10%, w/v) in Tris-HCl buffer 10mM, pH 7.4) and centrifuge (3000g for 10 min at 4[degrees]C) to separate the protein from the liver homogenate. GSH was determined in a suitable aliquot of protein free supernatant (200 [micro]l diluted with phosphate buffer (0.2 M, pH 8.0) to 1.0 ml) by reaction with 5, 5'-Dithio-bis(2-Nitro Benzoic Acid) (DTBN 2 ml, 0.6mM, dissolved in water) at a final concentration of 0.4mM in phosphate buffer (0.2 M, pH 8.0) medium. The O.D. was recorded at 412 nm, against blank using a spectrophotometer (Uvikon-810, Kontron Instruments Switzerland). The reduced glutathine was used as standard dissolved in TCA (5%) (Table 5).

Immobilization (physical)-induced gastric ulceration in rats

After stress, the stomachs of all the animals were opened and examined with a magnifying lens for the degree of ulceration (Selye, 1936a). The ulcerogenic indices were scored according to Bhargava and Singh (1981), i.e. no ulcerogenic changes, shedding of epithelium, petichial and frank haemorrhages, one to four ulcers, more than four ulcers and perforated ulcers were assigned the score of 0, 10, 20, 30, 40 and 50, respectively. Severity and group score (Robert et al., 1968) were calculated and incidence rate was calculated as percentage of the ulceration in a group. The weighted score was taken as a measure of severity. The pooled group score was taken as the sum of scores over all the animals in a group (Table 6).

Chemical (GalN) stress-induced hepatic function in rats

Nine groups of rats were used (Table 7). The antistress activity of AF was monitored against hepatic stress produced by galctosamine (GalN) 300 mg/kg, subcutaneously, prophylactic study (Decker and Keppler, 1974; AL-Tuwaijiri et al., 1981; Kumar et al., 1992; Zbigniew 1994) in all the animals except groups 1 and 3 (group 1 served as vehicle control without stress and group 3 served as per se effect of the drug without stress). The normal control animals received an equal volume of normal saline, as vehicle. The different doses of test drug AF or vehicle (normal saline) were administered once daily for 13 days and on the 14 day 2 h before and 6 h after galctosamine (GalN) 300 mg/kg, subcutaneously-induced stress.

Blood was collected from the orbital sinus from all the animals 18 h after the last dose of drug administered i.e. on day 15, and serum separated for different estimations: (AST) aspartate aminotransferase, (ALT) alanine aminotransferase (Reitman and Frankel, 1957), (ALP) alkaline phosphatase (Klaus and Christian, 1974), bilirubin (Malloy and Evelyn, 1937) and triglycerides (Neri and Frings, 1973). All the animals were then killed by decapitation, their livers were quickly excised, cleaned of adhering tissue, weighed and homogenized in chilled phosphate buffer saline for the analysis of hepatic glycogen (Hawk et al., 1978), lipid peroxidation (Buege and Aust, 1978) and GSH (Ellman, 1959; David et al., 1987).

In vitro free radical scavenger activity

In vitro free radical scavenger activity was determined by the method of Lamaison et al. (1991) using DPPH reaction mixture in a final volume of 2.5 ml contained test material and DPPH (0.6 X [10.sup.-4] M) in methanol. Rutin and Quercetin were dissolved in methanol and AF was dissolved in distilled water. After 30 min of incubation at room temperature the colour was extracted in toluene and absorbance was recorded using UVIKON-922 spectrophotometer at 517 nm. Percent decolourization was taken as free radical scavenger activity. Data reported here are mean values of two experiments carried out in triplicate (Table 8).

Anti-stress activity (A) was calculated by the following equation:

A = [1 - (TS - V/VS - V)] X 100,

where V is the vehicle, TS and VS are the drug + stress and vehicle + stress treated groups of animals, respectively.

Acute toxicity studies

Using different doses (100-3000 mg/kg, p.o.) of AF given to the groups of 10 mice for each dose, while one group with the same number of mice served as control. The animals were observed continuously for 1 h and then half hourly for 4h for any gross behavioural changes for general motor activity, writhing, convulsion, response to tail pinching, gnawing, piloerection, pupil size, fecal output, feeding behaviour etc. and further up to 72h and finally up to 15 days for any mortality. Acute L[D.sub.50] values in mice were calculated by the method of Miller and Tainter (1944). Mortality of animals in all the groups used in different models for determining antistress activity during the period of treatment was also recorded as a rough index of sub-acute toxicity.

Statistical analysis

The data obtained were subjected to statistical analysis using ANOVA (Armitage and Berry, 1987) for comparing different groups and Dunnett's t-test for control and multiple test groups (Dunnett, 1964). The regression coefficient (slope b) correlation coefficient (r) with its p value and E[D.sub.50] with 95% confidence limit (CL) was determined by regression analysis using log dose and percent effect of adaptogenic activity (Swinscow, 1980). The two-tailed paired student t-test was used for comparing means before and after treatment and one tailed unpaired student t-test was used for comparing control and drug treated groups (Ghosh, 1984).

The p value of <0.05 or less was taken as the criterion of significance.

Results

Anti-inflammatory evaluation in rats

Adjuvant-induced polyarthritis in rats

The results are summarized in Table 2. AF significantly inhibited the primary and secondary reaction to adjuvant injection in the injected paw and increases the secondary reaction to adjuvant injection in the un-injected paw and fore limbs. The acute (primary) edema in the injected paw 18h after the adjuvant injection was inhibited in dose-related manner by AF given orally (b = 49.53; r = 0.92, p < 0.001) with an E[D.sub.50] value of 74.75 (95% CL = 65.45-85.40) mg/kg. ANOVA analysis showed a significant difference between control and drug treated groups [F (31.68 > 2.41); p < 0.05].

AF markedly inhibited the late (secondary) 21-day reaction in the injected paw (b = 51.50; r = 0.88, p < 0.001) with an E[D.sub.50] value of 58.70 (95% CL = 50.22-68.61) mg/kg. ANOVA analysis showed a significant difference between control and drug treated groups [F (31.68 > 2.41); p <0.05] (Table 2). The effect of AF appeared to be more marked in un-injected paw as immuno-stimulatory effect and the effect was dose-dependent (b = 54.50; r = 0.85, p < 0.001) with an E[D.sub.50] value of 15.63 (95% CL = 13.07-18.69) mg/kg. ANOVA analysis showed a significant difference between control and drug treated groups [F(31.68 > 2.41); p < 0.05]. The dose for AF is more effective than PBZ and standard.

Effect on humoral antibody synthesis in mice

Immuno-stimulant activity in normal mice

The results are summarized in Table 3. Oral administration of AF, standard and LEV to the normal immunocompetent mice for 7 days, postimmunization with SRBC (5 X [10.sup.9]/ml) showed significant influence on the primary antibody synthesis (b = 0.03; r = 0.81, p < 0.001) with an E[D.sub.50] value of 42.82 (95% CL = 35.22-52.07) mg/kg. A similar effect was observed with LEV (positive control). On continued oral administration of AF, standard and LEV for 7 days more after challenge with SRBC, the response on the secondary antibody titre was observed (b = 36.07; r = 0.52, p < 0.01) with an E[D.sub.50] value of 73.23 (95% CL = 54.68-98.05) mg/kg. Analysis by ANOVA showed a significant difference between control and drug treated groups [F (9.98 > 2.53); p < 0.05] and [F (4.95 > 2.53); p < 0.05], respectively.

Immuno-stimulant activity in immuno-suppressed mice

AF markedly influences the antibody synthesis in immunosuppressed mice. Oral administration of AF, standard and LEV to the immunosuppressed mice for 7 days, post immunization with SRBC (5 X [10.sup.9] /ml) showed significant influence on the antibody synthesis. (b = 54.59; r = 0.64, p < 0.001) with an E[D.sub.50] value of 22.72 (95% CL = 17.48-29.52) mg/kg. A similar effect was observed with LEV (positive control). Analysis by ANOVA showed a significant difference between control and drug treated groups [F (7.04>2.53); p < 0.05] (Table 3).

Delayed type of hypersensitivity (DTH) reaction in mice

The cell mediated immunity was significantly affected by AF and the effect was dose related (b = 91.74; r = 0.80, p < 0.001) with an E[D.sub.50] value of 22.72 (95% CL = 8.50-27.91) mg/kg. Analysis by ANOVA showed a significant difference between control and drug treated groups [F (13.54>2.53); p < 0.05] (Table 3).

Immobilization (physical)-induced stress in rats

Immobilization (physical)-induced biochemical parameters in rats

Exposure to restrain stress causes hypertrophy of the adrenal glands which is associated with significant depletion of adrenal contents of ascorbic acid, corticosterone and chlosterol (p < 0.01) (Table 4). Pretreatment with AF attenuated the restraint stress-induced hypertrophy effect on the adrenal gland (b = 48.29; r = 0.80, p < 0.001) and depletion of adrenal ascorbic acid (b = 9.52; r = 0.80, p < 0.001), corticosterone (b = 49.44; r = 0.72, p < 0.001) and cholesterol (b = 74.77; r = 0.60, p < 0.001). The E[D.sub.50] values were 22.80 (95% CL = 18.57-28.01) mg/kg; 24.15 (95% CL = 19.74-29.56) mg/kg; 35.22 (95% CL = 27.90-44.45) mg/kg and 24.47 (95% CL = 18.92-31.64) mg/kg, respectively. ANOVA analysis showed a significant difference between control and drug treated groups [F (50.52>2.54); p<0.05]; [F (25.04>2.54); p < 0.05]; [F (25.58>2.54); p < 0.05] and [F (7.59 > 2.54); p < 0.05].

The extract of Withania somnifera roots (a commercial preparation available locally) used as standard showed an anti-stress effect on all the parameters studied, in general the effect of AF was more pronounced on all the parameters when compared with the standard (Table 4).

Immobilization (physical)-induced stress hepatic function in rats

Stress causes a significant alteration in hepatic function and increase in serum levels of GPT, GOT, ALP, triglycerides and hepatic lipid peroxidation and decrease of the hepatic glycogen (p < 0.01). Pretreatment with AF and standard significantly reversed the altered values in a dose-dependent manner as evident from serum level of GPT (b = 42.60; r = 0.89, p < 0.001) with an E[D.sub.50] value of 37.94 (95% CL = 32.58-44.18) mg/kg, GOT (b = 38.95; r = 0.80, p < 0.001) with an E[D.sub.50] value of 25.51 (95% CL = 20.50-30.86) mg/kg, ALP (b = 68.96; r = 0.73, p < 0.001) with an E[D.sub.50] value of 18.62 (95% CL = 14.76-23.48) mg/kg, Bilirubin (b = 56.85; r = 0.82, p < 0.001) with an E[D.sub.50] value of 33.89 (95% CL = 27.84-41.27) mg/kg, and triglycerides (b = 58.60; r = 0.88, p < 0.001) with an E[D.sub.50] value of 54.21 (95% CL = 46.40-63.34) mg/kg, and hepatic (whole liver homogenate) glycogen (b = 54.22; r = 0.79, p<0.001) with an E[D.sub.50] value of 30.91 (95% CL = 25.10-38.06) mg/kg, lipid peroxidation (b = 2.55; r = 0.86, p < 0.001) with an E[D.sub.50] value of 14.68 (95% CL = 12.33-17.46) mg/kg, and GSH (glutathione) (b = 89.55; r = 0.87, p < 0.001) with an E[D.sub.50] value of 28.31 (95% CL = 24.02-33.36) mg/kg, p.o. Analysis by ANOVA showed a significant difference between control and drug treated groups, SGPT [F (130.79 > 2.34); p < 0.05]; SGOT [F (90.06 > 2.34); p < 0.05] ALP [F (21.34 > 2.34); p < 0.05]; Bilirubin [F (41.60>2.34); p < 0.05]; Triglycerides [F (100.15 > 2.34); p < 0.05]; hepatic glycogen [F (38.19 > 2.34); p < 0.05]; hepatic lipid peroxidation [F (39.02>2.34); p < 0.05], and GSH (glutathione) [F (26.95 > 2.34); p < 0.05], respectively. AF 100 mg/kg fed alone for the same schedule of treatment did not produce any per se effect when compared to normal vehicle control (Table 5).

Immobilization (physical)-induced gastric ulceration in rats

Pretreatment with AF showed significant concentration-dependent inhibition of ulcerogenic indices (b = 43.82; r = 0.82, p < 0.001) induced by immobilization stress with an E[D.sub.50] value of 69.92 (95% CL = 57.72-84.70 mg/kg. ANOVA analysis showed a significant difference between control and drug treated groups [F (16.57 > 2.54; p < 0.05] (Table 6).

Chemically (GalN)-induced stress hepatic function in rats

The results are summarized in Table 7. Administration of galactosamine (GalN) alone significantly raised the serum levels of GPT, GOT, ALP, bilirubin, triglycerides and hepatic (whole liver homogenate) lipid peroxidation and lowered hepatic glycogen and GSH (p<0.01). Pretreatment with AF and standard significantly reversed the altered values in a dose-dependent manner as evident from serum level of GPT (b = 44.83; r = 0.82, p < 0.001) with an E[D.sub.50] value of 29.20 (95% CL = 25.24-33.78 mg/kg; GOT (b = 39.88; r = 0.88, p<0.001) with an E[D.sub.50] value of 27.25 (95% CL = 23.23-31.97) mg/kg; ALP (b = 47.98; r = 0.74, p > 0.001) with an E[D.sub.50] value of 24.91 (95% CL = 19.77-31.38) mg/kg; bilirubin (b = 56.83; r = 0.92, p<0.001 with an E[D.sub.50] value of 35.31 (95% CL = 30.89-40.35) mg/kg; and triglycerides (b = 61.06; r = 0.87, p < 0.001) with an E[D.sub.50] value of 47.13 (95% CL = 40.00-55.53) mg/kg; and hepatic glycogen (b = 79.30; r = 0.90, p < 0.001) with an E[D.sub.50] value of 28.08 (95% CL = 24.32-32.42) mg/kg; lipid per oxidation (b = 60.48; r = 0.87, p < 0.001) with an E[D.sub.50] value of 18.49 (95% CL = 15.66-21.840) mg/kg; and GSH (b = 55.31; r = 0.67, p<0.001) with an E[D.sub.50] value of 21.77 (95% CL = 16.89-28.07) mg/kg; ANOVA analysis showed a significant difference between control and drug treated groups, SGPT [F (94.40 > 2.34); p<0.05], SGOT [F (122.08 > 2.34); p<0.05] ALP [F (31.21 > 2.34); p<0.05], Bilirubin [F (75.80 < 2.34); p<0.05 triglycerides [F (72.95>2.34); p < 0.05], hepatic glycogen [F (30.40<2.34); p<0.05 lipid peroxidation [F (38.24<2.34); p>0.05] and hepatic GSH [F (17.88>2.34); p < 0.05], respectively.

In vitro free radical scavenger activity

In vitro free radical scavenger activity determined using DPPH reaction mixture showed significant free radical scavenger activity in a dose-dependent manner and a similar effect was observed in Quercetin and Rutin used as standard (Table 8).

Acute toxicity studies

AF up to 3.0 g/kg did not cause any death and changes in gross behaviour in mice observed for 15 days. No mortality or abnormal signs were observed in animals in any of the groups, used in different models for determining anti-stress activity during the period of treatment.

Discussion

Stress causes significant depression in the immunological functions (Solomon et al., 1985; Harman, 1993). An adaptogen exerts a strong immunomodulatory influence in the healthy test subjects and can be considered a non-specific immunostimulant. Our studies showed that AF during stress rendered vital support to the immune system (Table 3). This is further supported by its effect on carrageenan-induced trauma (Singh et al., 2001) and adjuvant-induced arthritis in rats (Swingle et al., 1969; Swingle, 1974; Otternes and Bliven, 1985; Bowen and Fauci, 1988; Sternberg and Licinio, 1995). Inhibition of trauma induced by Friend's adjuvant may be achieved by stabilizing the blood vessel membranes that stabilizes the vascular permeability, neutralizing the chemical mediators (Kumar et al., 1992; Singh et al., 2001) and stimulating the immune mediated responses (humoral and cell mediated immunity) (Arrigoni-Martelli and and Brahm, 1975; Arrigoni-Martelli et al., 1976). That immuno-stimulant activity is associated with the enhancement of secondary lesions in adjuvant arthritis is further supported by its effects in models of DTH using SRBC as antigen (Arrigoni-Martelli et al., 1976). This indicates its adaptogenic activity and the effect was statistically significant with all the doses (Tables 2 and 3).

Stress is known to induce peptic ulcer and the severity of ulceration is entirely dependent upon the intensity of the stress (Bhargava and Singh, 1981). The mechanism is still unclear but the central nervous system plays an important role in the pathogenesis and prevention of restraint stress-induced ulcers in rats (Bhargava et al., 1980; Lu et al., 1988). The inhibition in the incidence and severity of gastric ulceration by AF in vivo during stress indicates its adaptogenic activity, and the effect was statistically significant with all the doses (Table 6).

The adrenal glands contain relatively large amounts of cholesterol, ascorbic acid and cortical steroids which are markedly decreased and cause hypertrophy when they are stimulated by stress or injection of adrenocorticotropin hormone of the anterior pituitary lobe (Brekhman, 1965; Landfield and Eldridge, 1991; Jacobson and Sapolsky, 1991). Pretreatment with AF significantly prevents the depletion of cholesterol, ascorbic acid and cortical steroids and hypertrophy of adrenal glands, indicating that Rasaayana drugs (NSIR) appear to have a corticosteroid sparing effect as observed in our study and reported by others (Brekhman, 1965; Bhargava and Singh, 1981; Solomon et al., 1985; Henriksson et al., 1992). However, AF per se has no effect on these parameters in adrenals (Table 4).

Our experimental study has conclusively demonstrated that AF changes the course of the primary physiological indicators of stress by reducing the activation of the adrenal cortex and sparing the corticosteroid effect.

Stress of any kind results in a progressive deterioration in most of the endocrine functions and enhances exogenous or endogenous stress associated with an impairment of hepato-pancreatic function (Kumar et al., 1992; Zbigniew, 1994), and results in a significant elevation of the activities of GPT, GOT, ALP, bilirubin, triglycerides in serum and liver glycogen, lipid-peroxidation and GSH, indicating considerable alteration in normal physiological functioning of the body. Oral administration of AF significantly reversed the disturbed levels of enzymes activities produced by both chemical and physical stress strongly pointing to the possibility of AF being able to combat adverse conditions. A considerable stabilization of serum bilirubin following the AF treatment is a further clear indication of the improvement of functional status of the hepatocytes.

GSH is the most important endogenous protective bio-molecule against adverse conditions. In this connection, the protective role of GSH against cellular lipid peroxidation has been well documented (Burk, 1983). A substantial increase in hepatic lipid peroxidation is evident from elevated MDA levels in liver homogenate with a concurrent fall in hepatic GSH and glycogen contents following chemical and physical stress is indicative that stress alters the physiological functioning of the body.

The potential adaptogen combat adverse conditions and restored altered level of these parameters to normal (Cao et al., 1993). The role of AF in attenuating these altered features may be visualized as a form of adaptation on the part of the GSH-dependent defense system against lipid peroxidation. This unique effect of the AF may be attributed to its anti-oxidant properties which may inhibit the deleterious effect of free radicals generated by diverse stress situations. This may be further supported by in vitro free radical scavenging activity (Tables 5,7 and 8).

Conclusion

At the cellular level, stress affects our ability to properly transform glucose into energy. [beta]-lipoproteins build up and inhibit the passage of energy through the cell walls. This reduced energy level not only affects our ability to perform physical functions, but also inhibits the proper functioning of the body organs-including the brain. Adaptogens provide the basis through which one can build up an energy reserve to be tapped when the body needs it most under extreme physical/chemical tension and during recovery from fatigue. After adaptogen administration performance of physical labour revealed that AF treated animals showed an improvement in their general physical and mental states i.e. coordination of movement and a reduction in the duration of the recovery period in all the test subjects. This clearly demonstrates the value of adaptogens for increasing stamina and accelerating recovery processes after physical/chemical exertion.

Acknowledgements

The authors recognize the valuable contributions as a technical assistance of Mrs. Reeta Bhat, Mr. Benjamin, and Mr. Karan Singh.

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B. Singh (a,*), B.K. Chandan (a), N. Sharma (a), S. Singh (a), A. Khajuria (a), D.K. Gupta (b)

(a) Departments of Pharmacology Regional Research Laboratory, Canal Road, Jammu--Tawi, India

(b) Natural Products Chemistry, Regional Research Laboratory, Canal Road, Jammu--Tawi, India

Received 19 September 2002; accepted 6 October 2003

*Corresponding author.

E-mail address: bsjaggi2001@yahoo.com (B. Singh).
Table 1. Grouping and treatment schedule of animals

 Day 1-15 at Day 15, 1 h after
Group n = 6 24 h interval treatment

Group I Control without Vehicle No stress
 stress
Group II Control + Vehicle Stress
 stress
Group III 'AF' per se 'AF' 100 mg/kg, p.o. No stress
Group IV 'AF' + stress 'AF' 12.5 mg/kg, p.o. Stress
Group V 'AF' + stress 'AF' 25 mg/kg, p.o. Stress
Group VI 'AF' + stress 'AF' 50 mg/kg, p.o. Stress
Group VII 'AF' + stress 'AF' 100 mg/kg, p.o. Stress
Group VIII Std + stress Standard 125 mg/kg, p.o. Stress
Group IX PBZ + stress Reference 50 mg/kg, p.o.

The extract of Withania somnifera roots extract (a commercial
preparation available locally) was used as standard (std.).
Vehicle: Normal saline or 0.2% Gum acacia in normal saline.
PBZ: Phenylbutazone.
Number of animals in each group is six (n = 6).
Treatment: given orally by gastric intubation.
AF: Active fraction (aqueous solution in normal saline).

Table 2. Effect of oral administration of Trichopus zeylanicus Gaerten.
'AF', Std. and PBZ on adjuvant-induced acute (+ 18) and (+ 21 day)
reaction in male rats

 Dose Injected limb
Treatment (mg/kg) + 18 h + 21 days

Vehicle only -- -- --
Vehicle + AA -- 1.05[+ or -]0.05 2.83[+ or -]0.06
AF per se 100 -- --
AF + AA 12.5 0.94[+ or -]0.03 (NS) 2.53[+ or -]0.04 (NS)
 (10.32[+ or -]3.79) (10.42[+ or -]1.46)
AF + AA 25 0.76[+ or -]0.03 (NS) 1.76[+ or -]0.07*
 (27.46[+ or -]3.25) (37.58[+ or -]6.23)
AF + AA 50 0.59[+ or -]0.03* 1.44[+ or -]0.03**
 (43.33[+ or -]2.91) (48.88[+ or -]0.96)
AF + AA 100 0.47[+ or -]0.02** 1.18[+ or -]0.06**
 (54.76[+ or -]2.05) (58.12[+ or -]2.12)
Std. + AA 125 0.77[+ or -]0.03 (NS) 1.83[+ or -]0.05*
 (26.19[+ or -]2.97) (35.10[+ or -]1.98)
PBZ + AA 50 0.62[+ or -]0.03* 1.27[+ or -]0.03**
 (40.47[+ or -]2.97) (55.00[+ or -]1.22)

 Contralateral limb
Treatment + 21 days

Vehicle only --
Vehicle + AA 0.91[+ or -]0.04
AF per se --
AF + AA 1.31[+ or -]0.03**
 (44.68[+ or -]3.09)[up arrow]
AF + AA 1.49[+ or -]0.04**
 (63.73[+ or -]4.48)[up arrow]
AF + AA 1.57[+ or -]0.03**
 (72.89[+ or -]3.48)[up arrow]
AF + AA 1.78[+ or -]0.06**
 (96.34[+ or -]7.26)[up arrow]
Std. + AA 0.49[+ or -]0.03**
 (46.15[+ or -]3.25)[down arrow]
PBZ + AA 0.33[+ or -]0.03**
 (63.00[+ or -]2.97)[down arrow]

Data shows edema in ml (mean[+ or -]SE, n = 6) and within parentheses,
the percent inhibition in inflammation of injected limb and secondary
reaction to adjuvant of un-injected limb (contralateral limb) by drug
treatment expressed as mean[+ or -]SE.
PBZ: Phenylbutazone.
AA: Adjuvant induced Arthritis.
[up arrow]: % increase of secondary lesions as compared to vehicle
treated animals.
[down arrow]: % inhibition of secondary lesions as compared to vehicle
treated animals.
**p<0.01; *p<0.05; (NS) not significant (Dunnett's t-test against the
respective control).

Table 3. Effect of oral administration of Trichopus zeylanicus Gaerten.
'AF', Std. and levamisole (LEV) on antigen (SRBC) induced humoral
antibody synthesis in normal and immuno-suppressed mice and delayed type
of hypersensitivity (DTH) reaction in male mice

 Normal immunocompetent mice
 Primary Secondary
 Dose antibody titre antibody titre
Treatment (mg/kg) Mean[+ or -]SE Mean[+ or -]SE

Control + -- 5.66[+ or -]0.42 5.83[+ or -]0.47
 SRBC
AF + SRBC 12.5 6.50[+ or -]0.43 (NS) 7.16[+ or -]0.47 (NS)
 (14.84[+ or -]7.56) (22.92[+ or -]8.18)
AF + SRBC 25 7.50[+ or -]0.42* 7.83[+ or -]0.47*
 (32.51[+ or -]7.56) (34.36[+ or -]8.18)
AF + SRBC 50 8.83[+ or -]0.47** 8.11[+ or -]0.47*
 (56.06[+ or -]8.43) (40.07[+ or -]8.18)
AF + SRBC 100 9.83[+ or -]0.47** 9.16[+ or -]0.60**
 (73.75[+ or -]8.43) (57.23[+ or -]10.30)
Std + SRBC 125 7.33[+ or -]0.49 (NS) 7.33[+ or -]0.42 (NS)
 (29.56[+ or -]8.73) (25.78[+ or -]7.23)
Lev. + SRBC 2.5 8.50[+ or -]0.42** 8.50[+ or -]0.42**
 (50.17[+ or -]7.56) (45.79[+ or -]7.34)

 Normal immunocompetent
 Immunosuppressed mice mice
 Humoral DTH response
 antibody titre Edema mm
Treatment Mean[+ or -]SE Mean[+ or -]SE

Control + 4.16[+ or -]0.30 0.38[+ or -]0.03
 SRBC
AF + SRBC 5.5[+ or -]0.42 (NS) 0.46[+ or -]0.03 (NS)
 (33.49[+ or -]9.44) (22.80[+ or -]8.77)
AF + SRBC 6.5[+ or -]0.42** 0.6[+ or -]0.03**
 (56.25[+ or -]10.29) (57.89[+ or -]9.61)
AF + SRBC 7.0[+ or -]0.36** 0.70[+ or -]0.03**
 (68.27[+ or -]8.78) (84.21[+ or -]9.61)
AF + SRBC 7.66[+ or -]0.42** 0.78[+ or -]0.04**
 (84.30[+ or -]10.14) (106.14[+ or -]12.55)
Std + SRBC 5.66[+ or -]0.33 (NS) 0.50[+ or -]0.02 (NS)
 (36.22[+ or -]8.01) (31.58[+ or -]6.79)
Lev. + SRBC 6.0[+ or -]0.51* 0.61[+ or -]0.04**
 (44.23[+ or -]12.41) (62.28[+ or -]12.56)

Increase of primary and secondary humoral antibody synthesis in normal
and immunosuppressed male mice and delayed type of hypersensitivity
(DTH) reaction in male mice expressed as the mean[+ or -]SE (n = 6), and
within parentheses, the percent increase of humoral primary and
secondary antibody synthesis and increase in delayed type of
hypersensitivity (DTH) reaction by drug treatment expressed as
mean[+ or -]SE against the respective control.
**p<0.01; *p<0.05; (NS) not significant (Dunnett's t-test against the
respective control).

Table 4. Effect of oral administration of Trichopus zeylanicus Gaerten.
'AF' and Std. on immobilization (physical) stress induced biochemical
parameters in adrenal glands in rats (a)

 Ascorbic acid Cholesterol (g/
 Dose (mg/100 g) of 100 g) of adrenal
Treatment (mg/kg) adrenal wt. wt.

Control -- 342.33[+ or -]7.85 3.96[+ or -]0.32
without
stress
Control + -- 191.08[+ or -]18.59** 2.11[+ or -]0.09**
stress
AF per se 100 341.80[+ or -]9.95 3.83[+ or -]0.24
AF + stress 12.5 229.60[+ or -]13.13 (NS) 2.55[+ or -]0.16 (NS)
 (25.46[+ or -]6.81) (27.78[+ or -]8.68)
AF + stress 25 275.68[+ or -]13.13** 3.10[+ or -]0.24 (NS)
 (54.94[+ or -]8.72) (53.51[+ or -]13.16)
AF + stress 50 302.52[+ or -]12.45** 3.61[+ or -]0.30**
 (73.67[+ or -]8.23) (81.44[+ or -]16.24)
AF + stress 100 339.36[+ or -]15.80** 3.76[+ or -]0.19**
 (99.06[+ or -]10.47 (89.54[+ or -]10.51)
Std. + 125 224.46[+ or -]13.77 (NS) 2.26[+ or -]0.16 (NS)
stress (22.52[+ or -]8.88) (3.24.9.66)

 Corticosterone Adrenal Weight
 ([micro]g/100 g) of (mg/100 g) of live
Treatment adrenal wt. wt.

Control 5.01[+ or -]0.28 14.16[+ or -]0.54
without
stress
Control + 1.62[+ or -]0.17** 33.63[+ or -]1.63**
stress
AF per se 4.91[+ or -]0.25 13.45[+ or -]0.47
AF + stress 2.51[+ or -]0.18 (NS) 26.28[+ or -]0.91**
 (26.43[+ or -]5.46) (37.72[+ or -]4.68)
AF + stress 3.10[+ or -]0.19** 23.36[+ or -]0.83**
 (43.65[+ or -]5.85) (52.71[+ or -]4.29)
AF + stress 3.65[+ or -]0.25** 21.17[+ or -]0.98**
 (59.87[+ or -]7.45) (64.34[+ or -]4.84)
AF + stress 4.01[+ or -]0.27** 17.60[+ or -]1.42**
 (70.69[+ or -]8.21) (82.33[+ or -]7.29)
Std. + 3.03[+ or -]0.21** 27.68[+ or -]1.04**
stress (49.55[+ or -]6.35) (30.54[+ or -]5.34)

*p< 0.05, **p<0.01. (NS) p>0.05 (not significant) Dunnett's t-test
against the respective control.
(a) Values represent mean[+ or -]SE (n = 6) and within parentheses
percent protection {[1-(TS-V/VS-V)] X 100}, where V is the control
without stress, TS and VS are the drug + stress and control + stress
treated groups of animals, respectively.

Table 5. Effect of oral administration of Trichopus zeylanicus Gaerten'
AF'. Std and Silymarin on physical (immobilization) induced stress
hepatic function in rats (a)

 Dose Serum
 mg/kg, parameters
Treatment p.o. GPT (units)

Vehical control -- 61.67[+ or -]9.38
Vehicle + stress -- 1079.56[+ or -]42.82
AF Per se 100 62.13[+ or -]11.01
AF + stress 12.5 799.05[+ or -]33.93 (NS)
 (27.56[+ or -]3.33)
AF + stress 25 629.65[+ or -]36.07 (NS)
 (44.20[+ or -]3.54)
AF + stress 50 500.08[+ or -]24.97**
 (56.93[+ or -]2.45)
AF + stress 100 403.91[+ or -]24.57**
 (66.31[+ or -]2.40)
Std. + stress 125 801.51[+ or -]33.27**
 (27.32[+ or -]3.27)
Silymarin + stress 50 508.80[+ or -]37.02**
 (56.07[+ or -]3.64)

 Hepatic
 parameter
Treatment GOT (units) ALP (a)

Vehical control 44.53[+ or -]5.36 20.71[+ or -]1.38
Vehicle + stress 944.89[+ or -]43.55 40.78[+ or -]1.15
AF Per se 45.67[+ or -]3.79 21.32[+ or -]1.72
AF + stress 633.61[+ or -]55.47 (NS) 32.69[+ or -]2.10 (NS)
 (34.57[+ or -]6.16) (40.29[+ or -]10.48)
AF + stress 444.63[+ or -]27.58** 29.79[+ or -]2.26**
 (55.56[+ or -]3.06) (54.75[+ or -]11.26)
AF + stress 391.72[+ or -]25.01** 24.40[+ or -]1.77**
 (61.44[+ or -]2.78) (81.61[+ or -]8.84)
AF + stress 299.18[+ or -]30.45** 20.59[+ or -]1.33**
 (71.72[+ or -]3.38) (100.58[+ or -]6.65)
Std. + stress 695.27[+ or -]32.28 (NS) 38.19[+ or -]1.11 (NS)
 (27.72[+ or -]3.58) (14.31[+ or -]4.79)
Silymarin + stress 477.36[+ or -]25.84** 33.51[+ or -]2.08*
 (51.85[+ or -]2.88) (39.27[+ or -]7.85)

Treatment Bilirubin (mg%) Triglycerides (mg%)

Vehical control 0.12[+ or -]0.01 7.58[+ or -]0.65
Vehicle + stress 0.45[+ or -]0.02 40.04[+ or -]0.77
AF Per se 0.13[+ or -]0.02 7.28[+ or -]0.36
AF + stress 0.37[+ or -]0.02 (NS) 35.68[+ or -]1.61 (NS)
 (22.23[+ or -]6.58) (13.40[+ or -]4.97)
AF + stress 0.30[+ or -]0.01* 31.00[+ or -]1.20 (NS)
 (44.44[+ or -]4.99) (27.85[+ or -]3.71)
AF + stress 0.23[+ or -]0.01** 23.46[+ or -]1.44**
 (65.66[+ or -]5.62) (51.07[+ or -]4.45)
AF + stress 0.21[+ or -]0.01** 19.09[+ or -]1.41**
 (72.22[+ or -]5.55) (64.52[+ or -]4.34)
Std. + stress 0.41[+ or -]0.01 (NS) 37.52[+ or -]1.43 (NS)
 (14.69[+ or -]3.32) (7.75[+ or -]4.41)
Silymarin + stress 0.35[+ or -]0.02* 30.88[+ or -]1.70 (NS)
 (30.31[+ or -]6.77) (28.21[+ or -]5.26)

Treatment Glycogen (b) Lipid Peroxidation (c)

Vehical control 5.67[+ or -]0.30 24.37[+ or -]1.86
Vehicle + stress 1.13[+ or -]0.16 58.62[+ or -]1.84
AF Per se 5.43[+ or -]0.30 24.68[+ or -]2.27
AF + stress 2.50[+ or -]0.20 (NS) 44.75[+ or -]2.53*
 (30.21[+ or -]4.52) (37.57[+ or -]7.02)
AF + stress 3.22[+ or -]0.29* 33.11[+ or -]1.81**
 (45.82[+ or -]6.48) (74.48[+ or -]5.30)
AF + stress 3.65[+ or -]0.23** 25.77[+ or -]1.84**
 (55.40[+ or -]5.02) (95.90[+ or -]5.38)
AF + stress 4.83[+ or -]0.34** 46.34[+ or -]2.40**
 (81.57[+ or -]7.50) (103.27[+ or -]3.86)
Std. + stress 1.42[+ or -]0.24 (NS) 23.25[+ or -]1.32*
 (8.35[+ or -]4.78) (35.86[+ or -]7.03)
Silymarin + stress 2.89[+ or -]0.31* 36.96[+ or -]2.09**
 (38.73[+ or -]6.82) (63.23[+ or -]6.12)

Treatment Glutathione (d)

Vehical control 6.86[+ or -]0.38
Vehicle + stress 2.65[+ or -]0.25
AF Per se 6.96[+ or -]0.37
AF + stress 3.48[+ or -]0.23 (NS)
 (19.75[+ or -]5.54)
AF + stress 4.47[+ or -]0.25*
 (43.30[+ or -]6.05)
AF + stress 5.68[+ or -]0.39**
 (71.93[+ or -]9.33)
AF + stress 6.86[+ or -]0.32**
 (100.12[+ or -]7.75)
Std. + stress 3.64[+ or -]0.29 (NS)
 (23.47[+ or -]6.97)
Silymarin + stress 5.17[+ or -]0.31**
 (59.81[+ or -]7.58)

Unit:[micro]mol pyruvate/min/l.
* p<0.05, ** p<0.01, (NS) p>0.05, [not significant] Dunnett's t-test
against the respective control.
e: is [micro]mol GSH/g Liver.
(a) Values represent the Mean[+ or -]SE of six animals (rats, wistar,
150-175 g body weight, male) in each group. The values with in
parentheses represent percent hepatoprotection.
(b) [micro]mol of p-nitrophenol formed/min/l.
(c) mg/g Liver.
(d) n moles MDA/g liver.

Table 6. Effect of oral administration of Trichopus zeylanicus Gaerten.
'AF' and Std. on gastric lesion subjected to Immoblization induced
stress gastric ulceration in rats

 (%) Incidence of ulcers
Treatment Dose (mg/kg) Mean [+ or -] SE

Control + stress -- 100.39
AF per se 100 0
AF + stress 12.5 82.55 [+ or -] 4.51 (b*)
AF + stress 25 70.79 [+ or -] 5.35 (b**)
AF + stress 50 56.33 [+ or -] 4.92 (b**)
AF + stress 100 43.03 [+ or -] 2.48 (b**)
Std + stress 125 74.97 [+ or -] 2.57 (b**)

 (%) Protection (a) Severity
Treatment Mean [+ or -] SE Mean [+ or -] SE

Control + stress 0 33.50 [+ or -] 1.05
AF per se 100 0
AF + stress 17.91 [+ or -] 4.52 27.66 [+ or -] 0.84 (bNS)
AF + stress 29.48 [+ or -] 5.32 25.16 [+ or -] 1.11 (b*)
AF + stress 43.90 [+ or -] 4.91 19.33 [+ or -] 0.95 (b**)
AF + stress 57.09 [+ or -] 2.46 16.66 [+ or -] 0.88 (b**)
Std + stress 25.32 [+ or -] 2.56 26.00 [+ or -] 1.65 (bNS)

Incidence of ulcers is expressed as (%) Mean [+ or -] SE against control
(n = 6).
Severity of ulcers is expressed as (%) Mean [+ or -] SE against control
(n = 6).
** p < 0.01; * p< 0.05; (NS) not significant (Dunnett's t-test against
the respective control.
(a) % protection of incidence of ulcers is expressed as (%) Mean
[+ or -] SE against control.
(b) Difference in relation to vehicle + stress treated rats.

Table 7. Effect of oral administration of Trichopus zeylanicus Gaerten.
'AF', Std and Silymarin (fed at 24 h interval for 13 days and, 2 h
before and 6 h after hepatotoxin on day 14th) on chemically (GalN 300
mg/kg, subcutaneously, prophylactic study) induced stress hepatic
function in rats (a)

 Dose Serum parameters
Treatment mg/kg, p.o. GPT (units)

Vehical control -- 79.06 [+ or -] 7.53
Vehicle + GalN -- 1316.68 [+ or -] 77.65
AF' per se 100 85.46 [+ or -] 7.69
AF' + GalN 12.5 892.28 [+ or -] 41.34 (NS)
 (34.29 [+ or -] 3.34)
AF' + GalN 25 763.77 [+ or -] 43.16*
 (44.67 [+ or -] 3.48)
AF' + GalN 50 538.42 [+ or -] 31.63**
 (62.98 [+ or -] 2.49)
AF' + GalN 100 410.74 [+ or -] 35.07**
 (73.20 [+ or -] 2.83)
Std. + GalN 125 957.98 [+ or -] 51.80 (NS)
 (28.98 [+ or -] 4.18)
Silymarin + GalN 50 694.62 [+ or -] 43.25**
 (50.26 [+ or -] 3.49)

 Serum parameters
Treatment GOT (units) ALP (b)

Vehical control 105.08 [+ or -] 11.63 19.67 [+ or -] 1.44
Vehicle + GalN 1144.88 [+ or -] 51.82 50.84 [+ or -] 2.26
AF' per se 101.66 [+ or -] 7.75 19.20 [+ or -] 1.49
AF' + GalN 788.43 [+ or -] 41.63 (NS) 40.0 [+ or -] 2.21 (NS)
 (34.28 [+ or -] 4.00) (34.77 [+ or -] 7.10)
AF' + GalN 613.44 [+ or -] 22.17** 34.85 [+ or -] 1.95**
 (51.11 [+ or -] 2.13) (52.21 [+ or -] 5.85)
AF' + GalN 496.68 [+ or -] 28.31** 31.13 [+ or -] 2.07**
 (62.34 [+ or -] 2.72) (63.23 [+ or -] 6.64)
AF' + GalN 410.89 [+ or -] 29.63** 26.12 [+ or -] 2.04**
 (70.59 [+ or -] 2.85) (79.28 [+ or -] 6.54)
Std. + GalN 632.24 [+ or -] 26.05* 42.33 [+ or -] 1.77 (NS)
 (49.20 [+ or -] 2.54) (27.31 [+ or -] 5.69)
Silymarin + GalN 550.45 [+ or -] 25.19** 35.01 [+ or -] 1.82**
 (57.16 [+ or -] 2.42) (50.78 [+ or -] 5.86)

 Serum parameters
 Bilirubin Triglycerides
Treatment (mg%) (mg%)

Vehical control 0.15 [+ or -] 0.01 9.94 [+ or -] 0.93
Vehicle + GalN 0.63 [+ or -] 0.02 45.23 [+ or -] 2.15
AF' per se 0.15 [+ or -] 0.01 9.64 [+ or -] 0.62
AF' + GalN 0.48 [+ or -] 0.01** 39.52 [+ or -] 1.70 (NS)
 (30.55 [+ or -] 3.94) (16.16 [+ or -] 4.83)
AF' + GalN 0.41 [+ or -] 0.02** 34.40 [+ or -] 1.64 (NS)
 (45.83 [+ or -] 4.13) (30.68 [+ or -] 4.65)
AF' + GalN 0.33 [+ or -] 0.01** 26.53 [+ or -] 2.00**
 (61.81 [+ or -] 2.78) (52.98 [+ or -] 5.69)
AF' + GalN 0.23 [+ or -] 0.01** 2.66 [+ or -] 1.51**
 (82.29 [+ or -] 3.22) (70.04 [+ or -] 4.57)
Std. + GalN 0.43 [+ or -] 0.01** 40.00 [+ or -] 1.26 (NS)
 (40.62 [+ or -] 3.56) (14.81 [+ or -] 3.59)
Silymarin + GalN 0.37 [+ or -] 0.01** 34.27 [+ or -] 1.60*
 (53.47 [+ or -] 3.72) (31.06 [+ or -] 4.55)

 Hepatic parameter
Treatment Glycogen (c) Lipid Peroxidation (d)

Vehical control 4.92 [+ or -] 0.44 30.10 [+ or -] 1.29
Vehicle + GalN 0.87 [+ or -] 0.10 67.74 [+ or -] 3.45
AF' per se 4.88 [+ or -] 0.40 29.88 [+ or -] 1.76
AF' + GalN 1.58 [+ or -] 0.23 (NS) 52.05 [+ or -] 2.21*
 (20.16 [+ or -] 4.99) (41.68 [+ or -] 5.88)
AF' + GalN 2.83 [+ or -] 0.14** 45.55 [+ or -] 1.27**
 (50.69 [+ or -] 3.70) (53.63 [+ or -] 3.39)
AF' + GalN 3.58 [+ or -] 0.23** 37.88 [+ or -] 1.98**
 (67.07 [+ or -] 5.71) (79.32 [+ or -] 5.25)
AF' + GalN 4.69 [+ or -] 0.26** 32.42 [+ or -] 1.77**
 (94.32 [+ or -] 6.59) (93.84 [+ or -] 4.71)
Std. + GalN 1.53 [+ or -] 0.16 (NS) 49.15 [+ or -] 1.75**
 (16.42 [+ or -] 3.96) (49.38 [+ or -] 4.61)
Silymarin + GalN 3.06 [+ or -] 0.36** 43.69 [+ or -] 2.10**
 (53.00 [+ or -] 9.25) (63.88 [+ or -] 5.58)

 Hepatic parameter
Treatment Glutathione (e)

Vehical control 7.18 [+ or -] 0.36
Vehicle + GalN 2.49 [+ or -] 0.31
AF' per se 7.04 [+ or -] 0.42
AF' + GalN 4.26 [+ or -] 0.32*
 (37.77 [+ or -] 6.92)
AF' + GalN 5.01 [+ or -] 0.39**
 (53.84 [+ or -] 8.33)
AF' + GalN 5.58 [+ or -] 0.42**
 (66.06 [+ or -] 9.13)
AF' + GalN 6.67 [+ or -] 0.54**
 (89.23 [+ or -] 11.49)
Std. + GalN 3.79 [+ or -] 0.28 (NS)
 (27.89 [+ or -] 6.17)
Silymarin + GalN 5.02 [+ or -] 0.29**
 (54.12 [+ or -] 6.26)

Unit: [micro]mol pyruvate/min/1.
* p < 0.05, ** p < 0.01, (NS) p > 0.05, [not significant] Dunnett's
t-test against the respective control.
(a) Values represent the mean + SE of six animals (rats, wistar, 150-175
g body weight, male) in each group. The values with in parentheses
represent percent hepatoprotection.
(b) [micro]mol of p-nitrophenol formed/min/1.
(c) mg/g Liver.
(d) nmol MDA/g liver.
(e) is [micro]mol GSH/g Liver.

Table 8. Effect of Trichopus zeylanicus Gaerten 'AF' Quercetin and Rutin
on in vitro free radical scavenger activity

 Final concentration
Test material ([micro]g/ml) Percent activity

AF 1.25 26.4 (NS)
 2.5 39.2*
 5 48.6**
 10 59.2**
 20 88.4**
 40 93.0**
Quercetin 0.8 30.8 (NS)
 1.6 59.9**
 2.4 86.4**
 3.2 97.8**
 4.0 98.6**
Rutin 4 61.6**
 8 89.9**

In vitro free radical scavenger activity was determined using DPPH.
Reaction mixture in a final volume of 2.5 ml contained test material
and DPPH (0.6 X [10.sup.-4] M) in methanol Rutin and Quercetin were
dissolved in methanol and 'AF' was dissolved in distilled water. After
30 min of incubation at room temperature the colour was extracted in
toluene and absorbance was recorded using UVIKON-922 spectrophotometer
at 517 nm. Percent decolorization was taken as free radical scavenger
activity. Data reported here are mean values of two experiments done in
duplicate.
* p<0.05, ** p < 0.01, (NS) p > 0.05, [not significant] Dunnett's t-test
against the respective control.
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Author:Singh, B.; Chandan, B.K.; Sharma, N.; Singh, S.; Khajuria, A.; Gupta, D.K.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Geographic Code:9INDI
Date:Jun 1, 2005
Words:10973
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