Phytochemical and therapeutic evaluation of leaf and In vitro derived callus and shoot of Solanum trilobatum L. (original) (raw)

Abstract

This study focuses on the phytochemical properties and the anti-hepatocarcinogenic effects of the leaf and in vitro-derived callus and shoot extracts of Solanum trilobatum. In the leaf, callus and shoot, the presence of sugar, proteins, alkaloids, flavonoids, saponins, tananins, cardiacglycoside, terpenoid and lipids was established by preliminary phytochemical screening. Surface-sterilized explants (0.5-1.0 cm) were placed on the MS basal medium supplemented with different concentrations of 2,4-dichlorophenoxyacetic acid (0.45, 2.26, 4.52, 11.31 and 12.56µM), naphthylacetic acid (NAA; 0.54, 1.34, 2.69, 5.37, 13.43 and 26.85µM) and 6-benzylaminopurine (BA; 0.44, 1.11, 2.22, 4.44, 8.88 and 13.32µM) for callus induction. Explants from node and callus culture were inoculated on the MS basal medium supplemented with varying concentrations of BA (0.44-22.20 µM) and NAA (0.54-10.74µM) for shoot multiplication. Rats were divided into five groups and administered with diethyl nitrosamine (DEN) and DEN (200mg/kg bwt) intraperitoneally along with methanol leaf and in vitro-derived callus and shoot extracts (250mg/kg bwt) orally for 3 months. A significant deviation (P<0.05) in marker enzymes such as alanine transaminase, aspartate transaminase, lactate dehydrogenase and total bilirubin was found in rats administered with DEN. The liver tissue was used for the analysis of glutathione reductase, lipid peroxidation, glutathione peroxidase, glutathione S-transferase, superoxide dismutase and catalase. DEN administration caused a significant elevation in serum enzymes and total bilirubin. Moreover, antioxidant enzymes were drastically inhibited with significant reduction in glutathione and increased lipid per oxidation. Increased glutathione level and reduced lipid peroxidation were also evident in S. trilobatum-treated rats. However, crude S. trilobatum and in vitro-derived callus and shoot extracts offered better protection against free radical toxicity induced by DEN.

Figures (10)

Male Wister rats weighing about 200+20g were used in the study. They were housed in a well-ventilated room with 12 h light/12h dark photoperiod. They were fed with standard animal feed (Lipton India, Ban galore, India) and water ad Libitum. Experiments were conducted in accordance with the institutional ethical committee guidelines (Biotech SBU.001/10). Fig. 2: Fresh weight and dry weight of callus of S. trilobatum. RESULTS

Male Wister rats weighing about 200+20g were used in the study. They were housed in a well-ventilated room with 12 h light/12h dark photoperiod. They were fed with standard animal feed (Lipton India, Ban galore, India) and water ad Libitum. Experiments were conducted in accordance with the institutional ethical committee guidelines (Biotech SBU.001/10). Fig. 2: Fresh weight and dry weight of callus of S. trilobatum. RESULTS

Fig. 1: Stages of callus grown in vitro: (A) emergence of callus from leaf explant; (B) callus growth in vitro; (C) shoot generation from the callus. leaves and standardized callus of S. trilobatum was taken and deposited into different organic solvents such as chloroform, methanol, and petroleum ether solution at a concentration of 10%w/v and allowed to stand overnight (table 5 a,b,c for concentration details). The extracts were filtered through Whatmann no.1 filter paper to remove cellular materials and other insoluble components. The extracts were allowed to concentrate using a rotary flash evaporator under reduced pressure. Qualitative tests for alkaloids, flavonoids, carbohydrates, glycosides, saponins and tannins, terpenoids, proteins and anthraquinone were performed. The tests for alkaloids showed positive. Mayers test, Wagners test and Dragendorff test were carried out using standard procedures. The results are presented in table 2.

Fig. 1: Stages of callus grown in vitro: (A) emergence of callus from leaf explant; (B) callus growth in vitro; (C) shoot generation from the callus. leaves and standardized callus of S. trilobatum was taken and deposited into different organic solvents such as chloroform, methanol, and petroleum ether solution at a concentration of 10%w/v and allowed to stand overnight (table 5 a,b,c for concentration details). The extracts were filtered through Whatmann no.1 filter paper to remove cellular materials and other insoluble components. The extracts were allowed to concentrate using a rotary flash evaporator under reduced pressure. Qualitative tests for alkaloids, flavonoids, carbohydrates, glycosides, saponins and tannins, terpenoids, proteins and anthraquinone were performed. The tests for alkaloids showed positive. Mayers test, Wagners test and Dragendorff test were carried out using standard procedures. The results are presented in table 2.

was adminis intraperitonea intraperitonea callus extract V was administered with intraperitonea ered with DE y) and co administered with methanol lea extract (250mg/kg bwt) orall was administered with DE y) and co ad (250mg/kg bwt) A D y) and co ad N (200mg/kg bwt single y for 3 months. Group IV N (200mg/kg bwt single ministered with methano orally for 3 months. Group EN (200mg/kg bwt single ministered with methano shoot extract (250mg/kg bwt) orally for 3 months. Fig. 3: Callus biomass in solid medium of a growing culture of S. trilobatum

was adminis intraperitonea intraperitonea callus extract V was administered with intraperitonea ered with DE y) and co administered with methanol lea extract (250mg/kg bwt) orall was administered with DE y) and co ad (250mg/kg bwt) A D y) and co ad N (200mg/kg bwt single y for 3 months. Group IV N (200mg/kg bwt single ministered with methano orally for 3 months. Group EN (200mg/kg bwt single ministered with methano shoot extract (250mg/kg bwt) orally for 3 months. Fig. 3: Callus biomass in solid medium of a growing culture of S. trilobatum

Table 1: Effect of plant growth regulators (PGRs) on callus induction Table 2: Preliminary phytochemical screening of leaf and callus of S. trilobatum

Table 1: Effect of plant growth regulators (PGRs) on callus induction Table 2: Preliminary phytochemical screening of leaf and callus of S. trilobatum

(table 1, fig. 1), wherein the callus was well developed, spongy and loosely arranged. Whereas, a very high concentration of 2,4-D (22.62uM) was not in favor of callus induction. The moisture content of callus was also high as compared to other auxin supplemented media. In NAA-supplemented medium, the callus was pale, yellowish green, more friable, hard and granular. In BA- supplemented medium, the callus was green, more compact, hard and granular. Fig. 2 shows that the maximum biomass yield was on day 24 in solid medium 82.3 and 7.8g DW/L), after which the biomass yield gradually decreased (fig. 2). The callus remained green up o 36 days and then started browning, being symptoms of aging. The plantlets were then transferred to paper cups diameter 6cm) containing red soil, vermiculite and farmyard manure in the ratio of 1:1:1 and allowed to harden. The plantlets were covered with polythene bags to prevent transpiration and to maintain a relative humidity

(table 1, fig. 1), wherein the callus was well developed, spongy and loosely arranged. Whereas, a very high concentration of 2,4-D (22.62uM) was not in favor of callus induction. The moisture content of callus was also high as compared to other auxin supplemented media. In NAA-supplemented medium, the callus was pale, yellowish green, more friable, hard and granular. In BA- supplemented medium, the callus was green, more compact, hard and granular. Fig. 2 shows that the maximum biomass yield was on day 24 in solid medium 82.3 and 7.8g DW/L), after which the biomass yield gradually decreased (fig. 2). The callus remained green up o 36 days and then started browning, being symptoms of aging. The plantlets were then transferred to paper cups diameter 6cm) containing red soil, vermiculite and farmyard manure in the ratio of 1:1:1 and allowed to harden. The plantlets were covered with polythene bags to prevent transpiration and to maintain a relative humidity

Values are mean +SE (n=5) significance **(P<0.05); *(P<0.01): Group I Vs Group II, II, 'V, V Table 3: Marker Enzyme level in the serum of different treatment group rats

Values are mean +SE (n=5) significance **(P<0.05); *(P<0.01): Group I Vs Group II, II, 'V, V Table 3: Marker Enzyme level in the serum of different treatment group rats

Values are expressed as GPx (nmol GSH oxidized/min/mg), GST (U/min/mg Protein), SOD (U/g Protein), CAT (nmol/min/mg Protein), GSH (nmol/g tissue) and LPO (nmol/mg) Values are mean +S.E (n=5). **(P<0.05): GroupI Vs Group II, II, IV, V Table 4: Antioxidant enzymes, lipid peroxidation and glutathione levels in the liver of experimental rats

Values are expressed as GPx (nmol GSH oxidized/min/mg), GST (U/min/mg Protein), SOD (U/g Protein), CAT (nmol/min/mg Protein), GSH (nmol/g tissue) and LPO (nmol/mg) Values are mean +S.E (n=5). **(P<0.05): GroupI Vs Group II, II, IV, V Table 4: Antioxidant enzymes, lipid peroxidation and glutathione levels in the liver of experimental rats

Table 5(a): Percentage yield of the sample extracts of Solanum trilobatum leaves. % Yield = (Weight of the Sample Extract *100) /Weight of Powdered Sample used (g) Overall Yield percentage = (2.25+4.56 - 8.03) =14.84%

Table 5(a): Percentage yield of the sample extracts of Solanum trilobatum leaves. % Yield = (Weight of the Sample Extract *100) /Weight of Powdered Sample used (g) Overall Yield percentage = (2.25+4.56 - 8.03) =14.84%

Table 5(b): Percentage yield of the sample extracts of Solanum trilobatum callus. % Yield = (Weight of the Sample Extract * 100) / Weight of Powdered Sample used (g) Overall Yield percentage = (2.56 +9.00+ 4.86) =16.82%

Table 5(b): Percentage yield of the sample extracts of Solanum trilobatum callus. % Yield = (Weight of the Sample Extract * 100) / Weight of Powdered Sample used (g) Overall Yield percentage = (2.56 +9.00+ 4.86) =16.82%

% Yield = (Weight of the Sample Extract * 100) / Weight of Powdered Sample used (g) Overall Yield percentage = (2.02 + 9.03 + 5.56) = 18.86% Table 5(c): Percentage yield of the sample extracts of Solanum trilobatum shoot.

% Yield = (Weight of the Sample Extract * 100) / Weight of Powdered Sample used (g) Overall Yield percentage = (2.02 + 9.03 + 5.56) = 18.86% Table 5(c): Percentage yield of the sample extracts of Solanum trilobatum shoot.

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