De Novo Transcriptome Profiling for the Generation and Validation of Microsatellite Markers, Transcription Factors, and Database Development for Andrographis paniculata.

Andrographis paniculata belongs to the family Acanthaceae and is known for its medicinal properties owing to the presence of unique constituents belonging to the lactones, diterpenoids, diterpene glycosides, flavonoids, and flavonoid glycosides groups of chemicals. Andrographolide, a major therapeutic constituent of A. paniculata, is extracted primarily from the leaves of this plant and exhibits antimicrobial and anti-inflammatory activities. Using 454 GS-FLX pyrosequencing, we have generated a whole transcriptome profile of entire leaves of A. paniculata. A total of 22,402 high-quality transcripts were generated, with an average transcript length and N50 of 884 bp and 1007 bp, respectively. Functional annotation revealed that 19,264 (86%) of the total transcripts showed significant similarity with the NCBI-Nr database and were successfully annotated. Out of the 19,264 BLAST hits, 17,623 transcripts were assigned GO terms and distributed into three major functional categories: molecular function (44.62%), biological processes (29.19%), and cellular component (26.18%) based on BLAST2GO. Transcription factor analysis showed 6669 transcripts, belonging to 57 different transcription factor families. Fifteen TF genes that belong to the NAC, MYB, and bHLH TF categories were validated by RT PCR amplification. In silico analysis of gene families involved in the synthesis of biochemical compounds having medicinal values, such as cytochrome p450, protein kinases, heat shock proteins, and transporters, was completed and a total of 102 different transcripts encoding enzymes involved in the biosynthesis of terpenoids were predicted. Out of these, 33 transcripts belonged to terpenoid backbone biosynthesis. This study also identified 4254 EST-SSRs from 3661 transcripts, representing 16.34% of the total transcripts. Fifty-three novel EST-SSR markers generated from our EST dataset were used to assess the genetic diversity among eighteen A. paniculata accessions. The genetic diversity analysis revealed two distinct sub-clusters and all accessions based on the genetic similarity index were distinct from each other. A database based on EST transcripts, EST-SSR markers, and transcription factors has been developed using data generated from the present study combined with available transcriptomic resources from a public database using Meta transcriptome analysis to make genomic resources available in one place to the researchers working on this medicinal plant.

Development of Novel Genomic Simple Sequence Repeat (g-SSR) Markers and Their Validation for Genetic Diversity Analyses in Kalmegh [Andrographis paniculata (Burm. F.) Nees].

Kalmegh (Andrographis paniculata (Burm. F.) Nees) is one of the most important medicinal plants and has been widely explored as traditional medicine. To exploit its natural genetic diversity and initiations of molecular breeding to develop novel cultivars or varieties, developments of genomic resources are essential. Four microsatellite-enriched genomic libraries-(CT)14, (GT)12, (AG)15 and (AAC)8-were constructed using the genomic DNA of A. paniculata. Initially, 183 recombinant colonies were screened for the presence of CT, GT, AG, and AAC microsatellite repeats, out of which 47 clones found positive for the desired simple sequence repeats (SSRs). It was found that few colonies had more than one desirable SSR. Thus, a sum of 67 SSRs were designed and synthesized for their validation among 42 A. paniculata accessions. Out of the 67 SSRs used for genotyping, only 41 were found to be polymorphic. The developed set of g-SSR markers showed substantial genetic variability among the selected A. paniculata accessions, with an average polymorphic information content (PIC) value of 0.32. Neighbor-joining tree analysis, population structure analysis, analysis of molecular variance (AMOVA), and principal coordinate analysis (PCoA) illustrated the considerable genetic diversity among them. The novel g-SSR markers developed in the present study could be important genomic resources for future applications in A. paniculata.

Antihepatotoxic activity of Euphorbia neriifolia extract against N-nitrosodiethylamine-induced hepatocarcinogenesis in mice.

OBJECTIVE: To scrutinize the protective role of hydroethanolic extract of Euphorbia neriifolia leaves (HEEN) against N-nitrosodiethylamine (DENA)-induced hepatocarcinogenesis in male Swiss albino mice. METHODS: Experimental mice were pretreated with 150 and 400 mg/kg of HEEN, or 0.5% and 1% of butylated hydroxyanisole (BHA) as a standard for 14 d prior to the administration of a single dose of 50 mg/kg of DENA. Levels of xenobiotic metabolizing enzymes such as cytochrome (Cyt) P450 and Cyt b5, activities of reduced glutathione (GSH), glutathione-S-transferase (GST), aspartate amino transaminase (AST), alanine amino transaminase (ALT) and alkaline phosphatase (ALP), and total protein and cholesterol content in the liver tissues were measured to determine the hepatotoxicity caused by DENA. RESULTS: The levels of Cyt P450 and Cyt b5 were significantly increased, and GST and GSH were significantly depleted after DENA treatment (P<0.01). The activities of AST, ALT and ALP, and the total protein content were also significantly dropped off (P<0.01). The total cholesterol level was markedly increased by DENA as compared with the normal group (P<0.01). However, the pre-supplementation of HEEN showed a remarkable amelioration of these abnormalities, and the levels of the antioxidant enzymes in the liver were significantly restored, which exhibited the dose-dependent protective effect against DENA-induced hepatotoxicity. CONCLUSION: HEEN exerts its chemopreventive effects by alleviating the xenobiotic enzymes and enhancing the levels of antioxidants and biochemical assays in DENA-induced carcinogenesis by reducing the formation of free radicals.

Renoprotective effects of Moringa oleifera pods in 7,12-dimethylbenz [a] anthracene-exposed mice.

OBJECTIVE: To investigate the potential of hydroethanolic extract of Moringa oleifera (MOHE) against 7, 12-dimethylbenz [a] anthracene (DMBA)-induced toxicity in male Swiss albino mice. METHODS: Experimental mice were respectively pretreated with 200 and 400 mg/kg of MOHE, and 0.5% and 1% of butylated hydroxyanisole (BHA) for two weeks prior to the administration of 15 mg/kg of DMBA, respectively. Levels of xenobiotic metabolizing enzymes such as cytochrome (Cyt) P450 and Cyt b5, activities of reduced glutathione (GSH) and glutathione-S-transferase (GST) and renal aspartate amino transaminase (AST), alanine amino transaminase (ALT) and alkaline phosphatase (ALP), and content of protein and total cholesterol were measured to determine the nephrotoxicity caused by DMBA and to elucidate the ameliorating role of M. oleifera. RESULTS: Single oral administration of 15 mg/kg of DMBA resulted in significant increases in Cyt P450 and Cyt b5 (P<0.01). The toxic effect of DMBA was justified by the significant decreases in the activities of GSH and GST in renal tissues (P<0.05). The levels of renal AST, ALT and ALP and protein content which are indicative of renocellular damage were also found decreased along with significant increase in total cholesterol content in DMBA-treated mice (P<0.01). The DMBA-induced alterations in the tissues were significantly reversed after pretreatment with 200 and 400 mg/kg of MOHE orally for 14 d (P<0.01). CONCLUSION: The effects of MOHE in enhancing the levels of antioxidants and enhancing the levels of biochemical assays in DMBA-induced carcinogenesis are by reducing the formation of free radicals. This study rationalizes the ethnomedicinal use of M. oleifera for the protection against nephrotoxicity induced by chemical carcinogens.

Chemopreventive efficacy of Moringa oleifera pods against 7, 12-dimethylbenz[a]anthracene induced hepatic carcinogenesis in mice.

Oxidative stress is a common mechanism contributing to initiation and progression of hepatic damage in a variety of liver disorders. Hence there is a great demand for the development of agents with potent antioxidant effect. The aim of the present investigation is to evaluate the efficacy of Moringa oleifera as a hepatoprotective and an antioxidant against 7, 12-dimethylbenz[a]anthracene induced hepatocellular damage. Single oral administration of DMBA (15 mg/kg) to mice resulted in significantly (p<0.001) depleted levels of xenobiotic enzymes like, cytochrome P450 and b5. DMBA induced oxidative stress was confirmed by decreased levels of reduced glutathione (GSH) and glutathione-S-transferase (GST) in the liver tissue. The status of hepatic aspartate transaminase (AST), alanine transaminase (ALT), and alkaline phosphatase (ALP) which is indicative of hepatocellular damage were also found to be decreased in DMBA administered mice. Pretreatment with the Moringa oleifera (200 and 400 mg/kg) orally for 14 days significantly reversed the DMBA induced alterations in the liver tissue and offered almost complete protection. The results from the present study indicate that Moringa oleifera exhibits good hepatoprotective and antioxidant potential against DMBA induced hepatocellular damage in mice that might be due to decreased free radical generation.

Chemopreventive effect of hydroethanolic extract of Euphorbia neriifolia leaves against DENA-induced renal carcinogenesis in mice.

The present study was conducted to investigate the chemopreventive effects of hydro-ethanolic extract of Euphorbia neriifolia (EN) on N-nitrosodiethylamine (DENA) induced renal cancer in male Swiss albino mice. Animals were pretreated with EN extract (150 and 400 mg/kg body weight; p.o) and butylated hydroxyanisole (BHA) as a standard (0.5% and 1% BHA p.o) both for two week prior to the administration of single dose of DENA (50 mg/kg body weight; p.o). Various in vivo antioxidant biochemical parameters like lipid peroxidation (LPO), superoxide dismutase (SOD), and catalase (CAT) were evaluated to determine the reno-protective and antioxidant activity of EN. DENA increased oxidative stress through increase in LPO and decrease in antioxidant enzymes (SOD, and CAT). The EN extract significantly restored the antioxidant enzyme level in the kidney and exhibited significant dose dependant protective effect against DENA induced nephrotoxicity, which can be mainly attributed to the antioxidant property of the extract. This study rationalized the ethno-medicinal use of EN for protection against renal cancer.