De novo transcriptome analysis unravels tissue-specific expression of candidate genes involved in major secondary metabolite biosynthetic pathways of Plumbago zeylanica: implication for pharmacological potential.

KEY MESSAGE: The present study provides comparative transcriptome analysis, besides identifying functional secondary metabolite genes of Plumbago zeylanica with pharmacological potential for future functional genomics, and metabolomic engineering of secondary metabolites from this plant towards diversified biomedical applications. ABSTRACT: Plumbago zeylanica is a widely used medicinal plant of the traditional Indian system of medicine with wide pharmacological potential to treat several disorders. The present study aimed to carry out comparative transcriptome analysis in leaf and root tissue of P. zeylanica using Illumina paired end sequencing to identify tissue-specific functional genes involved in the biosynthesis of secondary metabolites, contributing to its therapeutic efficacy. De novo sequencing assembly resulted in the identification of 62,321 "Unigenes" transcripts with an average size of 1325 bp. Functional annotation using BLAST2GO resulted in the identification of 50,301 annotated transcripts (80.71%) and GO assigned to 18,814 transcripts. KEGG pathway annotation of the "Unigenes" revealed that 2465 transcripts could be assigned to 242 KEGG pathway maps wherein the number of transcripts involved in secondary metabolism was distinct in root and leaf transcriptome. Among the secondary metabolite biosynthesis pathways, the cluster of "Unigenes" encoding enzymes of 'Phenylpropanoid biosynthesis pathway' represents the largest group (84 transcripts) followed by 'Terpenoid Backbone biosynthesis' (48 transcripts). The transcript levels of the candidate unigenes encoding key enzymes of phenylpropanoid (PAL, TAL) and flavanoid biosynthesis (CHS, ANS, FLS) pathways were up-regulated in root, while the expression levels of candidate "Unigenes" transcript for monoterpenoid (DXS, ISPF), diterpenoid biosynthesis (SPS, SDS) and indole alkaloid pathways (STR) were significantly higher in leaf of P. zeylanica. Interestingly, validation of differential gene expression profile by qRT-PCR also confirmed that candidate "Unigenes" enzymes of phenylpropanoid and flavonoid biosynthesis were highly expressed in the root, while the key regulatory enzymes of terpenoid and indole alkaloid compounds were up-regulated in the leaf, suggesting that (differences in) the levels of these functional genes could be attributed to the (differential) pharmacological activity (between root and leaf) in tissues of P. zeylanica.

Herbal medicines for liver diseases in India.

The use of natural remedies for the treatment of liver diseases has a long history, starting with the Ayurvedhic treatment, and extending to the Chinese, European and other systems of traditional medicines. The 21st century has seen a paradigm shift towards therapeutic evaluation of herbal products in liver diseases by carefully synergizing the strengths of the traditional systems of medicine with that of the modern concept of evidence-based medicinal evaluation, standardization of herbal products and randomized placebo controlled clinical trials to support clinical efficacy. The present review provides the status report on the scientific approaches made to herbal preparations used in Indian systems of medicine for the treatment of liver diseases. In spite of the availability of more than 300 preparations for the treatment of jaundice and chronic liver diseases in Indian systems of medicine using more than 87 Indian medicinal plants, only four terrestrial plants have been scientifically elucidated while adhering to the internationally acceptable scientific protocols. In-depth studies have proved Sylibum marianum to be anti-oxidative, antilipidperoxidative, antifibrotic, anti-inflammatory, immunomodulating and liver regenerative. Glycyrrhiza glabra has been shown to be hepatoprotective and capable of inducing an indigenous interferon. Picrorhiza kurroa is proved to be anti-inflammatory, hepatoprotective and immunomodulatory. Extensive studies on Phyllanthus amarus have confirmed this plant preparation as being anti-viral against hepatitis B and C viruses, hepatoprotective and immunomodulating, as well as possessing anti-inflammatory properties. For the first time in the Indian systems of medicine, a chemo-biological fingerprinting methodology for standardization of P. amarus preparation has been patented.

Efficacy of fluconazole and itraconazole in the treatment of oral candidiasis in HIV patients.

A total of 46 strains of Candida were collected from HIV infected patients, of which 25 strains were isolated from patients with oral candidiasis, and 21 strains were from mouthwash samples of asymptomatic carriers. The most common species isolated was Candida albicans (73.9%), followed by Candida tropicalis (21.7%). In vitro susceptibility of the strains to fluconazole and itraconazole was tested using minimum inhibitory concentration (MIC) studies by agar dilution technique. Out of the 18 strains of C. albicans isolated from mouthwash samples, four were resistant to fluconazole whereas only two were resistant to itraconazole. Out of 16 strains of C. albicans isolated from oral lesions, one was resistant to fluconazole where as all were sensitive to itraconazole. Among the other species of Candida tested, C. tropicalis gave higher MIC values to both drugs than other species such as Candida guillermondii and Candida krusei. In vitro MIC values correlated well with in vivo responses in patients. Hence, itraconazole may be used as an alternative in the treatment of candidiasis, which does not respond to fluconazole therapy.

Region-specific alterations in the concentrations of catecholamines and indoleamines in the brains of young and old F344 rats after L-deprenyl treatment.

The effects of L-deprenyl, a monoamine oxidase-B (MAO-B) inhibitor, on the concentrations of norepinephrine (NE), dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), serotonin (5-hydroxytryptamine), and 5-hydroxyindoleacetic acid (5-HIAA) in medial basal hypothalamus (MBH), substantia nigra (SN), striatum (Str), and nucleus accumbens (NAc) of young (3 month) and old (21 month) male F344 rats were examined after a 7-day wash-out period following 1, 15, or 30 days of deprenyl treatment in young rats and a 9-day wash-out period after a 10-week deprenyl treatment in old rats. The brain areas were microdissected and the concentrations of neurotransmitters were measured by High Performance liquid chromatography with electrochemical detection (HPLC-EC). Deprenyl administration following the drug wash-out period increased the concentrations of DOPAC in the SN, Str, and in the NAc of young rats but it was decreased in the NAc of old rats. The concentration of HVA was lower in the Str of young deprenyl-treated rats, and in the Str and NAc of old deprenyl-treated rats, but it was higher in the SN of young deprenyl-treated rats. The concentration of 5-HIAA was increased in the MBH, SN, and in the NAc of young deprenyl-treated rats, but it was decreased in the Str and NAc of old deprenyl-treated rats. The concentration of NE was increased in the MBH, SN, Str, and in the NAc of young rats treated with deprenyl and in the MBH of old deprenyl-treated rats. The concentration of 5-HT was increased in the SN of young deprenyl-treated rats. The concentration of DA increased in the Str of both young and old deprenyl-treated rats. We concluded that a drug wash-out period after deprenyl treatment differentially affects the metabolism of catecholamines and indoleamine depending on the region of the brain and that this effect may be due to variation in the kinetics of MAO inhibition.

Effect of the extract of Annona muricata and Petunia nyctaginiflora on Herpes simplex virus.

Annona muricata (Annonaceae) and Petunia nyctaginiflora (Solanaceae) were screened for their activity against Herpes simplex virus-1 (HSV-1) and clinical isolate (obtained from the human keratitis lesion). We have looked at the ability of extract(s) to inhibit the cytopathic effect of HSV-1 on vero cells as indicative of anti-HSV-1 potential. The minimum inhibitory concentration of ethanolic extract of A. muricata and aqueous extract of P. nyctaginiflora was found to be 1 mg/ml.

Phyllanthus amarus suppresses hepatitis B virus by interrupting interactions between HBV enhancer I and cellular transcription factors.

The Phyllanthus amarus plant suppresses HBV mRNA transcription in vitro and exhibits therapeutic potential in chronic HBV carriers, although further work is necessary to define its mechanism of action. Analysis in HuH-7 cells with transfected plasmids using a luciferase reporter showed that P. amarus specifically inhibited HBV enhancer I activity. To identify the mechanism of this HBV enhancer I inhibition, liver-enriched cellular transcription factors were co-expressed in HuH-7 cells. The C/EBP alpha and beta, as well as HNF-3 alpha and beta transcription factors, significantly up-regulated the HBV enhancer I activity. In contrast, co-transfection of HNF-I alpha or beta had no effect upon the HBV enhancer I activity. Exposure to P. amarus inhibited C/EBP alpha- and beta-mediated up-regulation of HBV enhancer I activity in a dose-dependent manner, whereas HNF-3 alpha- and beta-mediated up-regulation of HBV enhancer I was unaffected. In vitro gel shifts showed that P. amarus inhibited complexing of C/EBP transcription factors to a consensus oligonucleotide sequence, whereas DNA binding of AP-1 and SP-1 transcription factors was unaffected. As P. amarus down-regulates HBV mRNA transcription by a specific mechanism involving interactions between HBV enhancer I and C/EBP transcription factors, purification and further analysis of the active P. amarus component will advance insights into its antiviral activity.

Phyllanthus amarus down-regulates hepatitis B virus mRNA transcription and replication.

The Phyllanthus amarus plant shows potential for treating hepatitis B virus. To define the mechanism of action of P. amarus, we used HepG2 2.2.15 cells, which support hepatitis B virus replication. P. amarus inhibited hepatitis B virus polymerase activity, decreased episomal hepatitis B virus DNA content and suppressed virus release into culture medium. To examine transcriptional control mechanisms, we used G26 hepatitis B virus transgenic mice, which produce serum HBsAg but neither HBcAg nor virion particles. When P. amarus was administered to transgenic mice, hepatic HBsAg mRNA levels decreased, indicating transcriptional or post-transcriptional down-regulation of the transgene. Increase in hepatitis B virus mRNA expression after stimulation of the glucocorticoid responsive element was also suppressed by P. amarus, suggesting involvement of the hepatitis B virus enhancer in this response. Disruption by P. amarus of hepatitis B virus polymerase activity, mRNA transcription and replication supports its role as an antiviral agent.

Inhibition of HBsAg secretion from Alexander cell line by Phyllanthus amarus.

Alexander cell line, an human hepatocellular carcinoma derived cell line which has the property of secreting HBsAg in the supernatant was used to study the antiviral property of phyllanthus amarus. Aquous extract of Phyllanthus amarus was evaluated for its in vitro ability to inhibit HBsAg secretion on a dose dependent manner. It was seen that P. amarus at 1mg/ml concentration on a single dose inhibited the secretion of HBsAg for a period of 48 hours. This experiment proved the anti hepatitis B virus property of P. amarus at cellular level and further confirmed its beneficial use in the treatment of acute and chronic hepatitis B and healthy carriers of HBV.

Autoimplantation technique in the treatment of anogenital warts: a clinico-immunological study.

An autoimplantation technique was adopted in the treatment of 50 cases of anogenital warts and was compared with the conventionally used podophyllin regimen in a matched group of 50 patients. They were assessed with 15 untreated subjects in a control group for the rate of clinical cure after 6 weeks, recurrence after 1 year follow up and for humoral and cell mediated immune responses before and after treatment. In the podophyllin group, 70% of patients were cured after 6 weeks while in autoimplantation, only 44% of patients were cured, and none in the control group had natural remission of warts without any treatment. After 1 year all the cured cases (100%) that completed follow up had recurrence of warts with podophyllin treatment, while none had recurrence of lesions in the autoimplantation group. Results of the humoral and cell mediated immune (CMI) response studies revealed that autoimplantation technique significantly augmented both humoral and CMI responses while there was not significant change in the immune status after podophyllin treatment (P > 0.001).

Underfeeding-induced suppression of mammary tumors: counteraction by estrogen and haloperidol.

The purpose of this study was to investigate the mechanism by which underfeeding induces regression of carcinogen-induced mammary tumors in the rat and to determine if tumor regression in underfed rats could be prevented on a chronic basis by maintaining elevated circulating levels of estrogen and/or prolactin (PRL) by treatment with estradiol benzoate (EB) and a dopamine receptor blocker, haloperidol (HAL). Female rats with 7,12-dimethylbenzanthracene-induced mammary tumors were fed ad libitum (full-fed), half-fed (HF), or half-fed and treated wtih EB (HF+EB), HAL (HF+HAL), or both (HF+EB+HAL) for 15 weeks. Tumor diameter, tumor number, and body weight were determined each week. At the end of the experiment, hypothalamic concentrations of catecholamines, indoleamines, and their metabolites were determined by high performance liquid chromatography. Tumor diameter, tumor number, and body weight increased progressively in the full-fed rats, but decreased significantly in the HF rats. Treatment of HF rats with EB, HAL, or both prevented tumor regression, but had no effect on body weight, which declined continuously. In the HF rats, there was an increase in the concentration of dopamine and a decrease in the concentration of serotonin in the hypothalamus, whereas treatment with HAL reversed these effects. EB had no effect on neurotransmitter concentrations in the HF rats, but treatment of HF+EB animals with HAL decreased the dopamine concentration. The changes in dopamine and serotonin observed in HF rats are known to inhibit PRL secretion, whereas HAL, which blocked these changes, is a well established stimulator of PRL secretion. Since the mammary tumors are dependent on PRL for development and growth, it is probable that the regression of these tumors in the HF rats was ultimately due to a decrease in PRL secretion, and the prevention of this regression in HF+HAL rats was ultimately due to an increase in PRL secretion. EB, a potent PRL stimulator, probably blocked tumor regression in HF+EB rats by increasing PRL secretion by a direct effect on the pituitary.

Antiviral properties of the seed extract of an Indian medicinal plant, Pongamia pinnata, Linn., against herpes simplex viruses: in-vitro studies on Vero cells.

Pongamia pinnata, Linn., an Indian medicinal plant used in the Ayurvedha and Siddha traditional medicine systems, for treatment of clinical lesions of skin and genitalia, was evaluated for antiviral properties against herpes simplex virus type-1 (HSV-1) and type-2 (HSV-2) by in-vitro studies in Vero cells. A crude aqueous seed extract of P. pinnata completely inhibited the growth of HSV-1 and HSV-2 at concentrations of 1 and 20 mg/ml (w/v), respectively, as shown by complete absence of cytopathic effect.

Effect of Phyllanthus amarus on duck hepatitis B virus replication in vivo.

Nine ducks congenitally infected with the duck hepatitis B virus (DHBV) were treated either orally (four ducks for 10 weeks) or intraperitoneally (five ducks for 12 weeks) with the Indian traditional herbal remedy Phyllanthus amarus. Compared to placebo-treated control ducks, these treatments did not result in a reduction of circulating viral DNA in the serum or in the level of viral DNA replication in the liver. In two of the five intraperitoneal-treated ducks, a reduction in the levels of duck hepatitis B surface antigenaemia (DHBsAg) was observed. The data strongly suggest that Phyllanthus amarus has no significant inhibitory effect on DHBV DNA replication and only a minor effect on DHBsAg production.

Hepatitis B virus and primary hepatocellular carcinoma: treatment of HBV carriers with Phyllanthus amarus.

A viricide capable of eliminating hepatitis B virus (HBV) from chronic carriers should, theoretically, decrease the risk of primary hepatocellular carcinoma. Extracts of Phyllanthus amarus have been shown to inhibit the DNA polymerase of HBV and woodchuck hepatitis virus (WHV) in vitro. Three of four recently infected WHV carriers treated i.p. with P. amarus extract lost WHV, animals infected for greater than or equal to 3 months showed a decrease in virus levels. Preliminary results in human carriers treated orally with P. amarus for 1 month indicated that approximately 60% of the carriers lost HBV during the observation period.

Hepatitis B virus and hepatocellular carcinoma--treatment of HBV carriers with Phyllanthus amarus.

Extracts of Phyllanthus amarus inhibit the DNA polymerase of HBV and related viruses. Woodchuck carriers of woodchuck hepatitis virus (WHV) were treated intraperitoneally with P. amarus extract. Three of four animals which had been recently infected lost the virus. Animals infected for about 3 months or more had a decrease in virus levels. Human carriers of HBV were treated orally for 1 month. About 60% of the carriers lost HBV, which did not return during the observation period. Fractions containing active principles are now being isolated and characterized.

Effect of Phyllanthus amarus on chronic carriers of hepatitis B virus.

In a preliminary study, carriers of hepatitis B virus were treated with a preparation of the plant Phyllanthus amarus for 30 days. 22 of 37 (59%) treated patients had lost hepatitis B surface antigen when tested 15-20 days after the end of the treatment compared with only 1 of 23 (4%) placebo-treated controls. Some subjects have been followed for up to 9 months. In no case has the surface antigen returned. Clinical observation revealed few or no toxic effects. The encouraging results of this preliminary study recommend continued evaluation of this plant and the active principles isolated from it.