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In vivo effects of traditional Ayurvedic formulations in Drosophila melanogaster model relate with therapeutic applications.

Dwivedi V, Anandan EM, Mony RS, Muraleedharan TS, Valiathan MS, Mutsuddi M, Lakhotia SC - PLoS ONE (2012)

Bottom Line: On the contrary, feeding larvae on normal food and adults on AR supplement had no effect on fecundity but a comparable regime of feeding on RS-supplemented food improved fecundity.RS feeding did not cause heavy metal toxicity.Thus, Drosophila, with its very rich genetic tools and well-worked-out developmental pathways promises to be a very good model for examining the cellular and molecular bases of the effects of different Ayurvedic formulations.

View Article: PubMed Central - PubMed

Affiliation: Cytogenetics Laboratory, Department of Zoology, Banaras Hindu University, Varanasi, India.

ABSTRACT

Background: Ayurveda represents the traditional medicine system of India. Since mechanistic details of therapy in terms of current biology are not available in Ayurvedic literature, modern scientific studies are necessary to understand its major concepts and procedures. It is necessary to examine effects of the whole Ayurvedic formulations rather than their "active" components as is done in most current studies.

Methods: We tested two different categories of formulations, a Rasayana (Amalaki Rasayana or AR, an herbal derivative) and a Bhasma (Rasa-Sindoor or RS, an organo-metallic derivative of mercury), for effects on longevity, development, fecundity, stress-tolerance, and heterogeneous nuclear ribonucleoprotein (hnRNP) levels of Drosophila melanogaster using at least 200 larvae or flies for each assay.

Results: A 0.5% (weight/volume) supplement of AR or RS affected life-history and other physiological traits in distinct ways. While the size of salivary glands, hnRNP levels in larval tissues, and thermotolerance of larvae/adult flies improved significantly following feeding either of the two formulations, the median life span and starvation resistance improved only with AR. Feeding on AR or RS supplemented food improved fecundity differently. Feeding of larvae and adults with AR increased the fecundity while the same with RS had opposite effect. On the contrary, feeding larvae on normal food and adults on AR supplement had no effect on fecundity but a comparable regime of feeding on RS-supplemented food improved fecundity. RS feeding did not cause heavy metal toxicity.

Conclusions: The present study with two Ayurvedic formulations reveals formulation-specific effects on several parameters of the fly's life, which seem to generally agree with their recommended human usages in Ayurvedic practices. Thus, Drosophila, with its very rich genetic tools and well-worked-out developmental pathways promises to be a very good model for examining the cellular and molecular bases of the effects of different Ayurvedic formulations.

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Related in: MedlinePlus

Feeding larvae on AR or RS supplemented food enhances the levels of Hrp36 in larval tissues.A–F Confocal projections show GFP (red, A–C) and DAPI (white, D–F) fluorescence in SG polytene nuclei from 110 hr old HRB87F-GFP/Hrb87F-GFP larvae. Histograms in G show that the mean levels of nuclear Hrp36 (GFP fluorescence) in polytene nuclei of SG (N = 100 for each sample) are significantly greater in AR or RS-fed larvae. Western blot in H shows the relative amounts of Hrp36 (detected by the P11 antibody) in total proteins from differently fed (control, AR and RS lanes) late 3rd instar larvae; the relative value of Hrp36 (ratio of the signal for Hrp36 and β-tubulin in a given blot) in normally fed (1st lane) larvae in each replicate was taken as 1. The normalized values of the mean (±S.E., N = 3) relative levels of Hrp36 in total larval proteins are noted below the AR and RS lanes. I–K are confocal projections of squashed SG polytene chromosomes from 110 hr old control (I) or AR- (J) or RS- (K) fed larvae immunostained with P11 antibody to localize Hrp36 (red fluorescence) and counterstained with DAPI (white fluorescence); the early ecdysone puffs at 75B and 74EF are marked in each case. L shows representative cut outs of the left arm of chromosome 3 (3L) from squash preparations as in I–K from salivary glands of 110 hr old larvae fed on regular (CON) or AR or RS supplemented food; some of the known puff sites [29] are marked on the top panel and the corresponding sites in the other two cut outs (middle and lower panels) are indicated by connecting yellow lines (the chromosome arm in these images was cut and re-positioned, as required, to provide a relatively straight orientation). Histograms in M show the mean fluorescence intensity for DNA and Hrp36 on the entire 3L in differently fed (noted on X-axis) larval salivary gland squash preparations as in I–K; inset shows the relative levels of Hrp36 in relation to the nuclear DNA content in the three sets of nuclei; the numbers of 3L examined for each sample is mentioned in parenthesis on the X-axis. The scale bar in A (5 µm) applies to A–F, while that in I (10 µm) applies to I–K.
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pone-0037113-g005: Feeding larvae on AR or RS supplemented food enhances the levels of Hrp36 in larval tissues.A–F Confocal projections show GFP (red, A–C) and DAPI (white, D–F) fluorescence in SG polytene nuclei from 110 hr old HRB87F-GFP/Hrb87F-GFP larvae. Histograms in G show that the mean levels of nuclear Hrp36 (GFP fluorescence) in polytene nuclei of SG (N = 100 for each sample) are significantly greater in AR or RS-fed larvae. Western blot in H shows the relative amounts of Hrp36 (detected by the P11 antibody) in total proteins from differently fed (control, AR and RS lanes) late 3rd instar larvae; the relative value of Hrp36 (ratio of the signal for Hrp36 and β-tubulin in a given blot) in normally fed (1st lane) larvae in each replicate was taken as 1. The normalized values of the mean (±S.E., N = 3) relative levels of Hrp36 in total larval proteins are noted below the AR and RS lanes. I–K are confocal projections of squashed SG polytene chromosomes from 110 hr old control (I) or AR- (J) or RS- (K) fed larvae immunostained with P11 antibody to localize Hrp36 (red fluorescence) and counterstained with DAPI (white fluorescence); the early ecdysone puffs at 75B and 74EF are marked in each case. L shows representative cut outs of the left arm of chromosome 3 (3L) from squash preparations as in I–K from salivary glands of 110 hr old larvae fed on regular (CON) or AR or RS supplemented food; some of the known puff sites [29] are marked on the top panel and the corresponding sites in the other two cut outs (middle and lower panels) are indicated by connecting yellow lines (the chromosome arm in these images was cut and re-positioned, as required, to provide a relatively straight orientation). Histograms in M show the mean fluorescence intensity for DNA and Hrp36 on the entire 3L in differently fed (noted on X-axis) larval salivary gland squash preparations as in I–K; inset shows the relative levels of Hrp36 in relation to the nuclear DNA content in the three sets of nuclei; the numbers of 3L examined for each sample is mentioned in parenthesis on the X-axis. The scale bar in A (5 µm) applies to A–F, while that in I (10 µm) applies to I–K.

Mentions: In situ distribution of Hrp36 in intact SG cells in Hrb87F-GFP larvae reared under different feeding regimens was examined by confocal microscopy. The Hrp36 in SG was present in nucleoplasm and on specific chromatin regions in all the three cases (Fig. 5A–L). Interestingly, the SG nuclei from either of the formulation-fed larvae showed much higher nuclear GFP fluorescence, especially at certain chromatin regions than in corresponding tissues from larvae reared on regular food. Although the very intense signal from certain chromatin regions in nuclei from formulation-fed larvae appeared reminiscent of the strong presence of hnRNPs at the 93D site in heat shocked cells [27], a significant difference from the stressed cells is the continued presence of Hrp36 on other chromosome regions and in nucleoplasm (Fig. 5A–F). Parallel observations on polytene chromosome spreads revealed that the chromosome sites showing very intense Hrp36-GFP fluorescence in intact glands actually correspond to the early ecdysone puffs at 74DE and 75B on 3L, rather than the 93D puff (see below and Fig. 5L). Measurement of total nuclear GFP fluorescence in the distal salivary gland nuclei clearly showed that the levels of nuclear Hrp36 are significantly increased in AR- or RS- fed larvae (Fig. 5G). Comparison of the levels of Hrp36 in total proteins from Oregon R+ larvae by western-blotting using the Hrp36-specific P11 antibody [28], confirmed that the relative levels of this proteins were significantly higher in the body of formulation-fed larvae (Fig. 5H).


In vivo effects of traditional Ayurvedic formulations in Drosophila melanogaster model relate with therapeutic applications.

Dwivedi V, Anandan EM, Mony RS, Muraleedharan TS, Valiathan MS, Mutsuddi M, Lakhotia SC - PLoS ONE (2012)

Feeding larvae on AR or RS supplemented food enhances the levels of Hrp36 in larval tissues.A–F Confocal projections show GFP (red, A–C) and DAPI (white, D–F) fluorescence in SG polytene nuclei from 110 hr old HRB87F-GFP/Hrb87F-GFP larvae. Histograms in G show that the mean levels of nuclear Hrp36 (GFP fluorescence) in polytene nuclei of SG (N = 100 for each sample) are significantly greater in AR or RS-fed larvae. Western blot in H shows the relative amounts of Hrp36 (detected by the P11 antibody) in total proteins from differently fed (control, AR and RS lanes) late 3rd instar larvae; the relative value of Hrp36 (ratio of the signal for Hrp36 and β-tubulin in a given blot) in normally fed (1st lane) larvae in each replicate was taken as 1. The normalized values of the mean (±S.E., N = 3) relative levels of Hrp36 in total larval proteins are noted below the AR and RS lanes. I–K are confocal projections of squashed SG polytene chromosomes from 110 hr old control (I) or AR- (J) or RS- (K) fed larvae immunostained with P11 antibody to localize Hrp36 (red fluorescence) and counterstained with DAPI (white fluorescence); the early ecdysone puffs at 75B and 74EF are marked in each case. L shows representative cut outs of the left arm of chromosome 3 (3L) from squash preparations as in I–K from salivary glands of 110 hr old larvae fed on regular (CON) or AR or RS supplemented food; some of the known puff sites [29] are marked on the top panel and the corresponding sites in the other two cut outs (middle and lower panels) are indicated by connecting yellow lines (the chromosome arm in these images was cut and re-positioned, as required, to provide a relatively straight orientation). Histograms in M show the mean fluorescence intensity for DNA and Hrp36 on the entire 3L in differently fed (noted on X-axis) larval salivary gland squash preparations as in I–K; inset shows the relative levels of Hrp36 in relation to the nuclear DNA content in the three sets of nuclei; the numbers of 3L examined for each sample is mentioned in parenthesis on the X-axis. The scale bar in A (5 µm) applies to A–F, while that in I (10 µm) applies to I–K.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3351451&req=5

pone-0037113-g005: Feeding larvae on AR or RS supplemented food enhances the levels of Hrp36 in larval tissues.A–F Confocal projections show GFP (red, A–C) and DAPI (white, D–F) fluorescence in SG polytene nuclei from 110 hr old HRB87F-GFP/Hrb87F-GFP larvae. Histograms in G show that the mean levels of nuclear Hrp36 (GFP fluorescence) in polytene nuclei of SG (N = 100 for each sample) are significantly greater in AR or RS-fed larvae. Western blot in H shows the relative amounts of Hrp36 (detected by the P11 antibody) in total proteins from differently fed (control, AR and RS lanes) late 3rd instar larvae; the relative value of Hrp36 (ratio of the signal for Hrp36 and β-tubulin in a given blot) in normally fed (1st lane) larvae in each replicate was taken as 1. The normalized values of the mean (±S.E., N = 3) relative levels of Hrp36 in total larval proteins are noted below the AR and RS lanes. I–K are confocal projections of squashed SG polytene chromosomes from 110 hr old control (I) or AR- (J) or RS- (K) fed larvae immunostained with P11 antibody to localize Hrp36 (red fluorescence) and counterstained with DAPI (white fluorescence); the early ecdysone puffs at 75B and 74EF are marked in each case. L shows representative cut outs of the left arm of chromosome 3 (3L) from squash preparations as in I–K from salivary glands of 110 hr old larvae fed on regular (CON) or AR or RS supplemented food; some of the known puff sites [29] are marked on the top panel and the corresponding sites in the other two cut outs (middle and lower panels) are indicated by connecting yellow lines (the chromosome arm in these images was cut and re-positioned, as required, to provide a relatively straight orientation). Histograms in M show the mean fluorescence intensity for DNA and Hrp36 on the entire 3L in differently fed (noted on X-axis) larval salivary gland squash preparations as in I–K; inset shows the relative levels of Hrp36 in relation to the nuclear DNA content in the three sets of nuclei; the numbers of 3L examined for each sample is mentioned in parenthesis on the X-axis. The scale bar in A (5 µm) applies to A–F, while that in I (10 µm) applies to I–K.
Mentions: In situ distribution of Hrp36 in intact SG cells in Hrb87F-GFP larvae reared under different feeding regimens was examined by confocal microscopy. The Hrp36 in SG was present in nucleoplasm and on specific chromatin regions in all the three cases (Fig. 5A–L). Interestingly, the SG nuclei from either of the formulation-fed larvae showed much higher nuclear GFP fluorescence, especially at certain chromatin regions than in corresponding tissues from larvae reared on regular food. Although the very intense signal from certain chromatin regions in nuclei from formulation-fed larvae appeared reminiscent of the strong presence of hnRNPs at the 93D site in heat shocked cells [27], a significant difference from the stressed cells is the continued presence of Hrp36 on other chromosome regions and in nucleoplasm (Fig. 5A–F). Parallel observations on polytene chromosome spreads revealed that the chromosome sites showing very intense Hrp36-GFP fluorescence in intact glands actually correspond to the early ecdysone puffs at 74DE and 75B on 3L, rather than the 93D puff (see below and Fig. 5L). Measurement of total nuclear GFP fluorescence in the distal salivary gland nuclei clearly showed that the levels of nuclear Hrp36 are significantly increased in AR- or RS- fed larvae (Fig. 5G). Comparison of the levels of Hrp36 in total proteins from Oregon R+ larvae by western-blotting using the Hrp36-specific P11 antibody [28], confirmed that the relative levels of this proteins were significantly higher in the body of formulation-fed larvae (Fig. 5H).

Bottom Line: On the contrary, feeding larvae on normal food and adults on AR supplement had no effect on fecundity but a comparable regime of feeding on RS-supplemented food improved fecundity.RS feeding did not cause heavy metal toxicity.Thus, Drosophila, with its very rich genetic tools and well-worked-out developmental pathways promises to be a very good model for examining the cellular and molecular bases of the effects of different Ayurvedic formulations.

View Article: PubMed Central - PubMed

Affiliation: Cytogenetics Laboratory, Department of Zoology, Banaras Hindu University, Varanasi, India.

ABSTRACT

Background: Ayurveda represents the traditional medicine system of India. Since mechanistic details of therapy in terms of current biology are not available in Ayurvedic literature, modern scientific studies are necessary to understand its major concepts and procedures. It is necessary to examine effects of the whole Ayurvedic formulations rather than their "active" components as is done in most current studies.

Methods: We tested two different categories of formulations, a Rasayana (Amalaki Rasayana or AR, an herbal derivative) and a Bhasma (Rasa-Sindoor or RS, an organo-metallic derivative of mercury), for effects on longevity, development, fecundity, stress-tolerance, and heterogeneous nuclear ribonucleoprotein (hnRNP) levels of Drosophila melanogaster using at least 200 larvae or flies for each assay.

Results: A 0.5% (weight/volume) supplement of AR or RS affected life-history and other physiological traits in distinct ways. While the size of salivary glands, hnRNP levels in larval tissues, and thermotolerance of larvae/adult flies improved significantly following feeding either of the two formulations, the median life span and starvation resistance improved only with AR. Feeding on AR or RS supplemented food improved fecundity differently. Feeding of larvae and adults with AR increased the fecundity while the same with RS had opposite effect. On the contrary, feeding larvae on normal food and adults on AR supplement had no effect on fecundity but a comparable regime of feeding on RS-supplemented food improved fecundity. RS feeding did not cause heavy metal toxicity.

Conclusions: The present study with two Ayurvedic formulations reveals formulation-specific effects on several parameters of the fly's life, which seem to generally agree with their recommended human usages in Ayurvedic practices. Thus, Drosophila, with its very rich genetic tools and well-worked-out developmental pathways promises to be a very good model for examining the cellular and molecular bases of the effects of different Ayurvedic formulations.

Show MeSH
Related in: MedlinePlus