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

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

<i>Oregon R⁺</i> flies reared on AR or RS supplemented food show significantly improved thermotolerance in the knockdown assay.

*<p>Values significantly different (P<0.001) from the corresponding control values.</p>**<p>Values not significantly different from corresponding control values).</p

Differential effects of AR or RS feeding during larval or adult stage on fecundity.

*<p>P<0,001 when compared with corresponding control.</p>**<p>P>0.05 when compared with corresponding control.</p

Comparison of the observed effects of dietary supplement of AR or RS on different parameters in <i>Drosophila melanogaster</i>.

<p>Comparison of the observed effects of dietary supplement of AR or RS on different parameters in <i>Drosophila melanogaster</i>.</p

Feeding larvae on AR or RS supplemented food enhances the levels of Hrp36 in larval tissues.

<p><b>A</b>–<b>F</b> Confocal projections show GFP (red, <b>A–C</b>) and DAPI (white, <b>D–F</b>) fluorescence in SG polytene nuclei from 110 hr old <i>HRB87F-GFP/Hrb87F-GFP</i> larvae. Histograms in <b>G</b> 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 <b>H</b> shows the relative amounts of Hrp36 (detected by the P11 antibody) in total proteins from differently fed (control, AR and RS lanes) late 3^rd instar larvae; the relative value of Hrp36 (ratio of the signal for Hrp36 and β-tubulin in a given blot) in normally fed (1^st 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. <b>I</b>–<b>K</b> are confocal projections of squashed SG polytene chromosomes from 110 hr old control (<b>I</b>) or AR- (<b>J</b>) or RS- (<b>K</b>) 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. <b>L</b> shows representative cut outs of the left arm of chromosome 3 (3L) from squash preparations as in <b>I</b>–<b>K</b> from salivary glands of 110 hr old larvae fed on regular (CON) or AR or RS supplemented food; some of the known puff sites <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037113#pone.0037113-Ashburner1" target="_blank">[29]</a> 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 <b>M</b> 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 <b>I</b>–<b>K</b>; 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 <b>A</b> (5 µm) applies to <b>A</b>–<b>F</b>, while that in <b>I</b> (10 µm) applies to <b>I</b>–<b>K</b>.</p

Feeding on 0.5% AR or RS supplemented food during larval period increases dimensions of salivary glands of late 3^rd instar larvae.

*<p>P<0.001) when compared with corresponding control.</p

Levels of Hrp40 (Squid) are increased in formulation fed <i>Squid-GFP</i> larvae.

<p>Confocal projection images of GFP fluorescence (green, <b>A</b>–<b>C</b> and <b>G</b>–<b>I</b>) in polytene nuclei from late third instar larval salivary glands (<b>A</b>–<b>C</b>); DAPI-stained chromatin (<b>D</b>–<b>F</b>) is seen in red. Histograms in <b>G</b> show mean intensity of Squid-GFP fluorescence in SG polytene nuclei (N = 100 for each sample) from differently fed larvae. Western blot in <b>H</b> shows relative levels of Squid-GFP protein (Squid) and β-tubulin (β-tub) in whole proteins from differently fed (CON, RS or AR) larvae challenged with GFP antibody: the values below each lane indicate the relative levels (ratio of Squid∶β-tubulin in the blot with the value for larvae reared on regular food (CON) taken as 1.0) of Squid-GFP protein. The scale bar in <b>A</b> represents 5 µm and applies to <b>A</b>–<b>F</b>.</p

Median pupation and adult eclosion times following rearing on regular or AR or RS supplemented food.

<p>N = 200 for each feeding condition (8 replicates of 25 each).</p>*<p>P<0.001 when compared with the corresponding control (reared on regular food) set of flies;</p>**<p>P>0.05 when compared with the corresponding control.</p

Feeding of <i>Oregon R⁺</i> first instar larvae on food supplemented with 0.5% of AR or RS hastens pupation (A) and fly eclosion (B).

<p>The bars represent mean (± S.E.) of 8 replicates (N = 25 in each replicate; total N for each feeding regimen = 200). Numbers of pupae (<b>A</b>) or flies (<b>B</b>) in a given sample were monitored every two hours between 112–132 and 234–256 hr (X-axis), respectively, and expressed as % of larvae that had pupated or flies that had eclosed by the given time period. Control – regular food, AR – 0.5% AR supplemented food, AP- 0.143% Amalaki Powder supplemented food. RS – 0.5% RS supplemented food and H+G – Honey plus Ghee (same proportion as in 0.5% AR) supplemented food.</p

Larval salivary glands attain greater size following feeding on 0.5% AR (B) or RS (C) than in normally fed larvae (A) of same age (spiracle eversion stage).

<p>Individual polytene nuclei are also bigger in larvae reared on AR- (<b>E</b>) or RS-supplemented (<b>F</b>) food than in those fed on regular food (<b>D</b>); nuclei marked with arrows in <b>D</b>–<b>F</b> are shown at higher magnification in insets (also see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037113#pone-0037113-t002" target="_blank">Table 2</a>). Histograms in <b>G</b> show the mean (±S.E.) DAPI fluorescence (in arbitrary fluorescence units on Y-axis) in distal polytene nuclei in salivary glands from larvae reared on regular (CON) or formulation (AR or RS) supplemented food (numbers of nuclei examined in each case are indicated in parentheses above the respective bars. Scale bar in <b>B</b> applies to <b>A</b>–<b>C</b> while that in <b>D</b>, applies to <b>D</b>–<b>F</b>.</p