Rheum Emodi Research Articles

HPLC and HPTLC Methods by Design for Quantitative Characterization and in vitro Anti-oxidant Activity of Polyherbal Formulation Containing Rheum emodi

Safoof-e-Pathar phori (SPP) is a traditional polyherbal formulation and has been used since long time for its anti-urolithiatic activity. It contains three plant constituents Didymocarpous pedicellata, Dolichous biflorus and Rheum emodi. Emodin and chrysophanic acid were selected as chemical markers for SPP and quantified using simultaneous HPTLC and RP-HPLC methods in R. emodi and in SPP. The simultaneous methods were found linear r(2) = 0.991 in a wide range (10-800 ng spot(-1) with HPTLC, 5-500 µg mL(-1) with HPLC) precise, accurate and robust for both the drugs. Anti-oxidant activity of SPP, R. emodi as well as standard emodin and chrysophanic acid were determined by using DPPH (2,2-diphenyl-1-picryl hydrazyl radical), which showed better activity of R. emodi (IC50 = 12.27) extract when compared with SPP (IC50 = 32.99) and standard drugs (IC50 = 66.81). The robustness of methods were proved by applying the Box-Behnken response surface design software and other validation parameters evaluated were satisfactorily met; hence, the developed method found suitable for application in the quality control of several formulations containing emodin and chrysophanic acid.

Acceleration of pro-caspase-3 maturation and cell migration inhibition in human breast cancer cells by phytoconstituents of Rheum emodi rhizome extracts.

The aggressive nature of estrogen receptor (ER)-negative breast cancer subtype obligates for innovative targeted therapies. The present study aimed to investigate the phytoconstituents and specific anticancer activities of Rheum emodi rhizome, a known food source used locally to treat various ailments. Petroleum ether extracts (hot [PHR] and cold [PCR]) of R. emodi, exhibited significant free radical scavenging potentials through DPPH and reducing power assays, rendering them as good sources of antioxidants. The extracts, PHR and PCR had shown significant (P < 0.05) cancer-cell-specific cytotoxicity in the assayed cells (MDA-MB-231 [breast carcinoma] and WRL-68 [non-tumoral]) at 100 μg/ml, and 50 and 100 μg/ml concentrations respectively. Extracts also induced fervent apoptosis in ER-negative cells (MDA-MB-231) compared to ER-positive subtype (MCF-7), and found to involve CPP32/caspase-3 in its apoptosis induction mechanism. Moreover, extracts had an inevitable potential to inhibit the migration of metastatic breast cancer cells (MDA-MB-231) in vitro. Further, the active principles of extracts were identified through HPLC and GC-MS analysis to reveal major polyphenolics, 4,7-Dimethyl-(octahydro)indolo[4,3-fg]quinolin-10-one, 5-Oxo-isolongifolene, Valencene-2, and other quinone, quinoline and anthraquinone derivatives. The extracts are thus good candidates to target malignant ER-negative breast cancer, and the identified phytoconstituents could be eluted in further exploratory studies for use in dietary-based anti-breast cancer therapies.

Preparation of novel antiproliferative emodin derivatives and studies on their cell cycle arrest, caspase dependent apoptosis and DNA binding interaction

Emodin (1) is the major bioactive compound of several herb species, which belongs to anthraquinone class of compound. As a part of our drug discovery program, large quantities of emodin (1) was isolated from the roots of Rheum emodi and a library of novel emodin derivatives 2–15 were prepared to evaluate their antiproliferative activities against HepG2, MDA-MB-231 and NIH/3T3 cells lines. The derivatives 3 and 12 strongly inhibited the proliferation of HepG2 and MDA-MB-231 cancer cell line with an IC50 of 5.6, 13.03 and 10.44, 5.027, respectively, which is comparable to marketed drug epirubicin (III). The compounds 3 and 12 were also capable of inducing cell cycle arrest and caspase dependent apoptosis in HepG2 cell lines and exhibit DNA intercalating activity. These emodin derivatives hold promise for developing safer alternatives to the marketed epirubicin.

New Chromatographic Methods (HPTLC and RP-HPLC) for Simultaneous Estimation of Emodin and Chrysophanic Acid in Safoof-e-Pathar phori and R. emodi

Safoof-e-Pathar phori is a Unani polyherbo-mineral formulation and has been used in Unani system of medicine for its anti-urolithiatic activity [1]. It contains six different constituents including Pathar phori (Didymocarpous pedicellata) and Revand chini (Rheum emodi) [2 – 4]. Simultaneous assays of emodin and chrysophanic acid in Safoof-e-Pathar phori and Rheum emodi were carried out as chemical markers using HPTLC and RP-HPLC. Toluene: ethyl acetate: formic acid (10:10:1, v/v/v) was used as mobile phase in HPTLC, whereas methanol and 0.1% ortho-phosphoric acid (80:20, v/v) was used for RP-HPLC. The developed simultaneous HPTLC and RP-HPLC methods for emodin and chrysophanic acid showed good linearity (r2 > 0.997) at a wavelength of 430nm. The methods were validated as per ICH guidelines and found accurate (% recovery 95.28 – 105.46%), precise (% RSD 0.50 – 2.09%), robust and reproducible (% RSD 0.54 – 2.03%), which were applied for analysis of emodin and chrysophanic acid in R. emodi and herbal formulations containing these ingredients. Acknowledgments: Thanks go to the Central Council of Research in Unani Medicine, Department of AYUSH, Ministry of Health and Family Welfare, New Delhi, India.

English

Seed germination of two species of the Himalayan rhubarb (Rheum acuminatum&nbsp;andRheum australe) growing in high altitudes was simulated by different fluctuating temperatures. The germination experiments in different temperature regimes (15/5, 20/5, and 25/10&deg;C) of fluctuating light regime of 12 h day and 12 h night, and same temperature regimes of constant darkness showed that both species germinated better in light than in complete darkness. An overall percentage of germination was higher for&nbsp;R. acuminatumthan for&nbsp;R. australe&nbsp;both in light and complete darkness. The optimum temperature for&nbsp;R. acuminatum&nbsp;was 15/5&deg;C light (around 87.08%) and lowest was 15/5&deg;C dark (9.62%). Similarly,&nbsp;R.&nbsp;australe&nbsp;germinated well at 20/5&deg;C light (61.39%) and least at 15/5&deg;C dark (4.71%). When testing effects of different factors on the germination, it was found that there was a positive effect of seed mass and also interaction of light and temperature. Looking at patterns of germination, smaller seeds of&nbsp;R. acuminatum&nbsp;germinated faster than bigger seeds of&nbsp;R. australe&nbsp;in all temperature regimes except 25/10&deg;C dark conditions. Seeds of both species will germinate better if shifted to lower altitudes where temperatures are higher than at their actual habitats. Since both species lack in seed bank, it is recommended that seed sowing should be carried out in spring rather than before winter for better germination. &nbsp; Key words:&nbsp;Himalayan region, rhubarb,&nbsp;Rheum acuminatum,&nbsp;Rheum australe, seed germination.

A protocol to remove colored metabolites and other inhibitors from plant tissues to facilitate RNA isolation suitable for downstream applications

This work developed a protocol to remove colored metabolites and other interfering substances to facilitate RNA isolation. These metabolites otherwise hinder RNA isolation and downstream applications. The developed protocol used sodium dodecyl sulphate, ethylenediamine tetra-acetic acid, and ethanol in a definite ratio that removed the said metabolites from the tissue and aided isolation of RNA using the existing methods. The protocol was developed for red colored roots of Arnebia euchroma and was extended to other colored tissues [rhizome of Rheum australe (Himalayan Rhubarb) and taproot of Daucus carota (purple carrot)] with success. Without inclusion of our protocol, the existing methods could not isolate good quality RNA from these tissues. RNA isolated by the developed protocol had A(260/280) ratio of 1.88-1.93, A(260/230) ratio of 1.94-2.0, and RNA integrity number of 6.3-8.0. RNA was amenable to downstream applications such as reverse-transcription polymerase chain reaction and primer extension assay.

Activity-guided isolation of antioxidants from the roots of Rheum emodi

An activity-guided isolation and purification process was used to identify the DPPH free radical scavenging components of Rheum emodi. The activity-guided isolation revealed that eugenol, gallic acid, quercetin, rutin, epicatechin, desoxyrhapontigenin, rhapontigenin and mesopsin are the major phenolic compounds responsible for the antioxidant activity of the roots of R. emodi.

Effect of mixed-cropping and water-stress on macro-nutrients and biochemical constituents of rhizomatous medicinal plants in Central Himalaya, India

Plants in the alpine zone mainly depend on the reserved food materials stored in their rhizomes for the next growing season. We investigated the effect of mixed cropping (Phaseolus vulgaris L. var. Pinto) with four rhizomatous medicinal plants, i.e., Angelica glauca, Arnebiabenthamii, Rheum emodi and Pleurospermum angelicoides as well as three levels of water stress treatment under two conditions (shade net and open field) on macronutrients (NPK) and biochemicals (carbohydrates and protein). The experiment was conducted by completely randomized design (CDR). The data were analyzed with ANOVA as well as CDR. The experimental results show that in all the species shade conditions with sever water stress (SSWS) increased the level of macronutrients (NPK). However, (N) concentration was highest under shade with mixed cropping (SMIX). Under SMIX, carbohydrate content was highest than open field control conditions (CONT). This investigation results demonstrate that mixed cropping of medicinal plants with Phaseolus vulgaris could be a good livelihood option in the mountainous regions of Indian Central Himalaya. And the water-stress conditions along with mixed cropping could improve the biochemical constituents in the rhizome of these species.

Antimicrobial activity of wool yarn dyed with Rheum emodi L. (Indian Rhubarb)

This work is an attempt to examine the effect of Rheum emodi L. as dye and its dyed wool yarns against two bacterial (Escherichia coli and Staphylococcus aureus) and two fungal (Candida albicans and Candida tropicalis) species. The dyeing was carried out using 5% and 10% o.w.f. dye concentration in presence and absence of ferrous sulphate, stannous chloride and alum mordants. The colour strength, CIELab values and fastness properties of dyed samples were also assessed. FTIR spectra of untreated, mordanted and dyed wool yarn were investigated to study the interaction between fibre, mordant and dye. The structural morphology of wool yarn was investigated by Scanning Electron Microscopy (SEM). The susceptibility tests for R. emodi L. were carried out in terms of disc diffusion, growth curve and viability assays against all the tested microorganisms. Dyed samples showed very effective antimicrobial properties showing more than 90% microbial reduction in both bacterial as well as fungal population.

Conservation of aromatic medicinal plant Rheum emodi through improved seed germination

Conservation of aromatic medicinal plant Rheum emodi through improved seed germination

Pharmacognostical and Phytochemical Evaluation of Rheum emodi Wall.

Pharmacognostical and Phytochemical Evaluation of Rheum emodi Wall.

Active constituents in Rheum acuminatum and Rheum australe (Polygonaceae) roots: A variation between cultivated and naturally growing plants

The concentrations of five anthraquinones (chrysophanol, emodin, aloe-emodin, physcion and rhein) and two stilbenes (piceatannol and resveratrol) present in the roots of Rheum acuminatum and Rheum australe were determined and compared in cultivated and naturally grown plants. The roots of naturally grown plants used for experiments were collected from populations in the Gosaikunda area of Nepal, while artificially cultivated roots were grown in Průhonice in the Czech Republic. The extracts of powdered roots were analyzed using HPLC. Aloe-emodin and rhein were nearly absent in cultivated roots of both species, and the concentrations of emodin, physcion and chrysophanol were slightly higher in roots from natural habitats than from cultivated sites. Piceatannol and resveratrol were present in almost the same amounts in cultivated and natural samples from both species. Higher concentrations of emodin and physcion were present in R. acuminatum than in R. australe, while the opposite trend was observed for chrysophanol. The other compounds studied were equally common in both species. The results suggest that the two species could be used interchangeably. The cultivated plants can be used when high levels of piceatannol and resveratrol are desired, whereas plants from natural habitats can be used to obtain higher levels of the other compounds.

Inhibition of Cytochrome P450 Enzymes by Quinones and Anthraquinones

In silico docking studies and quantitative structure-activity relationship analysis of a number of in-house cytochrome P450 inhibitors have revealed important structural characteristics that are required for a molecule to function as a good inhibitor of P450 enzymes 1A1, 1A2, 2B1, and/or 2A6. These insights were incorporated into the design of pharmacophores used for a 2D search of the Chinese medicine database. Emodin, a natural anthraquinone isolated from Rheum emodi and known to be metabolized by cytochrome P450 enzymes, was one of the hits and was used as the lead compound. Emodin was found to inhibit P450s 1A1, 1A2, and 2B1 with IC(50) values of 12.25, 3.73, and 14.89 μM, respectively. On the basis of the emodin molecular structure, further similarity searches of the PubChem and ZINC chemical databases were conducted resulting in the identification of 12 emodin analogues for testing against P450s 1A1-, 1A2-, 2B1-, and 2A6-dependent activities. 1-Amino-4-chloro-2-methylanthracene-9,10-dione (compound 1) showed the best inhibition potency for P450 1A1 with an IC(50) value of 0.40 μM. 1-Amino-4-chloro-2-methylanthracene-9,10-dione (compound 1) and 1-amino-4-hydroxyanthracene-9,10-dione (compound 2) both inhibited P450 1A2 with the same IC(50) value of 0.53 μM. In addition, compound 1 acted as a mechanism-based inhibitor of cytochrome P450s 1A1 and 1A2 with K(I) and K(inactivation) values of 5.38 μM and 1.57 min(-1) for P450 1A1 and 0.50 μM and 0.08 min(-1) for P450 1A2. 2,6-Di-tert-butyl-5-hydroxynaphthalene-1,4-dione (compound 8) directly inhibited P450 2B1 with good selectivity and inhibition potency (IC(50) = 5.66 μM). Docking studies using the 3D structures of the enzymes were carried out on all of the compounds. The binding modes of these compounds revealed the structural characteristics responsible for their potency and selectivity. Compound 1, which is structurally similar to compound 2 with the presence of an amino group at position 1, showed a difference in the mechanism of inhibition toward P450s 1A1 and 1A2. The mechanism-based inhibition seen for compound 1 may be attributed to the presence of the methyl group at the 2-position, in close proximity to the amino group. Compound 2, which is otherwise similar, lacks that methyl moiety and did not show mechanism-based inhibition.

In VitroConservation of Twenty-Three Overexploited Medicinal Plants Belonging to the Indian Sub Continent

Twenty-three pharmaceutically important plants, namely, Elaeocarpus spharicus, Rheum emodi, Indigofera tinctoria, Picrorrhiza kurroa, Bergenia ciliata, Lavandula officinalis, Valeriana wallichii, Coleus forskohlii, Gentiana kurroo, Saussurea lappa, Stevia rebaudiana, Acorus calamus, Pyrethrum cinerariaefolium, Aloe vera, Bacopa monnieri, Salvia sclarea, Glycyrrhiza glabra, Swertia cordata, Psoralea corylifolia, Jurinea mollis, Ocimum sanctum, Paris polyphylla, and Papaver somniferum, which are at the verge of being endangered due to their overexploitation and collection from the wild, were successfully established in vitro. Collections were made from the different biodiversity zones of India including Western Himalaya, Northeast Himalaya, Gangetic plain, Western Ghats, Semiarid Zone, and Central Highlands. Aseptic cultures were raised at the morphogenic level of callus, suspension, axillary shoot, multiple shoot, and rooted plants. Synseeds were also produced from highly proliferating shoot cultures of Bacopa monnieri, Glycyrrhiza glabra, Stevia rebaudiana, Valeriana wallichii, Gentiana kurroo, Lavandula officinalis, and Papaver somniferum. In vitro flowering was observed in Papaver somniferum, Psoralea corylifolia, and Ocimum sanctum shoots cultures. Out of 23 plants, 18 plants were successfully hardened under glasshouse conditions.

Impact of BAP and different auxins on Invitro shoot proliferation of Rheum emodi Wall.

Impact of BAP and different auxins on Invitro shoot proliferation of Rheum emodi Wall.

Anthraquinone derivatives based natural dye from Rheum emodi as a probe for thermal stability of proteins: Spectroscopic and chromatographic studies

Rheum emodi is a storehouse of a large number of anthraquinone derivatives which are known for a large number of biological activities of significant potency. In this work, a study on the interactions between anthraquinone derivatives based natural dye isolated from R. emodi and different proteins has been reported for the first time, revealing the use of dye as an extrinsic probe to determine the stability of these proteins alone and in the presence of additives. The stability parameters have been evaluated as a change in the fluorescence intensity of the dye as a function of temperature due to the differential interaction of the dye with various conformations of a protein. Also, the effect of the change in polarity of the solvent on the fluorescence emission spectra of dye was studied where high quantum yield was observed in alcohol as compared to water. The RP-HPLC characterization of the dye revealed the presence of anthraquinones glycosides as main compounds in it. Thus, natural dyes may be used as biosensors to follow the conformational changes in proteins.

An RNA isolation system for plant tissues rich in secondary metabolites

BackgroundSecondary metabolites are reported to interfere with the isolation of RNA particularly with the recipes that use guanidinium-based salt. Such interference was observed in isolation of RNA with medicinal plants rheum (Rheum australe) and arnebia (Arnebia euchroma). A rapid and less cumbersome system for isolation of RNA was essential to facilitate any study related to gene expression.FindingsAn RNA isolation system free of guanidinium salt was developed that successfully isolated RNA from rheum and arnebia. The method took about 45 min and was successfully evaluated on twenty one tissues with varied secondary metabolites. The A260/280 ratio ranged between 1.8 - 2.0 with distinct 28 S and 18 S rRNA bands visible on a formaldehyde-agarose gel.ConclusionsThe present manuscript describes a rapid protocol for isolation of RNA, which works well with all the tissues examined so far. The remarkable feature was the success in isolation of RNA with those tissues, wherein the most commonly used methods failed. Isolated RNA was amenable to downstream applications such as reverse transcription-polymerase chain reaction (RT-PCR), differential display (DD), suppression subtractive hybridization (SSH) library construction, and northern hybridization.

Quality Control Analysis of Safoof-e-Pathar Phori, A Unani Herbo-mineral Formulation

Safoof-e-Pathar Phori is a Unani polyherbo-mineral formulation and has been used in the Unani system of medicine for its anti-urolithiatic activity [1]. It is a powdered formulation, which contains six different plant/mineral constituents: Pathar phori (Didymocarpous pedicellata), Kulthi (Dolichous biflorus), Revand chini (Rheum emodi), Namak turb (Raphanus sativus), Jawakhar (Potassium carbo-nate) and Shora qalmi (Potassium nitrate) [2, 3, 4]. The formulation was prepared and quality control studies were carried out as per WHO and AYUSH guidelines. The formulation was evaluated for its pharmacognostic parameters (macroscopy and microscopy), physicochemical parameters (loss on drying, ash value, extractive value, successive extraction), total phenolic and flavonoid content as well as chromatographic profiling (TLC, HPTLC and HPLC). The safety evaluation was also done for heavy metal analysis. Aflatoxin and pesticides data generated can be used for quality control of Safoof-e-Pathar Phori, whereas chromatographic fingerprints generated can be used for its identification of formulation. Acknowledgements: Authors are grateful to CCRUM-AYUSH, Ministry of Family Welfare, Government of India, for providing financial assistance.

Photosynthetic Characteristics of Indian Rhubarb (Rheum emodi Wall.): An Endangered Medicinal Plant

Rheum emodi Wall. ex Meissn (Polygonaceae) is a perennial herb distributed in altitudes ranging from 2800 to 3800m in the temperate and subtropical regions of Himalayas. The herb has been traditionally used to treat pathological ailments like fevers, ulcers, bacterial and fungal infections, jaundice and liver disorders. In addition to that, roots of R. emodi are reported to have biological activities such as laxative, diuretic, and in vivo inhibitory effect towards P388 leukemia in mice models. Due to commercial exploitation and its unscientific extraction from nature, Rheum emodi has been categorized as an endangered and top-priority species for conservation by Threatened Plants Species Committee of the Survival (TPSSC) of International Union for Conservation of Nature (IUCN). In this regard, strategies are being developed for the conservation and cultivation of this species at comparatively lower altitudes. In the present study, photosynthetic characteristics of R. emodi Wall were studied to assess its conservation and cultivation possibility at comparatively lower altitude. Plants of R. emodi, collected from its natural habitat (Tungnath, 3600m altitude), grown at lower altitude (550m) were examined for their growth and photosynthetic characteristics at different light (0, 100, 600, 900, 1200, 1500 and 2000µmol m-2 s-1), and temperature (10, 15, 20, 25, 30 oC) and CO2 (350 and 700µmol mol-2) concentrations. Optimum temperature for photosynthesis in this species was observed at 25 oC, which appears to be much higher than the average temperature of its natural habitat (3600m) during active growth period. A gradual increase in the rate of photosynthesis with light intensity up to 2000µmol m-2 s-1 at both (ambient and elevated) levels of CO2 indicates its adaptability potential to survive under high irradiance. Under optimum conditions, a considerable increase (˜ 30%) in its water use efficiency was observed when exposed to elevated CO2 concentration. Since lower altitude is normally characterized with higher temperature and CO2 conditions as compared to alpines, this species can perform much better at lower altitudes as it is able to acclimate under higher temperature and CO2 conditions. Within its limits, this study suggests that R. emodi can be cultivated at lower altitudes in sun-exposed sites for its conservation and cultivation to meet its ever increasing industrial demand.

Distribution pattern and conservation of threatened medicinal and aromatic plants of Central Himalaya, India

A study was conducted to examine the distribution pattern of four rhizomatous medicinal and aromatic plant species (MAPs) viz., Angelica glauca, Pleurospermum angelicoides, Rheum emodi and Arnebia benthamii in different forest stands in Central Himalaya. Results show that A. glauca and P. angelicoides had a higher (50%) frequency at Chipkoan, Garpak and Phagati forest, R. emodi had a higher (60%) frequency at Rishikund, Suki and Himtoli, and A. benthamii had a higher (70%) frequency at Suki and Khambdhar The densities of A. glauca (0.6 plants·m−2) and P. angelicoides (0.5 plants·m−2) were higher at Chipkoan and Garpak sites than at other micro-sites, while densities of R. emodi (0.8 plants·m−2) and A. benthamii (1.0 plants·m−2) were higher at Suki and Khambdhar sites. A. glauca had highest total basal covers (TBC) (1.2 cm2·m−2) at Chipkoan, P. angelicoides had highest TBC (0.92 cm2·m−2) at Lati kharak site, A. benthamii had the highest TBC (6.48 cm2·m−2) at Khambdhar, and R. emodi had highest TBC (4.53 cm2·m−2) at Rishikund. For the four studied species, A. glauca showed a contagious distribution, P. angelicoides and R. emodi showed the random and A. benthamii showed the regular type of distribution.