Hexane Extract of Rhizomes of Curcuma Longa , Zingiber
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Hexane Extract of Rhizomes of RESEARCH Curcuma Longa , Zingiber Officinale and Curcumine Life Span Extension in Caenorhabditis Elegans By Reducing Fat in Intestine. Parveen Gazala *1, Basavan Duraiswamy1, Ansari Firoz2 1 Department of Pharmacognosy, J.S.S. College of Pharmacy Ooty-643001, India 2 Department of Emergency, National Medical College, Birgunj, Nepal Date of Submission : Feb 11, 2018 Received in Revised Form : March 05, 2018 Date of Acceptance : March 20, 2018 Date of Publishing : July 30, 2018 ABSTRACT Curcuma longa and Zingiber officinale provides Background: Curcumine is obtained from longevity and decreases the fat content in curcuma longa and we examined the effects of C.elegans. curcumin, hexane extracts of Curcuma longa and Zingiber officinale on the lifespan and Keywords Curcumin, Curcuma longa, Zingiber aging in Caenorhabditis elegans and found that officinale, Lifespan, Aging, Caenorhabditis it responded to curcumin, hexane extracts of elegans Curcuma longa and Zingiber officinale with an increased lifespan and reduced intracellular *Corresponding Author: Dr. Gazala Perveen, reactive oxygen species during aging. Department of Pharmacognosy, J.S.S. College of Pharmacy Ooty-643001, India, E-mail: Methods: MutantStrains, culture, Curcumin and [email protected] Hexane extract of Rhizomes of Curcuma longa and Zingiber officinale treatment of C. Elegan. INTRODUCTION Curcumin, a yellow colouring agent present in Results: Curcumin and hexane extracts of the spice turmeric (Curcuma longa) and Zingiber Curcuma longa and Zingiber officinale increased officinale that belongs to the ginger (Zingiberaceae) the life span and life cycle of the N2 wild type familu is the pharmacologically active substance and Zdls-5 worms. On comparison, curcumin in turmeric. Traditional Indian medicine considers was found to be the most effective followed curcumin an effective drug for several disorders by the hexane extracts of Curcuma longa and such as GI upset, flatulence, dysentery, ulcers, hexane extracts of Zingiber officinale. Hexane jaundice, arthritis, sprains, wounds, acne, skin extracts of Zingiber officinale were found to be and eye infections.1 Currently, many lines of least effective. Sudan black staining exhibited evidence indicate that curcumin exhibits anti- that stored contents of fat in C.elegans decreased inflammatory, anticarcinogenic, antiaging and as the concentration of the drug increased. antioxidant properties.2,3,4 Today, ginger root is widely used as a digestive aid for mild stomach Conclusions: Our study has established that upset and is commonly recommended by health curcumin and hexane extract of rhizomes of care professionals to prevent or treat nausea MED PHOENIX : An Official Journal of NMC, Birgunj, Nepal, Volume (3), Issue (1), July, 2018, 21-28 21 Gazala et al. and vomiting associated with motion sickness, bacterial lawn was made because it allows for pregnancy, cancer chemotherapy and also used easier observation and better mating of the as an antiaging agent.5,6,7 The nematode animal worms. The LB media were stored at room model, Caenorhabditis elegans (C. elegans) has temperature and inoculated to overnight at 37°C. been increasingly utilized for biological and The E. coli OP50 streak plate and liquid culture medical studies and >65% of the genes relating were stored at 4°C. The E.coli was taken into to human diseases are conserved in C. Elegans.8 NGM media for feeding worms.14 Three hundred and five genes in C. elegans have been shown to be involved in reducing body fat and Method for preparation of NGM Petri plates.15 112 genes are involved in increasing fat storage as NaCl 3g, 17 g agar, and 2.5 g peptone was mixed in demonstrated by RNAi and Sudan black staining, a 2 litre Erlenmeyer flask. Water 975 ml was added. Nile Red staining.9 C. elegans is well-suited to The mouth of flask was covered with aluminium obesity studies because deposits of fat for energy foil and autoclaved for 50 min. The flask was storage can be found along its intestinal tract and the cooled in 55°C water bath for 15 min. CaCl2 1 ml bodies of C. elegans are transparent.10 Moreover, of 1 M, 1 ml in 5 mg/ml cholesterol in ethanol, 1 C. elegans and humans share similar aspects of ml of 1 M MgSO4 and 25 ml of 1 M KPO4 buffer ageing.11 It is for these reasons that C. elegans is was mixed. Swirling was done to mix well. Using highly-valued for identifying compounds, genes, sterile procedures, the NGM solution was dispensed and mechanisms that may extend the longevity into Petri plates using a peristaltic pump. Plates of humans.12 To our best knowledge, the uptake were filled with 2/3 full of agar. Plates were left at of curcumin and other pharmacological active room temperature for 2-3 days before use to allow compounds or extracts of plant origin into the for detection of contaminants and to allow excess worms has not been studied yet. To investigate moisture to evaporate. Plates were stored in an air- whether curcumin can delay aging and prolong tight container at room temperature. the lifespan of a whole organism, we used the nematode C. elegans . Seeding NGM plates Using sterile technique approximately 0.05 ml of Herein, we study that the effect of curcumin, E. coli OP50 liquid culture in small or medium hexane extracts of Curcuma longa and Zingiber NGM plates or 0.1 ml to large NGM plates using a officinale treatment on resistance to oxidative pipette. Spreading will create a larger lawn which stress and adult lifespan of C. elegans. can aid in visualizing the worms. The worms tend to spend most of the time in the bacteria. E. MATERIALS AND METHODS coli OP50 lawn was allowed to grow overnight Curcumin (Sigma–Aldrich) was dissolved in at room temperature or at 37°C for 8 hours (cool dimethyl sulfoxide (DMSO, Sigma–Aldrich). plates to room temperature before adding worms). A final DMSO concentration of 0.1% (v/v) was Seeded plates were stored in an air-tight container maintained under all conditions. which could be used for 2-3 weeks. Mutant Strains, culture, Curcumin and Hexane Culturing C. elegans on petri plates extract of Rhizomes of Curcuma longa and C. elegans, single worms between the fourth Zingiber officinale treatment of C. elegan larvae and adult stages: for synchronization maintained on NGM were picked with a platinum C. elegans two strains N2 Wild type and Zd Is 5 wire (heated in a flame red hot and cooled) and stain: sk4005 were collected from national centre transferred to new NGM plates. for biological sciences Banglore. Assays Maintenance of C. elegans Experiments to test the tolerance of C.elegans Preparation of bacterial food source to Curcumin (10, 20, 50, 100 and 500 µg/ml in C. elegans were grown in the laboratory using 0.1% DMSO) was conducted in the absence and E.coli strain OP50 as a food source.13 A limited presence of E.coli in liquid NGM. Single colonies 22 MED PHOENIX : An Official Journal of NMC, Birgunj, Nepal, Volume (3), Issue (1), July, 2018, 21-28 Life Span Extension of E.coli growing on LB plates were transferred the yield of hexane extract of Zingiber officinale to test tubes containing 3ml of liquid NGM was found to be 15gm. Extracts were obtained medium. Ten worms growing on NGM plates by silica gel column chromatography of were transferred to these tubes. The worms were Curcuma longa and Zingiber officinale, using observed periodically after shaking the test tubes. hexane, Chloroform : hexane (1:1), chloroform, The time taken for the worms to become immobile chloroform : Methanol (1:1) as a solvent. The was noted. The sensitivity of these worms to touch total yield of material and % of hexane extract after transfer to solid NGM-media was monitored. of these fractions were found to be 13, 0.404, O O 0.888, 0.338, 2.574, 0.690, 0.497, 0.388, O O H3C CH3 1.005gm and 100, 3.1, 6.8, 2.6, 19.8, 5.3, 3.8, Curcumin 2.9, 7.7 % for Curcuma longa and 15, 0.348, HO OH (1E,6E)-1,7-Bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione 0.215,0.294, 1.921, 0.560, 0.785, 0.299, 1.464 gm and 100, 2.3, 1.4, 1.9, 12.8, 3.7, 5.2, 1.9, Life span of C.elegans in solid medium 9.7% for Zingiber officinale.19 Synchronized worms N2 wild, zdis-5 were incubated for 24 hrs on NGM/OP50 plates16 Tolerance of C.elegans to Curcumin several plates were seeded with ten worms each. Tolerance of C. elegans to Curcumin at different The plates were then maintained at 200C, 250C or concentration (10, 20, 50,100 and 500 µg/ml at 370C. The adult worms were picked daily and in 0.1% DMSO) was conducted in the absence transferred to new plates for determination of life and presence of E.coli in liquid NGM. All the span. The ability of the worms to respond to touch worms were found dead on the third day of the was monitored daily. A worm was considered experiment. dead if it did not respond to touch and lay eggs. Experiment life span was determined in the Life span in solid medium absence of E.coli too. Experiments were carried Life span in solid medium without E.coli at out with the incorporation of 0.5, 1.0, 1.5 and 2.0 200C, 250C for N2 wild type and zdls-5 worms µg/ml of curcumin and the hexane extracts from was performed in all the concentrations (0.5, Curcuma longa and Zingiber officinale (dissolved 1.0, 1.5 and 2.0 µg/ml).