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IN-VITRO ANTIOXIDANT AND ANTIDIABETIC EVALUATION OF FRACTIONATED HYDROALCOHOLIC EXTRACT OF LEAVES OF TAGETES ERECTA. LINN

SYNOPSIS FOR

M. PHARM. DISSERTATION

SUBMITTED TO

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES

KARNATAKA



BY

DEEPIKA N.P

I M.Pharm

Department of Pharmacognosy

AL-Ameen College Of Pharmacy

Bangalore – 560 027

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, KARNATAKA, BANGALORE

ANNEXURE – II

PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

1.

NAME OF THE CANDIDATE AND ADDRESS

DEEPIKA N. P

CESELIA, #104

NEAR JYOTHI NIVAS COLLEGE

KORAMANGALA, BANGALORE

2.

NAME OF THE INSTITUTION

AL-AMEEN COLLEGE OF PHARMACY

HOSUR ROAD, BANGALORE – 560 027


3.

COURSE OF STUDY AND SUBJECT

M. PHARM: PHARMACOGNOSY


4.

DATE OF ADMISSION

AUGUST -2012

5.

TITLE OF TOPIC : -

IN-VITRO ANTIOXIDANT AND ANTIDIABETIC EVALUATION OF FRACTIONATED HYDROALCOHOLIC EXTRACT OF LEAVES OF TAGETES ERECTA. LINN




6.0

BRIEF RESUME OF THE INTENDED WORK

6.1

NEED FOR THE STUDY

Medicinal plants and derived medicine are widely used in traditional cultures all over the world and they are becoming increasingly popular in modern society as natural alternatives to synthetic chemicals. In the last few decades there has been exponential growth in the field of herbal medicine. It is getting popularized in developing and developed countries owing its natural origin and lesser side effects¹.

Herbal drugs referred as plants materials or herbalism, involves the use of whole plants or parts of plants, to treat injuries or illnesses. Herbal drugs are the oldest form of health care known to mankind. There are many herbal products offered that assert to treat the symptoms of a broad range of problems, from depression to cold and flu. WHO estimates that 80% of the world populations currently use herbal drugs for major healthcare12.

Tagetes erecta L. (Compositae) is commonly known as “Marigold”. The plant is widely distributed in India, asia, central Europe, USA and Africa3. Marigold is a common garden plant which is rather coarse, erect, branched and grows to about 1 meter high. However there is short or dwarf varieties as well. The leaves are very deeply incised and sharply toothed. Flower heads are solitary, long stalked and thickening upward. The flowers are bright yellow, brownish-yellow or crange3. Different parts of this plants including flower are used in folk medicine to cure various diseases. The leaves are reported to be effective against piles, kidney troubles, muscular pain, ulcers, wounds and earache. The pounded leaves are used as an external application to boils and carbuncles. It is reported to have antioxidant, antimycotic, analgesic activity and 18 active compounds are identified by GC-MS, many of them are terpenoids. The flower is useful in fevers; epileptic fits, astringent, carminative, stomachic, scabies, liver complaints, and also employed in diseases of the eyes. They are said to purify blood and flower juice is given as a remedy for bleeding piles and also used in rheumatism, cold and bronchitis.

Phytochemical studies of different parts have resulted in the isolation of various chemical constituents such as thiophene, flavanoids, carotenoids and triterpenoids. Plant T. erecta has been shown to contain quercetagetin, a glucoside of quercetagetin, phenolics, synergic acids, methyl-3,5-dihydroxy -4-methoxy benzoate, quercetin, thienyl and ethyl gallate2.



In the proposed study, Soxhlet extraction of dried leaves of Tagetes erecta using Water-Alcohol mixture will be carried out followed by fractionation of extract using various Non polar solvents. Each of the fractions will be subjected to qualitative chemical identification tests followed by in-vitro anti-oxidant and anti-diabetic evaluation, later based on the activity, bioactive fractions will be subjected to isolation followed by characterization of biologically active constituents with the help of spectroscopical methods viz. UV, IR, CNMR, HNMR and Mass spectroscopy.




6.2

REVIEW OF LITERATURE

  1. G Gopi et al., (2012) reviewed the importance of Medicinal plants to the health care needs of individuals and their communities, use of herbal preparations made from medicinal plants widespread in developing countries, healing powers of traditional herbal medicines etc. Authors also reviewed the pharmacological and phytochemistry of Tagetes erecta and its application in the treatment of various ailments like the use of flower parts as hepato-protective, insecticide, anti-oxidants and analgesic. This review also discusses the investigation made by various workers related to chemical constituents, pharmacological action and toxicological studies of this plant since years till date1.




  1. Kiranmai et al., (2012) carried out investigation of antibacterial effect of different extracts of leaves and flowers of Tagetes erecta Linn. After performing preliminary Phytochemical screening and thin layer chromatography, antibacterial study was evaluated according to the agar diffusion method by using gram positive B. cereus, S. aureus and gram negative E.coli, P. aeruginosa. Study was showed that petroleum ether extract of leaves and ethyl acetate extract of flower of Tagetes erecta significantly inhibited the growth of bacteria dose dependently2.

  2. Sandipan Chatterjee et al., (2011) compared the efficacy of hydroalcoholic extracts of leaves of Tagetes erecta (T. erecta) and aerial parts of Centella asiatica (C. asiatica) on Excision, Incision and Dead space wound models in albino rats. Extract of T. erecta and C. asiatica (P < 0.001) showed significant in-crease in rate of wound contraction, epithelization and formation of scar faster in excision wound model compare to control group. T. erecta extract (250 and 500 mg/kg) showed significantly increased the wound breaking strength in incision wound model and wet and dry granulation tissue weights, breaking strength in a dead space wound model compare to control and C. asiatica treated group (P < 0.001)3.

  3. Basavaraj Chivde et al., (2011) performed in Vitro antioxidant study on the ethanolic extract of Tagetes erecta flowers. During the study preliminary phytochemical analysis were carried out on ethanolic extract of flowers of Tagetes erecta and found the presence of Alkaloids, Flavonoids, Proteins, Steroids and tannins. For in Vitro antioxidant activity three different assays like DPPH, reducing power and super oxide radical scavenging activity at different concentrations were used. In all the three assay, Tagetes erecta showed better reducing power than the standard (i.e. ascorbic acid), and super oxide anion scavenging activity and DPPH antioxidant activity showed less than standard. However, ethanolic extract of Tagetes erecta demonstrated antioxidant property in all the in Vitro models4.




  1. Rodda Raghuveer et al., (2011) carried out studies on hydro alcoholic extract of Tagetes erecta its anti diabetic activity by inducing diabetes using single intra-peritoneal injection of streptozotocin (60 mg/kg b.w). Treatment with standard drug Glibenclamide, blood glucose rose at 30 min followed by subsequent fall up to 120 min. From present study It was observed that administration of Tagetes erecta extracts showed increase in glucose levels after 30 min and hypoglycaemia effect was observed only after 120 minutes5.




  1. Kiranmai et al., (2011) carried out the screening of wound healing activity of carbopol gels prepared from hydro alcoholic extracts of Gymnema sylvestere (GE) and Tagetes erecta Linn. (TE) in excision wound model and burn wound models, showed significant reduction in period of epithelization and wound contraction and combined gel showed accelerated wound healing activity may be because of synergism. The enhanced wound healing activity of hydro alcoholic extracts may be due to free radical scavenging action and the phytoconstituents (flavonoids) present in it which either due to their individual or additive effect fastened the process of wound healing. Presence of flavonoids in alcoholic extracts was confirmed by phytochemical investigation and TLC methods6.




  1. Rodda Raghuveer et al., (2011) investigated the anti hyperlipedemic activity of hydro alcoholic extract of Tagetes erecta in hyperlipedemic rats at a dose of 200 and 400 mg/kg. Hyperlipidemia was induced by cholesterol 25mg/kg/day. Lovastatin (10mg/kg/day) was used as standard. Blood samples were collected from rats in all the groups on 30th day and estimated for their serum cholesterol, serum triglyceride, serum HDL and serum LDL levels using standard procedures. From the study it was observed that administration of Tagetes erecta extracts significantly decreased all the hyperlipedemic parameters in rats7.




  1. Ranjan kumar et al., (2011) giving emphasis on use of many folk remedies from plant origin for their potential antioxidant and hepatoprotctive liver damage in experimental animal models, carried out hepatoprotective activity in Carbontetrachloride (CCl4)-induced hepatotoxicity model using 80% ethanolic soxhlet extract. In the experiment Wistar albino rats (150-250g) of either sex were used for the activity. The ethanolic extract of Tagetes erecta was found to show significant increase in serum ALT, AST, ALP and bilirubin levels in carbon tetrachloride intoxicated groups compared to the normal control group. Ethyl acetate fraction of T. ereta at the dose of 400mg/kg oraly significantly decreased the elevated serum marker enzymes and level of bilirubin almost to the normal levels compared to carbon tetrachloride intoxicated group8.




  1. Ioana-raluca Bunghed et al., (2011) demonstrated the importance of Marigold, a very useful species of medicinal plants with many uses in phyto-therapy and cosmetics. The pigment content was demonstrated and measured by thermal analysis, UV-VIS and FT-IR spectrometry. The results confirm the fact that this plant contains important pigments useful for our healthy life9.




  1. G Elango et al., (2011) assessed the ethyl acetate, acetone and methanol extracts of Andrographis paniculata, Eclipta prostrata and Tagetes erecta leaves for oviposition-deterrent, ovicidal and repellent activities against malaria vector, Anopheles subpictus Grassi (Diptera: Culicidae) and emphasized on Mosquito control facing a threat due to the emergence of resistance to synthetic insecticides and potential Insecticides of plant origin which may serve as suitable alternative biocontrol techniques in the future10.




  1. Agiang et al., (2011) carried out the investigation on effects of inclusion of aqueous extract of Bush marigold (Aspilia africana) leaf in quail diet using one hundred and fifty Japanese quail chicks in the study. In the 14-weeks feeding experiment, the birds were assigned to five treatments of; 0, 2.5, 5.0, 7.5 and 10% inclusion of aqueous extract of Bush marigold leaf. Each treatment had 3 replicates of 10 birds per replicate in a randomized complete block design. The results indicated that feeding Aqueous extract of Bush Marigold Leaf (AeBML) did not affect (p>0.05) daily weight gain, feed conversion ratio and carcass yield. Mortality and % cracked eggs were reduced (p<0.01). Feed intake and dressed weight of growing quails were significantly (p>0.05) affected by dietary supplementations with AeBML. Egg number and hen day production increased (p<0.001) as level of AeBML increased, though egg weight decreased (p<0.01). Feeding AeBML improved (p<0.001) albumen weight, shell thickness and yolk colour when compared to the control11.




  1. Bodhisattwa Maiti, et al., (2011) reviewed the therapeutic effectiveness of various herbal medicines, adverse drug reactions, drug interactions, standardization and stability testing of herbal medicines, pharmacovigilance and regulatory status of herbal medicines. Herbal medicines have been used since the dawn of civilization to maintain health and to treat various diseases. To compete with the growing pharmaceutical market, there is an importance to use and scientifically authenticate more medicinally useful herbal products12.




  1. Rainer W Bussmann et al., (2010) carried out studies on antibacterial activity of medicinal plants of Northern Peru . In this communication, antibacterial assays for 165 plant species conducted under simple laboratory conditions in a private clinic in Trujillo, Peru has been reported. Extracts of samples of 148 species traditionally used as antibacterial were screened for activity against Staphylococcus aureus and Escherichia coli using an agar-diffusion method. In addition, 17 closely related species that were also part of the local pharmacopoeia, but only used for other purposes, were included for comparison. Sixteen species tested as traditional water extract and 96 species extracted in ethanol showed activity against at least one of the bacteria. The study confirms that simple laboratory methods are very well suited to assess the efficacy of traditionally used medicinal plants to inhibit bacterial growth13




  1. B P Muley et al., (2009) giving emphasis of multiple pharmacological activities Calendula officinalis Linn. (Asteraceae). It is used in traditional medicine, especially for wound healing, jaundice, blood purification, and as an antispasmodic. Chemical studies have underlined the presence of various classes of compounds, the main being triterpenoids, flavonoids, coumarines, quinones, volatile oil, carotenoids and amino acids. The extract of this plant as well as pure compounds isolated from it, have been demonstrated to possess multiple pharmacological activities such as anti-HIV, cytotoxic, anti-inflammatory, hepato-protective, spasmolytic and spasmogenic, amongst others. In this review, we have explored the phytochemistry and pharmacological activities of C. officinalis in order to collate existing information on this plant as well as highlight its multi-activity properties as a medicinal agent14.




  1. G Elango et al., (2009) assessed the acetone, chloroform, ethyl acetate, hexane, and methanol extracts of Aegle marmelos , Andrographis lineata , Andrographis paniculata , Cocculus hirsutus , Eclipta prostrata , and Tagetes erecta for activity against fourth-instar larvae of malaria vector, A. subpictus Grassi and Japanese encephalitis vector, C. tritaeniorhynchus Giles (Diptera: Culicidae). All plant extracts showed moderate larvicidal effects after 24 h of exposure at 1,000 ppm; however, the highest larval mortality was found in leaf ethyl acetate of A. marmelos, E. prostrata, hexane, methanol of A. paniculata and C. hirsutus against the larvae of A. subpictus and against the larvae of C. tritaeniorhynchus respectively. This study showed that leaf hexane extract of A. paniculata and ethyl acetate extract of E. prostrata have the potential to be used as an ideal eco-friendly approach for the control of the A. subpictus and C. tritaeniorhynchus.15




  1. Chakraborthy et al., (2009) carried out the investigation of quercetin in T erecta using HPTLC. In this plant reported constituents are flavonoids, glycosides and saponins. It is considered to contain Quercetin, which was confirmed by TLC and qualitative test. Thus it was quantified using HPTLC. The method was carried out by using with silica gel 60 GF as stationary phase using solvent system as chloroform: methanol (9: 1) with Rf value of 0.43. Quantitative analysis was carried out in the absorbance at 366 nm. The linearity regression for the calibration showed r = 0.6548 and 0.999 with respect to height and area in a range of 0.5-5.0 g per spot. Thus the method developed can be used for routine analysis of marker compounds in crude drugs as well as in herbal formulations as an ingredient16.




  1. Koon-Hui wang et al., (2007) carried out the studies on nematocidal activity of marigold. This plant produces number of potentially bioactive compounds, among which α-therthienyl is recognized as one of the most toxic. This sulfur-containing compound is abundant in marigold tissues, including roots. It has nematicidal, insecticidal, fungicidal, antiviral, and cytotoxic activities, and it is believed to be the main compound responsible for the nematicidal activity of marigold. Nematicidal compounds apparently permeate from marigold root tissues into nematodes attached to the root, but they are also believed to kill nematodes found in the rhizosphere, the soil near marigold roots. Thus, marigold is believed to be most effective in suppressing plant-parasitic nematodes .17




  1. T I olabiyi et al., (2007) carried out the studies on field experiment to control nematode pests of cowpea (Ife brown variety) with marigold (Tagetes erecta) as inter plants were planted with in the rhizoplane and rhizosphere of cowpea grown on naturally nematode infested field . Nematode pest of cowpea encountered on the experimental field were Meloidogyne spp., Prantylenchus spp., and Helecotelynchus spp. The experiment was laid out in a randomized complete block design with five treatments replicated five times. cowpea is susceptible to nematode pests, and marigold that were planted with in the rhizoplane and rhizosphere significally controlled the nematode pests there by enhanced the growth rate per cowpea plant18.




  1. P. O. owino et al., (1992) assessed the Marigold leaf extract and captafol significantly inhibited fungal parasitism of Meloidogyne incognita and M. javanica eggs. Captafol treated eggs were the least parasitised. The highest concentration of extract had the second most significant inhibitory effect on egg parasitism. No significant difference in fungal egg parasitism of the two nematodes was detected in all treatments except at the lowest marigold extract concentration. Fungal parasitism of M. javanica eggs was significantly higher at this extract concentration. Fusarium so/ani and F. oxysporum differed in their ability to parasitize eggs. The former fungus was more parasitic than the latter19.

6.3

OBJECTIVE OF STUDY




Aim: To Carry out “In-Vitro Antioxidant and Antidiabetic Evaluation of Fractionated Hydroalcoholic Extract of Leaves of Tagetes erecta. Linn

The objectives are:

  1. Collection and authentification of plant material.

  2. Hydro alcoholic extraction of leaves of Tagetes erecta Linn.

  3. Phyto chemical screening of leaves of Tagetes erecta Linn.

  4. Fractionation of hydroalcoholic extract of Tagetes erecta Linn. In different solvents.

  5. Invitro Antioxidant testing of fractions of hydroalcoholic extract.

  6. Antidiabetic testing of fractions of hydroalcoholic extract.

  7. Isolation & Characterization of bioactive constituents.




7.0

MATERIALS AND METHODS

7.1

SOURCES OF DATA

  1. Information Centre, Al-Ameen College of Pharmacy, Bangalore.

  2. Search on MEDLINE, PUBMED, etc.

  3. Google search.

  4. Journals and publications.




7.2

METHOD OF COLLECTION OF DATA


PLACE OF STUDY:

Olive Life Sciences, Tumkur



  1. Leaves of Tagetes erecta Linn. will be collected and authenticated.

  2. Pharmacognostical evaluation viz., preliminary phytochemical screening of the plant will be carried out by extracting the dried plant leaves with Hydro-Alcoholic solvent by soxhlation followed by identification by performing qualitative chemical tests for the various secondary constituents.

  3. Fractionation of the Hydroalcoholic extract of leaves of T. erecta will be carried out using various solvents.

  4. Different fractions prepared will be subjected to activity evaluation for finding out Antioxidant activity and Antidiabetic activity using various in-vitro assays like DPPH for antioxidant and α-Glucosidase inhibition assay for antidiabetic evaluation.

  5. The fraction showing maximum biological activity in antidiabetic activity evaluation will be subjected to further purification through column chromatography, followed by Flash chromatography and monitored with TLC (and or with the assist of other chromatographic techniques). The purified bioactive components will be characterized with the help of spectroscopical methods viz. UV, IR, CNMR, HNMR and Mass spectroscopy.




7.3

PLACE OF WORK:


Olive Lifesciences Pvt. Ltd.Tumkur

7.4

Does the study require any investigations or interventions to be conducted on patient or other humans or animals? If so, please describe briefly.

-NO-


7.5

Has ethical clearance been obtained from your institution in case of the above query?

-NA- [



8.0

REFERENCES:-

  1. Gopi G, Elumalai A, Jayashri P. A concise review on Tagetes erecta (L). International Journal of Phytopharmacy and Research 2012; 3(1): 16-19.

  2. Kiranmai M, Ibrahim M. Antibacterial potential of different extracts of Tagetes erecta (L). International Journal of Pharmacy 2012; 2(1): 90-96.

  3. Sandipan Chatterjee, Prakash T, Kotrsha D, Rao RN, Goli D. Comparative Efficacy of Tagetes erecta and Centella asiatica Extracts on Wound Healing in Albino Rats. Scientific Research 2011; 2: 138-142.

  4. Chivide VB, Birder VK , Shirma SR , Manoj VK.Invitroantioxidant activity studies on the flowers of Tagetes erecta L. International Journal of Pharma and Bio sciences 2011; 2(3): 223-229




  1. Raghuveer Rodda, Kota A , Sreeja K, Ch R ,Valya N. Antidiabetic potential of Tagetes erecta whole plant in streptozotocin induced diabetic rats. Journal of Pharmacy Research 2011; 4(11): 4032-4034




  1. Kiranmai M, kazim S M , Ibrahim M. Combined wound healing activity of Gymnema stylvestere and Tagetes erecta L. International Journal of Pharmaceutical Applications 2011; 2(2): 135-140.




  1. Rodda Raghuveer, Sreeja K, Sinduri T, Kumar AS. Antihyperlipidemic Effect of Tagetes erecta in cholesterol fed hyperlipedemic rats. Scholars Research Library 2011; 3(5): 266-270.




  1. Ranjan Kumar Giri, Bose A, Mishra K S. Hepato-protective activity of Tagetes erecta against carbon tetra chloride induced hepatic damages in rats. Acta Poloniae Pharmaceutica-Drug Research 2011; 68: 999-1003.

  2. Ioana-raluca Bunghed, Ion MR. Complex spectral characterization of active principles from marigold (Calendula officinalis). Journal of Science and Arts 2011; 59-64.

  3. Elango G, Zahir AA, Bagavan A, Kamaraj C, Rajakumar G, Santhoshkumar T, Marimuthu S, Rahuman AA. Efficacy of indigenous plant extracts on the malaria vector Anopheles subpictus Grassi (Diptera: Culicidae). Indian J Med Res. 2011; 134: 375-383.




  1. Agiang EA, Oko Ook , Essien GE . Quails Response to Aqueous Extract of Bush Marigold (Aspilia africana) Leaf. American Journal of Animal and Veterinary Sciences 2011; 6 (4): 130-134.

  2. Bodhisattwa Maiti, Nagori BP, Singh R, Kumar P, Upadhyay N. Recent trends in herbal drugs a review. Int. J .Drug Res. Tech. 2011; 3(1): 17-25.

  3. Rainer W Bussmann, Glenn A, Sharon D. Antibacterial activity of medicinal plants of Northern Peru – Can traditional applications provide leads for modern medicine. Indian Journal of Traditional Knowledge 2010; 9(4): 742-753.

  4. Muley BP, Khadabadi SS, Banarase NB. Phytochemical Constituents and Pharmacological Activities of Calendula officinalis Linn (Asteraceae): A Review. Tropical Journal of Pharmaceutical Research 2009; 8 (5): 455-465.




  1. Elango G, Rahuman AA, Bagavan A, Kamaraj C, Zahir AA , Venkatesan C. Laboratory study on larvicidal activity of indigenous plant extracts against Anopheles subpictus and Culex tritaeniorhynchus. Parasitol Res. 2009; 1381-1388.




  1. Chakraborthy GS. Determination of quercetin by HPTLC in Tagetes erectus extract. Journal of Global Pharma Technology 2009; 1(1): 67-70

  2. Wanga Koon-Hui, Hooksa Cerruti, and Ploeg Antoon. Protecting Crops from Nematode Pests: Using Marigold as an Alternative to Chemical Nematicides. Plant Disease 2007; 1-6.

  3. Olabiyi TI, Oyedunmade EEA . Marygold (Tagetes erecta L) as interplant with cowpea for the control of nematode pests. African Crop Science Conference Proceedings 2007; 8: 1075-1078.

19. Owino P. Effect of Marigold leaf extract and captafol on fungal parasitism of root knot nematode eggs-kenyan isolates. Nematol. Medit. 1992; 20: 211-213.

20. Kurian JC. Plants that Heal. Oriental watchman publishing house 2004; 1: 135.






9.

SIGNATURE OF THE CANDIDATE




10.

REMARKS OF THE GUIDE

RECOMMENDED FOR RESEARCH AND SUBMISSION OF DISSERTATION

11.

NAME AND DESIGNATION OF THE GUIDE

Prof. B. G SHIVANANDA

Principal & Professor

Department of Pharmacognosy,

Al-Ameen College of Pharmacy, Bangalore.



11.1

SIGNATURE




11.2

CO-GUIDE (INSTITUTE)

Mr. TRIPATHY AMRUTANAND. S

Department of Pharmacognosy,

Al-Ameen College of Pharmacy, Bangalore.


11.3

SIGNATURE




11.4

CO-GUIDE (INDUSTRY)

Dr. H. N SHIVAPRASAD

Vice president –Research and Technical Affairs

Olive Lifesciences Pvt. Ltd.Bangalore


11.5

SIGNATURE




11.6

HEAD OF THE DEPARTMENT

Dr. SALMA KHANAM

Professor & HOD

Department of Pharmacognosy,

Al-Ameen College of Pharmacy, Bangalore.



11.7

SIGNATURE

.

11.8

PRINCIPAL


Prof. B. G. SHIVANANDA

Al-Ameen College of Pharmacy,



Hosur road, Bangalore.

11.9

REMARKS OF THE PRINCIPAL

RECOMMENDED FOR RESEARCH

12

SIGNATURE








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