|Rajiv Gandhi University of Health Sciences
“Evaluation of Some Indian Medicinal Plants for Antimicrobial, Antioxidant
and Cytotoxic Activity”
A Protocol submitted to Rajiv Gandhi University of Health Sciences
in partial fulfillment of the requirement for the award of
MASTER OF PHARMACY
Department of Pharmacognosy,
National College of Pharmacy,
A. Brief resume of the work:
A.1.Need of the study:
Herbal medicine is the oldest form of health care known to mankind. Herbs had been used by all the cultures throughout the history. It was an integral part of the development of modern civilization. The world health organization (WHO) estimates that 80% of the world populations presently use herbal medicine for some aspects of their primary health care. Herbal medicine is a major component of all indigenous peoples traditional medicine1 and a common element in Ayurvedic, Homoeopathic, Naturopathic, Traditional oriental and Native American Indian medicine. WHO notes that of 119 plants derived pharmaceutical medicines, about 74% are used in modern medicine.
Numerous screening of medicinal plants has been carried out for antimicrobial, cytotoxic and antioxidant activity. Some of the plants have shown satisfactory antimicrobial activity on certain Gram positive and Gram negative bacteria as well as anti oxidant property against certain free radicals. Many a times, Carcinogenesis is associated with the generation of reactive oxygen species (ROS) result from cell metabolism2 as well as extra cellular process. Although these ROS posses a necessary physiological function in homoeostasis but when produced in excess, they play role in pathogenesis of cancer. ROS exert detrimental effects such as damaging of cell macromolecules i.e. DNA, lipid and proteins. Among these targets, lipid peroxidation is particularly damaging because it leads to facile propagation of free radical reactions. It is very interesting to observe that, some compounds with highest antioxidant activity have also shown highest cytotoxic activity as well as antitumor promoting activity. These plants are of special interest.
The best approach of the phytochemist is, would be to screen for biological activity, the active extracts selected, fractions directed with bioassay and only the bioactive compounds isolated and identified. For this, three readily available technical methodologies are,
1) Separation techniques (Chromatography)
2) Structural elucidation (Spectral studies)
3) Simple bioassays
Two simple bioassay procedures have been evolved by the scientists of, Department of medicinal chemistry and pharmacognosy, school of pharmacy and pharmaceutical sciences, Purdue University.3
One of these is Brine Shrimp Lethality test, a general bioassay which detects a broad range of biological activities and a diversity of chemical structures. The basic for this method is that toxicology is simply pharmacology at a higher dose (or pharmacology is simply toxicology at a lower dose). Thus if a toxic compound is found, a lower non toxic dose might elicit a useful, pharmacology perturbation on a physiologic system.
The National Cancer Institute (USA) collected about 35000 plants from 20 countries and has screened around 114000 extracts for their anticancer activity4. Of the 92 anticancer drugs commercially available prior to 1983 in the US and among worldwide approved anticancer drugs between 1983 and 1994, 60% are of natural origin. The discovery of Paclitaxel from Pacific yew Taxus bravifolia and Camptothecin from Chinese ornamental plant Camptotheca acuminate has opened a new horizon for the treatment of carcinogenesis. There are more that 2,70,000 higher plants existing on this Earth, but a small portion so far has been explored phytochemically. So it can be anticipated that plants can provide potential bioactive compounds for the development of new ‘Leads’ to combat cancer disease.
Henceforth in the present study, it was decided to evaluate some of the Indian medicinal plants for their cytotoxic and antimicrobial property as well as evaluation of the responsible phytoconstituents. The plants will be selected based on their traditional uses or previous scientific data which would provide us new molecule to fight Cancer.
A.2.Review of the articles:
Cytotoxicity of antioxidant constituents from Hypericum triquetrifolium Turra has been reported. I3-IIB-biapigenin was isolated from the methanolic extract of H.triquetrifolium having potent antioxidant activity ( IC50=5.73 µgm/ml) has manifested strong cytotoxic activity against four human cancer cell lines by Sulforodamine B(SRB) assay. Methanoic extract and pure compounds were tested against Large cell lung carcinoma cell line (COR-L 23), heptocellular carcinoma cell line(HepG2),renal cells adenocarcinoma(ACHN) and the amelanotic melanoma cell lines(C32) and normal human foetal ling cell lines (MCR5)3.
Several plant derived agents being used for the treatment of Cancer including a number of promising agents such as Flavopiridol, Roscovitine, Combretastatin A-4, Betalinic acid and Silvestrol has been reported.4
Ethanolic and methanolic extracts of 16 Algirian plants has shown antibacterial activity against 3 Gram positive bacteria, 3 Gram negative bacteria and 3 Yeast species and their cytotoxic activity against FL-Cells.5
Cytotoxic activity of methanolic extracts of 35 plants including 28 traditionally used plants of Bangladesh was evaluated by Brine Shrimp Lethality bioassay method. 19 plant extracts among them found to show significant toxicity of brine shrimps with LC50<10 µgm/ml.6
Antioxidant, antimicrobial and cytotoxic activity of Garcinia eugenifolia and Calophyllum enervosum has been reported. Phytochemical investigation of these plants has yielded 5 known compound namely Combogin, Epicatechin, Osajaxanthone, Rubraxanthone, Isocowanol and one novel compound Enervosanone. Antimicrobial assay was performed using disc diffusion method, antioxidant evaluation using DPPH method by Electron spin resonance and cytotoxicity was evaluated by MTT assay against MCF-7 cell lines. Enervosanone and Rubraxanthone was found to be active against Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus with MIC value 26.82/60.97 µM, 26.82/30.48 µM, 26.82/60.97 µM and 26.82/60.97 µM. Rubraxanthone and Epicatechin exhibited antioxidant activities with IC50 of 0.89 µM and 2.6 µM respectively. The cytotoxicity assay on MCF-7 lines showed Enervosanone to be active in inhibiting cell proliferation with IC50 of 1.07 µM.7
Screening of 20 Thai local plants for their antimicrobial and cytotoxic activity has been reported. Methanolic extract of Cassia siamea, Limnophila aromatica, and Polygonum odoratum has exhibited strong antioxidant and antimicrobial activities. They found to be sensitive against Bacillus cereus, Listeria monocytogens, Staphylococcus aureus. Highest phenolic content was found in P.odoratum extract was identified by HPLC method has showed antioxidant value EC50 = 315.35 µM/ mg of DPPH. It was found to be moderately active against human brest adeno carcinoma (MCF-7) cell lines.8
8-hydroxy psoralen has been isolated from Wampee (Clausena lansium) and identified based on 1H and 13C NMR and and ESI-IR .It has exhibited good radical scavenging activity against DPPH radical and Superoxide anion as well as significant reducing power. It also has shown proliferation inhibitory activity against Human hepatocellular liver cell lines (HEPG2) Human lung adenrcinoma epithelial cell line (A549) and Human cervical carcino cell line(HELA).9
Screening of ethanolic extracts of seeds of 41 species of Euphorbiaceace has been reported. 18 of these extracts displayed toxicity (LC50 <1000 PPm) in brine shrimp assay results were compared with 9KB & 3PS cytotoxicity studies.10
Anti tumor promoting activity of a polyphenolic fraction isolated from Grape seeds(GSP) has been reported. 7-12-dimethyl benz(a)anthracene (DMBA) and 12-O-tetradecanoyl phorbol 13 acetate (TPA) promoted SENCAR mouse skin two stage carcinogenesis was employed as model system. Following tumor initiation with DMBA , topical application of GSP at a dose of 0.5 and 1.5mg/mouse/application to the dorsal initiated mouse skin showed highly significant inhibition of TPA tumor promotion.11
B. Materials and methods:
B.1.Objectives of the study:
a) Selection of plant with probable antioxidant or cytotoxic activity based on traditional use and literature survey.
b) Collection and authentication of the plant material.
c) Extraction of the plant material with various solvents.
d) Evaluation of antioxidant, antimicrobial and cytotoxic activity (BSL bioassay).
e) Active extracts will be subjected for fractionation.
f) Fractions will be subjected for the above said activities.
g) Active fractions will be subjected for detailed phytochemical investigations.
B.2.Source of data:
The required data will obtained from Electronic data (internet).
Published research papers.
Review and research articles from journals.
Library of IISC, National College Of Pharmacy, Shimoga.
B.3.Plan of Work:
Collection of the plant material: Plants will be identified with the help of botanist and will be collected from natural sources in and around Shimoga (Karnataka).Plants will be dried under shade and coarsely powdered and stored in air-tight containers until use.
Preliminary Extraction: Different plants/plant parts will be successively extracted with different solvents by soxhlet extractor.
Evaluation of anti-oxidant activity will be done by:
DPPH radical scavenging activity
Super oxide radical scavenging activity
Determination of reducing power
Determination of bioactivity
Determination of antimicrobial activity will be performed by cup plate method on both Gram positive and Gram negative bacteria and on fungi for antifungal activity.
Determination of cytotoxic activity: Cytotoxic activity will be determined using Brine Shrimp Lethality (BSL) bioassay developed by Meyer at al 1982 using eggs of Brine Shrimp Artemia salina leach.
B.4.Does this study requires any investigation to be conducted on patients or animals?
No, this study does not require any human or animal activity.
B.5.Has ethical clearance been obtained from your institution?
Conforti F, Loizzo MR, Statti AG and Menichini Z. Cytotoxic activity of antioxidant constituent from Hypericum triquetrifolium Turro. Natural Product Research; 2007; 21(1): 42-46.
Crown Gall Tumors On Potato Disc and Brine Shrimp Lethality : Two Simple Bioassay For Higher Plant Screening and Fractionation By McLaughlin JL ;5 :1
Shoeb M. Anticancer agents from medicinal plants. Bangladesh Journal Of Pharmacology. 2006; 1:35-41.
Akorum S, Dalila S and Korrichi L Antimicrobial, Anti oxidant and Cytotoxic activities and Phytochemical screening of some Algerian Plants European Journal Of Scientific Research 2009;31(2):289-295.
Rehman S, Begum B, Chawdhury R, Khondakar M and Rashid MA. Preliminary cytotoxicity screening of some medicinal plants of Bangladesh. Dhaka University Journal Of Pharmaceutical Sciences. 2008; 7(1):49-52.
Taher MD, Idris SM and Dayer A. Antimicrobial, antioxidant and cytotoxic activities of Garcinia eugenifolia. Iranian Journal Of Pharmacology and Therapeutics 2007; 6(1): 93-98.
Nanasombat S, Teckchuen N. Antimicrobial, antioxidant and anticancer activities of Thai local vegetables. Journal Of Medicinal Plant Research 2009;3(5): 443-449.
Prasad KN, Xie H, Hao J, Yang B, Qiu S, Chen F and Jiang Y. Antioxidant and anticancerous activities of 8-hydroxy psoralen isolated from Wampee [Clausena lansium(Lour)skeels] peels. Food Chemistry 2009; 118: 62-68.
Mayer BN, Forrigni NR, McLaughlin JL. Brine Shrimp: A Convenient General Bioassay For Active Plant Constituents. Planta Medica.1982; 45:31-34.
Lahiri-chatterjee M, Katiyar SK, Mohan RR and Agarwal R. A flavonoid antioxidant, Silymarin affords exceptionally high protection against tumor promotion in SENCAR mouse skin tumorigenesis model. Cancer Research.1999; 59: 622-632.