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3_AJPR_2_1_2012

3_AJPR_2_1_2012

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Asian J. Pharm. Res. 2012; Vol. 2: Issue 1, Pg 19-31
[AJPRes.]
19
 
ISSN- 2231–5683 (Print)
www.asianpharmaonline.org
 ISSN- 2231–5691 (Online)0974-3618
  RESEARCH ARTICLE
Secondary Metabolite screening, Bioactive compound extraction, andDisrupting Mitotic Activity of Wild Cabbage [Brassicaceae] towardsCancer Management.
A. Thenmozhi
1
, Dr. U.S Mahadeva Rao
2
*
1
Assistant Professor, PG Department of Biochemistry, SRM Arts and Science College, Chennai-603203, India.
2
Associate Professor, Department of Biochem., Faculty of Medicine and Health Science, Universiti Sultan ZainalAbidin, Malaysia.*Corresponding Author E-mail:
raousm@gmail.com
ABSTRACT:
 
Objective: The present study has been formulated with an objective to establish the preliminary phytochemicalanalysis and antimitotic activity of Brassica oleracea. Method: Brassica oleracea was collected, homogenized andextracted with different solvents. Antimitotic activity of Brassica oleracea was evaluated using the meristamatic cellsfrom the root of Allium cepa. Experiments were carried out with incorporation of folic acid in the extract. Results: Thepreliminary study revealed the presence of flavonoids, steroids, alkaloids, saponins, polyphenols and glycosides. Folicacid inhibited the antimitotic activity of Brassica oleracea extract. The antimitotic activity obtained was compared withmethotrexate-a referred anticancer drug. Discussion: The results obtained from the present study pinpoint thatantimitotic activity of Brassica oleracea may be due to the presence of flavonoids, steroids, alkaloids, polyphenols andsaponins. Conclusion: Hence Brassica oleracea is a promising source of phytochemicals which promote human healthby strengthening the human immune system, inactivate cancer-causing substances, protect the heart and eyes fromdisease, boost enzyme activity to increase the benefits of the various protective enzymes, reduce bad cholesterol levels,and anti-aging.
KEYWORDS:
Antimitotic; Brassica oleracea; Allium cepa; Polyphenols; Saponins.
INTRODUCTION:
The “functional food” industry has produced and marketedfoods enriched with bioactive compounds, but there are nouniversally accepted criteria for judging efficacy of thecompounds or enriched foods. The lack of understandingbioactive compounds and their health benefits should notserve to reduce research interest but should insteadencourage plant and nutritional scientists to work togetherto develop strategies for improvement of health throughfood
1
.Cruciferous vegetables are an excellent dietary source of phytochemicals including glucosinolates (and glucosinolatebreakdown products), phenolics and other antioxidants likevitamins(C, K1, etc.), as well as dietary essential minerals(Ca, Mg, Na, K, Fe, Zn, etc.). Dietary antioxidants (i.e.vitamins, flavonoids) present in broccoli may decrease therisk of certain cancers
2
.
Received on 24.12.2011 Accepted on 10.02.2012© Asian Pharma Press All Right Reserved
Asian J. Pharm. Res. 2(1)
: Jan.-Mar. 2012; Page 19-31
 
Broccoli is a plant of the cabbage family
Brassicaceae
(formerly
Cruciferae).
It is classified in the Italica cultivargroup of the species
 Brassica oleracea
. Broccoli has largeflower heads, usually green in color, arranged in a tree-likefashion on branches sprouting from a thick, edible stalk.The mass of flower heads is surrounded by leaves. Broccolimost closely resembles cauliflower, which is a differentcultivar group of the same species
3
.Research on
 Brassica
vegetables has been focused on theedible parts. However, scarce information is availableregardingthe corresponding by-products, which are in fact agood source of phenolic compounds of this unusual foodproduct (i.e. cauliflower leaf as by-product) with possibleuses as a dietary or food antioxidant
4
.The cancer-protective properties of 
 Brassica
(i.e. broccoli)consumption are most likely mediated through “bioactivecompounds” that induce a variety of physiological functionsincluding acting as direct or indirect antioxidants,regulating enzymes and controlling apoptosis and the cellcycle.
 
Asian J. Pharm. Res. 2012; Vol. 2: Issue 1, Pg 19-31
[AJPRes.]
20
The antimitotic activity was screened using
 Allium cepa
 root meristamatic cells which have been used extensively inscreening of drugs with antimitotic activity
5,6
. The roots of all plants have distinguished regions, one of them being theregion of cell division that lies beyond the root cap andextends a few millimeters after that. Cells of this regionundergo repeated divisions. The fate of cell division ishigher in this region compared to that of the other tissues.This region is called the meristamatic region (meristos:divided)
7
. This division is similar to the above mentionedcancer division in humans. Hence, these meristamatic cellscan be used for preliminary screening of drugs withanticancer activity. Even though doubts can be raised aboutextrapolation of results from plant tissue to animals andfinally to humans, Khilman has noted that plant cells are1000 times more resistant to colchicines which is a potentanti-carcinogen and which acts by inhibiting themicrotubule formation. Thus, it is possible that chemicalsthat affect plant chromosomes will also affect animals
8
.The American Cancer Society recommends eating morebroccoli and other cruciferous vegetables because theycontain anti-cancer phytochemicals. The Americangovernment has been endowing a number of researchprojects aimed at exploring
 Brassica oleracea
facts and thepotential of using
 Brassica oleracea
to reduce cancers andother degenerative diseases.
 Brassica oleracea
facts andnutrition information explains that this plant is one of themost important vegetable in the world because of its cancerfighting ability.
MATERIALS AND METHODS:
 Collection of plant material:
The
 Brassica oleracea
were purchased from RelianceFresh, Chennai. Botanical identification was made fromHerbarium of Department of Plant Biotechnology,Pachaiyappa's college, Chennai and voucher specimen wassubmitted in the herbarium. Fresh floret and stem werehomogenized with solvents and then extracted
. Allium cepa
 bulbs (red variety) were purchased from the local marketand stored for the entire study. Carmine stain was procuredfrom Sigma Aldrich, Bangalore. Other solvents used forextraction were of LR grade and were distilled before usefor greater purity.
Preparation of extracts:
The floret and stem (3g each) were homogenized withsolvents. The sample was kept in shaker for 30 minutes toget the aqueous extract. The extract obtained wasconcentrated and dried under controlled temperature (60
0
C).The dried powder was successively extracted with othersolvents ethanol, methanol, Chloroform, and petroleumether. Finally it was concentrated and made up to particularvolume. Extraction with each solvent was done in a waterbath for 60 minutes with a reflux condenser. Each timebefore extracting with the next solvent the marc was driedin an air oven below 50
o
C. Each extract was concentratedand evaporated to dry extract. Extracts of desiredconcentrations were prepared for further study using thesedried extracts.
Table.1
:
Qualitative Analysis of the phytochemicals of aqueous and organic extracts of 
 Brassica
 
 oleracea
 
S.NoPhytochemicaltestsMethanol Petroleum ether Aqueous Chloroform Ice cold water EthanolFloret Stem Floret Stem Floret Stem Floret Stem Floret Stem Floret Stem1 Flavanoids +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++2 Steriod(Salkowski)+++ ++ +++ ++ +++ ++ +++ ++ +++ ++ +++ ++3 Steriod(Libermann’s)++ + ++ + ++ - - +++ +++ +++ +++ +++ ++4 Alkaloids(Ehrlich’ s)- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -5 Alkaloids(Mayer’s)+++ ++ +++ ++ +++ ++ +++ ++ +++ ++ +++ ++6 Saponins(froth)+++ +++ - - - - - - +++ +++ - - - - - - +++ +++ - - - - - -7 Tannins - - - - - - - - - - - - - - - - - - - -8 Phenol(Ferricchloride)- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -9 Phenol(Lead acetate)+++ ++ ++ ++ +++ ++ +++ ++ +++ ++ +++ ++10 Protein(Biuret)+++ ++ + + - - - - - - - - - - - - - - - - - - - - - - - -11 Proteins(Million’s)+ + - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -12 ReducingCarbohydrate(Benedict’s)- - - - - - - - - - - - - - - - - - +++ +++ +++ +++ +++ +++13 Cardiacglycosides- - - - - - - - - - - - - - - - - - - - - - - - - -14 Glycosides -cardiac activeaglycones(Legal)+++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ ++++++ Maximum presence; ++ Moderate presence; + Minimum presence; - - - Nil
 
Asian J. Pharm. Res. 2012; Vol. 2: Issue 1, Pg 19-31
[AJPRes.]
21
Phytochemical analysis:
These studies were performed according to the standardmethods
9
. The aqueous and organic extracts were subjectedto preliminary phytochemical characterization, whichrevealed the presence of the phytochemicals-alkaloids,phenols, flavonoids, sterol, saponin glycosides, reducingsugars, proteins, cardio active aglycones and cardinolides,saponin glycosides.
Antimitotic activity
:
 A. cepa
was sprouted in tap water for 48 hours at roomtemperature. The bulbs that developed uniform root wereused for the experiment. These roots were treated withabove prepared extracts of 10 concentrations. Water wasused as medium/vehicle dilution. The different fractionsused have been mentioned in Table 1. A blank with waterwas used as control. Methotrexate was used as a standardcontrol. After 3hours of treatment, the root tips were fixedwith fixing solution of acetic acid and alcohol. Squashpreparations were made by staining the treated roots withacetocarmine stain and observed. The mitotic index wascalculated asMitotic Index = Number of dividing cells/Total number of cells x 100Folic acid added to the solution of methotrexate, aqueousextract and organic extract of 
 Brassica oleracea
. A similarexperiment was undertaken to find out the probablemechanism of action through which the extracts andmethotrexate act. Squash preparations made as above fromthe treated roots were observed.
 Separation of sterols by thin layer chromatography:
Slurry of silica gel GF
254
was made with distilled water.This slurry was then applied on glass plates (12.5 x 12.5cm)with the aid of a TLC spreader to obtain preparative silicagel plates having thickness of about 0.5mm. The plateswere dried in an oven at 105
o
C and activated 2hrs beforeuse.
Extraction of sterol from sample
:Three gms of floret and stem of 
 Brassica oleracea
werehomogenized and extracted with extraction solvent [eitherethyl ether: ethanol (3:1) or chloroform: methanol (2:1)].The contents were mixed vigorously and allowed to standtill the two phases were completely separated. The lowerorganic layer was drained out, which contained the sterol.The solvents were evaporated. Concentrated extracts werespotted on the plate. The plates were developed in thesolvent system consisting of petroleum ether or hexane:ethyl ether: glacial acetic acid (80: 20: 5) till the solventtravelled up to 1 cm from the opposite side of the plate. theplate were allowed to air dry 50% sulphuric acid wassprayed and heated in an oven at 110 º C for 10 min. Theplates were visualized and the spots were marked. Then theresults were compared with standard sterol.
Plate: 1
Plate: 1 Treatment of Allium cepa roots with different extract of Brassica oleracea and solutions
1. Standard – Methotrexate, 2. Methotrexate + Folic acid, 3. Water, 4. Ice cold water (floret), 5. Icecold water(stem), 6.Aqueous(floret), 7.Aqueous (Stem), 8. Methanol(floret), 9. Methanol(stem), 10. Chloroform (florate), 11. Aqueous(florate) + Folic acid, 12. Aqueous(florate) +Folic acid, 13. Methanol (florate) + , 14. Methanol(stem)+ Folic acid, 15. Chloroform (florate)+ Folic acid, 16. Ice cold water (floret) + Folic acid

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