INTRODUCTION:

The “functional food” industry has produced and marketed foods enriched with bioactive compounds, but there are no universally accepted criteria for judging efficacy of the compounds or enriched foods. The lack of understanding bioactive compounds and their health benefits should not serve to reduce research interest but should instead encourage plant and nutritional scientists to work together to develop strategies for improvement of health through food¹.

Cruciferous vegetables are an excellent dietary source of phytochemicals including glucosinolates (and glucosinolate breakdown products), phenolics and other antioxidants like vitamins(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 the risk of certain cancers².

Broccoli is a plant of the cabbage family Brassicaceae (formerly Cruciferae). It is classified in the Italica cultivar group of the species Brassica oleracea. Broccoli has large flower heads, usually green in color, arranged in a tree-like fashion on branches sprouting from a thick, edible stalk. The mass of flower heads is surrounded by leaves. Broccoli most closely resembles cauliflower, which is a different cultivar group of the same species³.

Research on Brassica vegetables has been focused on the edible parts. However, scarce information is available regardingthe corresponding by-products, which are in fact a good source of phenolic compounds of this unusual food product (i.e. cauliflower leaf as by-product) with possible uses as a dietary or food antioxidant⁴.

The cancer-protective properties of Brassica (i.e. broccoli) consumption are most likely mediated through “bioactive compounds” that induce a variety of physiological functions including acting as direct or indirect antioxidants, regulating enzymes and controlling apoptosis and the cell cycle.

The antimitotic activity was screened using Allium cepa root meristamatic cells which have been used extensively in screening of drugs with antimitotic activity^5,6. The roots of all plants have distinguished regions, one of them being the region of cell division that lies beyond the root cap and extends a few millimeters after that. Cells of this region undergo repeated divisions. The fate of cell division is higher 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 mentioned cancer division in humans. Hence, these meristamatic cells can be used for preliminary screening of drugs with anticancer activity. Even though doubts can be raised about extrapolation of results from plant tissue to animals and finally to humans, Khilman has noted that plant cells are 1000 times more resistant to colchicines which is a potent anti-carcinogen and which acts by inhibiting the microtubule formation. Thus, it is possible that chemicals that affect plant chromosomes will also affect animals⁸.

The American Cancer Society recommends eating more broccoli and other cruciferous vegetables because they contain anti-cancer phytochemicals. The American government has been endowing a number of research projects aimed at exploring Brassica oleracea facts and the potential of using Brassica oleracea to reduce cancers and other degenerative diseases. Brassica oleracea facts and nutrition information explains that this plant is one of the most important vegetable in the world because of its cancer fighting ability.

MATERIALS AND METHODS:

Collection of plant material:

The Brassica oleracea were purchased from Reliance Fresh, Chennai. Botanical identification was made from Herbarium of Department of Plant Biotechnology, Pachaiyappa's college, Chennai and voucher specimen was submitted in the herbarium. Fresh floret and stem were homogenized with solvents and then extracted. Allium cepa bulbs (red variety) were purchased from the local market and stored for the entire study. Carmine stain was procured from Sigma Aldrich, Bangalore. Other solvents used for extraction were of LR grade and were distilled before use for greater purity.

Preparation of extracts:

The floret and stem (3g each) were homogenized with solvents. The sample was kept in shaker for 30 minutes to get the aqueous extract. The extract obtained was concentrated and dried under controlled temperature (60⁰C). The dried powder was successively extracted with other solvents ethanol, methanol, Chloroform, and petroleum ether. Finally it was concentrated and made up to particular volume. Extraction with each solvent was done in a water bath for 60 minutes with a reflux condenser. Each time before extracting with the next solvent the marc was dried in an air oven below 50^oC. Each extract was concentrated and evaporated to dry extract. Extracts of desired concentrations were prepared for further study using these dried extracts.

Table.1: Qualitative Analysis of the phytochemicals of aqueous and organic extracts of Brassica oleracea

S. No	Phytochemical tests	Methanol		Petroleum ether		Aqueous		Chloroform		Ice cold water		Ethanol
S. No	Phytochemical tests	Floret	Stem	Floret	Stem	Floret	Stem	Floret	Stem	Floret	Stem	Floret	Stem
1	Flavanoids	+++	+++	+++	+++	+++	+++	+++	+++	+++	+++	+++	+++
2	Steriod (Salkowski)	+++	++	+++	++	+++	++	+++	++	+++	++	+++	++
3	Steriod (Libermann’s)	++	+	++	+	++	- -	+++	+++	+++	+++	+++	++
4	Alkaloids (Ehrlich’ s)	- - -	- - -	- - -	- - -	- - -	- - -	- - -	- - -	- - -	- - -	- - -	- - -
5	Alkaloids (Mayer’s)	+++	++	+++	++	+++	++	+++	++	+++	++	+++	++
6	Saponins (froth)	+++	+++	- - -	- - -	+++	+++	- - -	- - -	+++	+++	- - -	- - -
7	Tannins	- - -	- - -	- - -	- - -	-	-	-	-	-	-	-	-
8	Phenol(Ferric chloride)	- - -	- - -	- - -	- - -	- - -	- - -	- - -	- - -	- - -	- - -	- - -	- - -
9	Phenol (Lead acetate)	+++	++	++	++	+++	++	+++	++	+++	++	+++	++
10	Protein (Biuret)	+++	++	+	+	- - -	- - -	- - -	- - -	- - -	- - -	- - -	- - -
11	Proteins (Million’s)	+	+	- - -	- - -	- - -	- - -	- - -	- - -	- - -	- - -	- - -	- - -
12	Reducing Carbohydrate (Benedict’s)	- - -	- - -	- - -	- - -	- - -	- - -	+++	+++	+++	+++	+++	+++
13	Cardiac glycosides	- - -	- - -	- -	- -	- -	- -	- -	- -	- -	- -	- -	- -
14	Glycosides -cardiac active aglycones (Legal)	+++	+++	+++	+++	+++	+++	+++	+++	+++	+++	+++	+++

+++ Maximum presence; ++ Moderate presence; + Minimum presence; - - - Nil

Phytochemical analysis:

These studies were performed according to the standard methods⁹. The aqueous and organic extracts were subjected to preliminary phytochemical characterization, which revealed the presence of the phytochemicals-alkaloids, phenols, flavonoids, sterol, saponin glycosides, reducing sugars, proteins, cardio active aglycones and cardinolides, saponin glycosides.

Antimitotic activity:

A. cepa was sprouted in tap water for 48 hours at room temperature. The bulbs that developed uniform root were used for the experiment. These roots were treated with above prepared extracts of 10 concentrations. Water was used as medium/vehicle dilution. The different fractions used have been mentioned in Table 1. A blank with water was used as control. Methotrexate was used as a standard control. After 3hours of treatment, the root tips were fixed with fixing solution of acetic acid and alcohol. Squash preparations were made by staining the treated roots with acetocarmine stain and observed. The mitotic index was calculated as

Mitotic Index = Number of dividing cells/Total number of cells x 100

Folic acid added to the solution of methotrexate, aqueous extract and organic extract of Brassica oleracea. A similar experiment was undertaken to find out the probable mechanism of action through which the extracts and methotrexate act. Squash preparations made as above from the treated roots were observed.

Separation of sterols by thin layer chromatography:

Slurry of silica gel GF₂₅₄ 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 silica gel plates having thickness of about 0.5mm. The plates were dried in an oven at 105^oC and activated 2hrs before use.

Extraction of sterol from sample:

Three gms of floret and stem of Brassica oleracea were homogenized and extracted with extraction solvent [either ethyl ether: ethanol (3:1) or chloroform: methanol (2:1)]. The contents were mixed vigorously and allowed to stand till the two phases were completely separated. The lower organic layer was drained out, which contained the sterol. The solvents were evaporated. Concentrated extracts were spotted on the plate. The plates were developed in the solvent system consisting of petroleum ether or hexane: ethyl ether: glacial acetic acid (80: 20: 5) till the solvent travelled up to 1 cm from the opposite side of the plate. the plate were allowed to air dry 50% sulphuric acid was sprayed and heated in an oven at 110 º C for 10 min. The plates were visualized and the spots were marked. Then the results were compared with standard sterol.

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

RESULTS AND DISCUSSION:

The results and discussion were discussed under two phases.

Phase I : Phytochemical analysis

Phase II : Antimitotic activity

Phase I:

The aqueous and organic extracts of Brassica oleracea were subjected to preliminary Phytochemical characterization, which revealed the presence of many phytochemicals-alkaloids, phenols, flavonoids, sterol, saponin glycosides, reducing sugars, proteins, cardio active aglycones and cardinolides. The most important property of this phytochemicals is antimitotic activity.Saponins are found to have numerous health benefits. They lyse blood cells. Recent studies have illustrated saponins effects which have been beneficial on the control of blood cholesterol levels, bone health, cancer, and building up of the immune system. Saponin stromatolytic solution is being used for treating malaria.¹⁰The most important advantage of using saponin is that it is completely renewable, biodegradable material which can be put on to the compost heap once it gets spent. Saponins are allergy free and are especially beneficial for babies and children who have sensitive skin. People suffering from allergies and dermatitis will be benefited by using liquid soap solution prepared from saponin¹¹.

Anticancer activity has been reported for a number of triterpene and steroid saponins^12,13,14. Steroid saponin-containing plant materials gained commercial significance in 1950’s as raw materials for the production of steroid hormones and drugs. The synthesis of progesterone from the sapogenin diosgenin obtained from Mexican Yam by Marker et al. in 1940’s¹⁵was the beginning of a remarkable era in steroid research culminating in the synthesis of the first oral contraceptive in 1951.

Saponins have been used as immunological adjuvants in veterinary vaccine formulations due to their immune enhancing properties since 1950’s¹⁶. The wealth of information on the biological activity of saponins and aglycones from a variety of sources is providing leads for the development of drugs. The chemo preventive and chemotherapeutic activities of ginseng dammarane sapogenins have prompted the development of anticancer drugs for the various stages of development of cancer¹⁷.

Pharmaceutical compositions or plant extracts containing saponins have been patented for the prevention and treatment of a variety of conditions such as inflammation,infection¹⁸, alcoholism pre- and post- menopausal symptoms¹⁹, cardiovascular and cerebrovascular diseases such as coronary heart disease and hypertension^20,21, prophylaxis, dementia²², ultraviolet damage including cataract, and carcinoma cutaneum²³, gastritis, gastric ulcer, and duodenal ulcer²⁴. The use of saponins in pharmaceutical preparations as adjuvants to enhance absorption of pharmacologically active substances or drugs has also been patented^25,26.

Flavonoids are polyphenolic compounds that are ubiquitous in nature. The flavonoids have aroused considerable interest recently because of their potential beneficial effects on human health, they have been reported to have antiviral, anti-allergic, antiplatelet, anti-inflammatory, antitumor and antioxidant activities, heart disease, asthma and stroke. They may also play a special role in protecting the brain.

Flavonoids are another large family of protective phytochemicals found in fruits and vegetables. Flavonoids, also called bioflavonoids, act as antioxidants. Antioxidants neutralize or inactivate highly unstable and extremely reactive molecules, called free radicals that attack the cells of our body every day. Free radical damage is believed to contribute to a variety of health problems, including cancer, heart disease and aging^27-29.

Phenolic compounds may reduce the risk of heart disease and certain types of cancer. Indoles may reduce the risk of certain types of cancer, including breast cancer. Indoles are found in cruciferous vegetables, such as broccoli^30-32.

Phase II: Antimitotic activity:

Antimitotic activities of the aqueous and organic extracts were comparable to the activity of methotrexate. The aqueous extract showed lowest mitotic index with highest activity among all the different fractionated extracts.. The phases were differentiated in each case and it was observed that the number of non-dividing cells increased in extract treated root tips than with folic acid added extracts.

The cell divisions were differentiated and the numbers of cells in each phase of cell division i.e. either prophase, metphase, anaphase, or telophases were recorded.

Plate: 2

Plate 2 showed that the mitotic activity was quiet high when treated with water alone. The numbers of dividing cells were high as there is no antimitotic principle in water. Hence it was considered as control

Plate-4

Plate 4 showed that the number of non-dividing cells were in the following order.

Ice cold water floret extract > Ice cold water floret extract +folic acid > water

Plate 5

Plate 5 showed that the number of non-dividing cells were in the following order.

Ice cold water stem extract > Ice cold water stem extract +folic acid > water

Plate 6

Plate 6 showed that the number of non-dividing cells were in the following order.

Aqueous floret extract > Aqueous floret extract + folic acid > Water

Plate 7 Plate 7 showed that the number of non-dividing cells were in the following order.

Aqueous stem extract > Aqueous stem extract + folic acid > Water

Plate 8

Plate 8 showed that the number of non-dividing cells were in the following order.

Methanol floret extract > Methanol floret extract + folic acid > Water

Plate 9

Plate 9 showed that the number of non-dividing cells were in the following order.

Methanol stem extract > Methanol stem extract + folic acid > Water

Plate 10

Plate 10 showed that the number of non-dividing cells were in the following order.

Chloroform floret extract > Chloroform floret extract + folic acid > Water

Plate 11

Plate 11 showed that the number of non-dividing cells were in the following order.

Chloroform stem extract > Chloroform stem extract + folic acid > Water

Table 2: Antimitotic activity after treatment of Allium cepa root with aqueous, organic extract of Brassica oleracea, methotrexate and aqueous extract+folic acid, organic extract+folic acid, methotrexate+folic acid

S. No.	Different solutions used for treatment	% of non-dividing cells	% of dividing cells				Mitotic index	Mitotic Index
S. No.	Different solutions used for treatment	% of non-dividing cells	P	M	A	T		Mean	SD	SEM
1	Methotrexate	77	22	-	-	-	22
2	Methotrexate+Folic acid	30	68	-	-	1	69	68.5	0.92	0.53
3	Water	14	20	22	19	24	85	85.7	0.17	0.09
4	Ice cold water (Floret)	47	15	10	13	13	52	52.4	0.75	0.43
5	Ice cold water(Stem)	41	25	8	11	13	58	58.3	0.57	0.32
6	Aqueous(Floret)	39	19	13	11	15	59	60.5	0.86	0.49
7	Aqueous(Stem)	28	22	24	7	16	71	71.1	0.28	0.16
8	Methanol(Floret)	41	16	12	12	16	57	58	0.63	0.36
9	Methanol(Stem)	33	25	14	8	17	65	66.1	0.40	0.23
10	Chloroform(Floret)	33	25	13	12	16	66	66.7	0.23	0.13
11	Aqueous(Floret)+Folic acid	18	31	21	18	10	81	81.5	0.05	0.02
12	Aqueous(Stem)+Folic acid	15	28	26	10	20	84	84.6	0.34	0.19
13	Methanol(Floret)+Folic acid	15	40	15	12	15	84	84.4	0.11	0.06
14	Methanol(Stem)+Folic acid	14	20	25	14	25	85	85.6	0.17	0.09
15	Chloroform(Floret)+Folic acid	11	32	17	18	19	88	88.4	0.23	0.13
16	Cold water(Floret)+Folic acid	17	28	16	17	19	82	82.1	0.17	0.09

Effect of folic acid on antimitotic activity of Brassica oleracea and methotrexate:

Analysis of data using a 3-way ANOVA showed that there was a significant effect of the pretreatment with folic acid on the antimitotic activity of Brassica oleracea and methotrexate. The mitotic index increased when folic acid was added to the extracts of Brassica oleracea and methotrexate solution which otherwise reduce the mitotic activity in the absence of folic acid. This however, did not increase with increase in folic acid concentration suggesting that the effect was not dose-dependent. Comparing the mitotic index of methotrexate and Brassica oleracea extracts, it was observed that incorporation of folic acid increased the mitotic index significantly in case of methotrexate, but not so in case of Brassica oleracea. Post-hoc analysis of the data showed that folic acid inhibited the anti-mitotic activity of methotrexate to a greater extent as compared to Brassica oleracea. Results from our study indicate that ice cold water extract of Brassica oleracea had excellent antimitotic activity that was comparable to the activity of methotrexate. Addition of folic acid inhibited the antimitotic activity of Brassica oleracea significantly but not completely. Thus, it may be hypothesized that the extract may be acting through the pathway inhibiting tetrahydrofolic acid and hence folic acid required for DNA synthesis that arrests cell division. Methotrexate is a known anticancer drug which competes with folic acid for the enzyme reductase. The total aqueous extracts of Brassica oleracea may also be competing with folic acid thus inhibit the DNA synthesis. Hence, addition of folic acid increases the mitotic index due to the availability of folic acid. However, the mitotic index does not increase significantly in case of Brassica oleracea as compared to that of methotrexate because the extract may be mediating its effects through other mechanisms also. The phytochemicals present in the extract may bind with different cell proteins which are responsible for cell division.

Cancer usually evolves over a long period of time, agents that inhibit or retard one or more of its stages could affect the overall course of the disease. Certain micronutrients like polyphenolic compound posses potent cancer-preventive abilities. The blocking and suppressing agents found in specific herbs and food are having anticancer activities. These compounds inhibit cancer formation by blocking or diverting carcinogenic material away from the cell, allowing it to be metabolized by the liver to a less toxic and more excretable substance. This may prevent cancerous substance reacting with the cells DNA before it can do any damage and hence it‘s excretion through metabolism. This may retard cancer promotion by decreasing or turning off promotional factors that would otherwise be used for cancer promotion and proliferation. One of the most important effects of blocking agents found in herbs and foods in the inhibition of tumor formation by curbing the arachidonic acid cascade. This effect is particularly evident in high quality fats. Herbs that contain antitumour alkaloids can directly inhibit cancer growth because of their inhibitory activity against reverse transcriptase of RNA tumour viruses. This mechanism of action occurs within the cell cycle process. A more direct antitumour mechanism of plant compound has to do with inhibiting tubulin polymerization. This is the way that most chemotherapies work. These antitumour compounds are called antimitotic agents³⁶.

CONCLUSION:

From this study, it could be suggested that Brassica oleracea have been shown to posses cancer chemo preventive effects within their diverse pharmacological properties. Phytochemical characterization revealed the presence of many phytochemicals-alkaloids, phenols, flavonoids, sterol, saponin glycosides, reducing sugars, proteins, cardio active aglycones and cardinolides. The results obtained from the present study pinpoint that antimitotic activity of Brassica oleracea may be due to the presence of flavonoids, steroids, alkaloids, polyphenols and saponins. Mechanism of action of herbs that contains these phytochemicals occurs with in the cell cycle process. Hence these anti tumour compounds are called antimitotic agents. Brassica oleracea showed commendable antimitotic activity which can be exploided as cancer therapy.

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Received on 24.12.2011 Accepted on 10.02.2012

Asian J. Pharm. Res. 2(1): Jan.-Mar. 2012; Page 19-31

¹Assistant Professor, PG Department of Biochemistry, SRM Arts and Science College, Chennai-603203, India.

²Associate Professor, Department of Biochem., Faculty of Medicine and Health Science, Universiti Sultan Zainal Abidin, Malaysia.

*Corresponding Author E-mail: raousm@gmail.com

1Assistant Professor, PG Department of Biochemistry, SRM Arts and Science College, Chennai-603203, India.

2Associate Professor, Department of Biochem., Faculty of Medicine and Health Science, Universiti Sultan Zainal Abidin, Malaysia.

*Corresponding Author E-mail: raousm@gmail.com

¹Assistant Professor, PG Department of Biochemistry, SRM Arts and Science College, Chennai-603203, India.

²Associate Professor, Department of Biochem., Faculty of Medicine and Health Science, Universiti Sultan Zainal Abidin, Malaysia.