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ISSN- 22315683 (Print) www.asianpharmaonline.org ISSN- 22315691 (Online) 0974-3618 RESEARCH ARTICLE

Secondary Metabolite screening, Bioactive compound extraction, and Disrupting Mitotic Activity of Wild Cabbage [Brassicaceae] towards Cancer Management.
A. Thenmozhi1, Dr. U.S Mahadeva Rao2*
1 2

Assistant Professor, PG Department of Biochemistry, SRM Arts and Science College, Chennai-603203, India. Associate Professor, Department of Biochem., Faculty of Medicine and Health Science, Universiti Sultan Zainal Abidin, Malaysia. *Corresponding Author E-mail: raousm@gmail.com

ABSTRACT:

Objective: The present study has been formulated with an objective to establish the preliminary phytochemical analysis and antimitotic activity of Brassica oleracea. Method: Brassica oleracea was collected, homogenized and extracted with different solvents. Antimitotic activity of Brassica oleracea was evaluated using the meristamatic cells from the root of Allium cepa. Experiments were carried out with incorporation of folic acid in the extract. Results: The preliminary study revealed the presence of flavonoids, steroids, alkaloids, saponins, polyphenols and glycosides. Folic acid inhibited the antimitotic activity of Brassica oleracea extract. The antimitotic activity obtained was compared with methotrexate-a referred anticancer drug. Discussion: 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. Conclusion: Hence Brassica oleracea is a promising source of phytochemicals which promote human health by strengthening the human immune system, inactivate cancer-causing substances, protect the heart and eyes from disease, 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.

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.

INTRODUCTION:

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.

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 The cancer-protective properties of Brassica (i.e. broccoli) consumption are most likely mediated through bioactive risk of certain cancers2. compounds that induce a variety of physiological functions including acting as direct or indirect antioxidants, Received on 24.12.2011 Accepted on 10.02.2012 regulating enzymes and controlling apoptosis and the cell Asian Pharma Press All Right Reserved Asian J. Pharm. Res. 2(1): Jan.-Mar. 2012; Page 19-31 cycle.

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The antimitotic activity was screened using Allium cepa root meristamatic cells which have been used extensively in screening of drugs with antimitotic activity5,6. 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.

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 50oC. Each extract was concentrated and evaporated to dry extract. Extracts of desired concentrations were prepared for further study using these dried extracts.

MATERIALS AND METHODS:

Table.1: Qualitative Analysis of the phytochemicals of aqueous and organic extracts of Brassica oleracea Phytochemical Methanol Petroleum ether Aqueous Chloroform Ice cold water S. No tests Floret Stem Floret Stem Floret Stem Floret Stem Floret Stem 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Flavanoids +++ +++ +++ +++ +++ +++ Steriod +++ ++ +++ ++ +++ ++ (Salkowski) Steriod ++ + ++ + ++ -(Libermanns) Alkaloids ------------(Ehrlich s) Alkaloids +++ ++ +++ ++ +++ ++ (Mayers) Saponins +++ +++ ----+++ +++ (froth) Tannins --------Phenol(Ferric ------------chloride) Phenol +++ ++ ++ ++ +++ ++ (Lead acetate) Protein +++ ++ + + ----(Biuret) Proteins + + --------(Millions) Reducing ------------Carbohydrate (Benedicts) Cardiac --------glycosides Glycosides +++ +++ +++ +++ +++ +++ cardiac active aglycones (Legal) Maximum presence; ++ Moderate presence; + Minimum presence; - - - Nil +++ +++ +++ --+++ ----+++ ----+++ -+++ +++ ++ +++ --++ ----++ ----+++ -+++ +++ +++ +++ --+++ +++ --+++ ----+++ -+++ +++ ++ +++ --++ +++ --++ ----+++ -+++

Ethanol Floret +++ +++ +++ --+++ ----+++ ----+++ -+++ Stem +++ ++ ++ --++ ----++ ----+++ -+++

+++

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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

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 105oC 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 C for 10 min. The Mitotic Index = Number of dividing cells/Total number of plates were visualized and the spots were marked. Then the cells x 100 results were compared with standard sterol. 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.

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

Plate: 1

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symptoms19, cardiovascular and cerebrovascular diseases 20,21 The results and discussion were discussed under two such as coronary heart disease and hypertension , 22 prophylaxis, dementia , ultraviolet damage including phases. cataract, and carcinoma cutaneum23, gastritis, gastric ulcer, Phase I : Phytochemical analysis and duodenal ulcer24. The use of saponins in pharmaceutical Phase II : Antimitotic activity preparations as adjuvants to enhance absorption of pharmacologically active substances or drugs has also been Phase I: 25,26 The aqueous and organic extracts of Brassica oleracea were patented . subjected to preliminary Phytochemical characterization, which revealed the presence of many phytochemicals- Flavonoids are polyphenolic compounds that are ubiquitous alkaloids, phenols, flavonoids, sterol, saponin glycosides, in nature. The flavonoids have aroused considerable interest reducing sugars, proteins, cardio active aglycones and recently because of their potential beneficial effects on cardinolides. The most important property of this human health, they have been reported to have antiviral, phytochemicals is antimitotic activity.Saponins are found to anti-allergic, antiplatelet, anti-inflammatory, antitumor and have numerous health benefits. They lyse blood cells. antioxidant activities, heart disease, asthma and stroke. Recent studies have illustrated saponins effects which have They may also play a special role in protecting the brain. been beneficial on the control of blood cholesterol levels, bone health, cancer, and building up of the immune system. Flavonoids are another large family of protective Saponin stromatolytic solution is being used for treating phytochemicals found in fruits and vegetables. Flavonoids, malaria.10 The most important advantage of using saponin is also called bioflavonoids, act as antioxidants. Antioxidants that it is completely renewable, biodegradable material neutralize or inactivate highly unstable and extremely which can be put on to the compost heap once it gets spent. reactive molecules, called free radicals that attack the cells Saponins are allergy free and are especially beneficial for of our body every day. Free radical damage is believed to babies and children who have sensitive skin. People contribute to a variety of health problems, including cancer, 27-29 suffering from allergies and dermatitis will be benefited by heart disease and aging . 11 using liquid soap solution prepared from saponin . Phenolic compounds may reduce the risk of heart disease and certain types of cancer. Indoles may reduce the risk of Anticancer activity has been reported for a number of Indoles are triterpene and steroid saponins12,13,14. Steroid saponin- certain types of cancer, including breast cancer. 30-32 containing plant materials gained commercial significance found in cruciferous vegetables, such as broccoli . in 1950s as raw materials for the production of steroid hormones and drugs. The synthesis of progesterone from Phase II: Antimitotic activity: the sapogenin diosgenin obtained from Mexican Yam by Antimitotic activities of the aqueous and organic extracts Marker et al. in 1940s15was the beginning of a remarkable were comparable to the activity of methotrexate. The era in steroid research culminating in the synthesis of the aqueous extract showed lowest mitotic index with highest activity among all the different fractionated extracts.. The first oral contraceptive in 1951. phases were differentiated in each case and it was observed Saponins have been used as immunological adjuvants in that the number of non-dividing cells increased in extract veterinary vaccine formulations due to their immune treated root tips than with folic acid added extracts. enhancing properties since 1950s16. The wealth of information on the biological activity of saponins and The cell divisions were differentiated and the numbers of aglycones from a variety of sources is providing leads for cells in each phase of cell division i.e. either prophase, the development of drugs. The chemo preventive and metphase, anaphase, or telophases were recorded. chemotherapeutic activities of ginseng dammarane sapogenins have prompted the development of anticancer Plate: 2 Plate 2 showed that the mitotic activity was quiet high when drugs for the various stages of development of cancer17. Pharmaceutical compositions or plant extracts containing treated with water alone. The numbers of dividing cells saponins have been patented for the prevention and were high as there is no antimitotic principle in water. treatment of a variety of conditions such as inflammation, Hence it was considered as control infection18, alcoholism pre- and post- menopausal

RESULTS AND DISCUSSION:

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Plate :2 Different stages of mitosis of A.cepa roots after treatment with water (Control)

Plate-3

Plate 3a

Plate 3b

Plate 3 Antimiotic activity of methotrexate, methotrexate + folic acid and the stages of cell division

Plate 4a

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Plate-4 : Antimitotic activity of ice cold floret, floret + folic acid extracts of Brassica oleracea and the stages of cell division

Plate 4b

Plate 5a

Plate 5b Plate 5: Antimiotic activity of ice cold water stem, ice cold water stem + folic acid extract of Brassica oleracea and the stages of cell division

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Plate 6a

Plate 6b Plate 6: Antimitotic activity of aqueous floret, aqueous floret + folic acie extracts of Brassica oleracea and the stages of cell division

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Plate 7a

Plate 7b Plate 7: Antimitotic activity of aqueous stem, aqueous stem+folic acid extracts of Brassica oleracea and the stages of cell division

Plate 8a

Plate 8b Plate 8: Antimitotic activity of methanol floret, methanol floret + folic acid extracts of Brassica oleracea and the stages of cell division

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Plate 9a

Plate 9b Plate 9: Antimitotic actifity of methanol stem, methanol stem + folic acid extracts of Brassica oleracea and the stages of cell division

Plate 3a showed that the mitotic activity was almost nil when the A.cepa root tips were treated with methotrexate. Maximum numbers of non-dividing cells were observed. Methotrexate-anticancer drug which competitively inhibits dihydrofolate reductase (DHFR), an enzyme that participates in the tetrahydrofolate synthesis. Methotrexate acts specifically during DNA and RNA synthesis, and thus it is cytotoxic during the S-phase of the cell cycle. Logically, it therefore has a greater toxic effect on rapidly dividing cells such as malignant and myeloid cells33,34.

Plate 3b showed that methotrexate + folic acid treated cells increased the mitotic activity to a certain extent. Folic acid is essential for the production of tertrahydrofolic acid (THF) which in turn required for synthesis of DNA and consequently for cell replication. Hence the dividing cells were high compared to that of the methotrexate treated cell35.

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10a

Plate 10: Antimitotic activity of chloroform floret, chloroform floret + folic acid extracts of Brassica oleracea and the stages of cell division.

10b

11a

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11b

Plate 11: Antimitotic activity of chloroform stem, chloroform stem + folic acid extracts of Brassica oleracea and the stages of cell division.

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. Different solutions used for treatment % of non- % of dividing cells Mitotic Mitotic Index No. dividing cells index 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

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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. Posthoc 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 its 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. REFERENCES:
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