Antiyeast, Antioxidant and Anticancer Activity of Tribulus terrestris Linn and Bougainvillea spectabilis Linn

 

Mukesh Kumar D. J.¹, Sonia K.²*, Madhan R.³, Selvakumar K. ³ and Kalaichelvan P.T.¹   

¹ Centre for Advance Studies in Botany, University of Madras, Chennai, India

²Department of Biochemistry, Periyar University, Salem, India

³Department of Applied Biology and Nanobiosciences, BioLim Centre for Life Science, Chennai, India

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

ABSTRACT:

Ethanolic extracts of leaves, fruits and stem of Tribulus terrestris and leaves of Bougainvillea spectabilis were prepared and assessed for anti-yeast activity on Candida albicans and Malassezia furfur at 50 µl and 100 µl concentrations, compared with Fluconazole anti-yeast agent. The antioxidant activity was examined by various methods such as DPPH, ferrous ion reducing antioxidant power, ferrous ion chelating ability assay and cerium (IV) ion reducing power assay and compared with ascorbic acid, α-tocopherol, rutin and quercitin. The extracts were also evaluated for anticancer activity such as decatenation assay and catalytic activity assay and compared with KDNA. It was observed that the fruit of T. terrestris and leaves of B. spectabilis has flavanoid and saponin compounds by Thin layer chromatography. From the results, it is evident that ethanolic extract of fruit of T. terrestris and leaves of B. spectabilis has maximum anti-yeast activity against candida albicans at 100 µl concentration and has a potent antioxidant and anticancer activity.

 

 

INTRODUCTION:

Medicinal plants, which are raw materials for many of the herbal formulations and popular nutrient supplements, are sold all over the country. In the recent past, there has been a steady growth in the number of patients visiting practitioners of complementary system of medicine for treatment. This growing popularity is partly due to the popular concept that “being natural in origin, herbs are safe”.¹

 

Despite advances in antifungal therapies, many problems remain to be soft for most antifungal drugs available. Fluconazole was mostly used azole drugs, and its use has resulted in clinically resistant strains of Candida albicans. Especially, 3.6–7.2% of vaginal isolates of Candida albicans from women with Candida vaginitis is resistant to Fluconazole.² This situation highlights the need for advent of safe, novel and effective antifungal compounds.

 

When generation of ROS overtakes the antioxidant defense of the cells, the free radicals start attacking the cell proteins, lipids and carbohydrates^3–5 and this leads to a number of physiological disorders.

 

Many plants often contain substantial amounts of antioxidants including vitamin C and E, carotenoids, flavanoids and tannins, etc. and thus can be utilized to scavenge the excess free radicals from human body.⁶

 

The sensitivity to α Topo II drug should be dependent on the cellular content of the topoisomerase enzyme i.e., the response of human cancers to Topo II therapy may be dependent on, and perhaps predicted by, tumor content of topoisomerase. In normal cells, the activity of Topo II α is highly regulated, while it is present at high levels in rapidly proliferating cancerous cells.⁷ Both the isoforms are shown to be differently regulated in cancerous tissue.⁸ Thus, both Topo II α and β assume significance as anticancer targets for development of cancer specific chemotherapeutics.

 

Tribulus terrestris Linn (Zygophyllaceae) is an annual plant distributed in warm regions of Asia, Africa, Europe, America and Australia.^9–11 Tribulus terrestris is used in folk medicine as tonic, aphrodisiac, analgesic, astringent, stomachic, anti-hypertensive, diuretic, lithotriptic and urinary anti-infective.¹²

 

Bougainvillea spectabilis has 14 species with three that are horticulturally important Bougainvillea spectabilis, B.glabra, B.peruvina. Bougainvillea spectabilis is a large climber with distinctive cured thorns and hair on stems and leaves.¹³ Bougainvillea spectabilis were highly effective in reducing okra yellow vein mosaic virus infection of okra.¹⁴

Bougainvillea spectabilis belongs to the family Nyctaginaceae. The family has 30 genera and 300 species. Flower remains throughout the year particularly from April to August.  Diabetes Mellitus (DM) is the commonest endocrine disorder that affects over 100 million people worldwide. The conventional treatment for DM is oral hypoglycemic agent/insulin therapy. However, a lot of herbs are now being used in the management of DM, although the active principles some of them have been isolated. One of such herbs is Bougainvillea spectabilis.¹⁵

 

In our present study we have evaluated the antiyeast, antioxidant and anticancer activity of Tribulus terrestris L. and Bougainvillea spectabilis L

 

MATERIALS AND METHODS:

Chemicals:

DPPH (1,1-Diphenyl-2-picryl-hydrazil), α- tocopherol, Potato Dextrose Agar (PDA), Proteinase K, bovine serum albumin (BSA), kDNA, were purchased from Sigma-Aldrich and Topo II crude enzyme is a kind gift from Dr.J.Angayarkanni, Assistant professor, Department of Microbial Biotechnology, School of Biotechnology and Genetic Engineering and Bharathiyar University Coimbatore.  All other chemicals and reagents were of analytical grade.

 

Strains:

Candida albicans (MTCC 1243) and Malassezia furfur (MTCC 1374), dandruff causing yeast were purchased from Institute of Microbial Technology, Chandigarh, India and grown on potato dextrose agar (PDA) plates at 28 ºC for about 7 days. Thereafter, the plates were maintained at 4 ºC until used. The yeast was sub-cultured every 3 months. Media was sterilized by autoclaving for 15 min at 121 ºC.

 

Plant Material and Extraction:

Tribulus terrestris and Bougainvillea spectabilis were collected from Periyar University campus, Salem district, Tamil Nadu, India. Leaves stem and fruits of Tribulus terrestris and leaves of Bougainvillea spectabilis were used in this study. Plant materials were washed with distilled water and dried under shade at room temperature. They were powdered and stored in sterile containers for further use.  50 g of each dried powdered plant material was evenly packed and extracted with   95 % ethanol for 24h in the soxhlet apparatus. About 10 ml of ethanol per gram of powder was used. The ethanol extract was dried under a reduced pressure at 40 ºC. The dried extracts were packed in airtight containers and used for the following experimental analysis such as antiyeast screening, antioxidant assay, DNA binding activity and anticancer activity.

 

Screening of Antiyeast Activity:

Antiyeast activity of leaves, fruits and stem of T. terrestris L. and leaves of B. spectabilis were screened against C. albicans and M. furfur using agar diffusion method. 3.9 g of potato dextrose agar was weighed and dissolved in 100 ml of distilled water. The medium was sterilized by autoclaving at 121 ºC for 15 min and allowed to cool at room temperature and poured 20 ml into petridishes in sterile condition. C. albicans and M. furfur were aseptically inoculated uniformly on petridishes.  100 and 50µl concentration of the extracts were added into the well in yeast culture inoculated petridishes. Then the plate was incubated at 30 ˚C for overnight. Inhibition of yeast growth by the extracts was determined by measuring the diameter of zone of inhibition. For negative control, growth was determined for each strain where pure solvent was used instead of plant extracts. The standard antiyeast agent used in this study was Fluconazole, has highest antiyeast activity against C. albicans.

 

Antioxidant Activity:

Ferric Ion Reducing/ Antioxidant Power Assay (FRAP):

The antioxidants present in the sample reduced the oxidant probe and the respective product interacted with some colouring agents to form a coloured complex. In this method, the antioxidants reduced the Fe³⁺ to Fe²⁺. This ion then conjugated with the ferricyanide ion to form a Prussian blue coloured product, which was spectrophotometrically measured at 700nm.¹⁶ About 2.5ml of the extract (100-1000mg/ml), added 2.5ml phosphate buffer (0.2 M, pH 6.6) and 2.5ml of 1% potassium ferricyanide. The mixture was boiled in a water bath at 50°C for 20 minutes, then rapidly cooled, mixed with 2.5ml of 10% trichloroacetic acid and centrifuged at 3000×g for 10 minutes. From this pipette out 2.5 ml of supernatant, 2.5ml of distilled water and 0.5 ml of 0.1% ferric chloride. Mixed well and allowed to stand for 10 minutes. The increase in the absorbance at 700 nm was used to measure the reducing power of the plant extract. Ascorbic acid and α- tocopherol acetate was used as a positive control.

 

Ferrous Ion Chelating Ability Assay:

Ferrozine [FZ, disodium salt of 3- (2-pyridyl) -5, 6-bis (4-phenylsulfonic acid) -1, 2, 4- triazine] is a complexing agent which is highly specific to Fe²⁺ ions. The resulting complex Fe (II)-(FZ) ₃, is a magenta coloured product which was spectrophotometrically measured at 562nm. In the presence of various antioxidants this complex formation is impeded and thereby there is a decreased colour will be noticed.¹⁷ About 2ml of the extract (100-1000mg/ml) was mixed with 0.1ml of 2mM FeCl₂ and 0.2ml of 5mM ferrozine solutions and allowed to react for 10 minutes at room temperature. The absorbance at 562 nm of the resulting solutions were measured and recorded. The ferrous ion chelating ability was expressed in percentage. The FeCl₂ and ferrozine acted as control. Ascorbic acid and α- tocopherol acetate was used as a positive control.

 

DPPH Radical Scavenging Assay:

The DPPH reacted with methanol or absolute ethanol to yield a purple colour DPPH radical. The presence of antioxidants which included polyphenolics and flavonoids in the sample will scavenge the formed DPPH radical and there by a decreased colour will be observed which is spectrophotometrically measured at 517nm.¹⁸ To 0.5ml of DPPH radical solution, added 2ml of the extract (100-1000mg/ml) and the reaction mixture was vortexed for 10sec and allowed to stand at room temperature for 30 minutes. The absorbance was recorded at 517nm by using (Beckman DU-530) UV–Vis Spectrophotometer and compared with the 75% ethanol which acted as control solution. The percentage of DPPH radical scavenging activity was expressed in percentage. Ascorbic acid was used as reference antioxidant compound.

 

Cerium (IV) Sulphate Ion Reducing Power Assay:

Determination of Ce (IV) reducing capacity also known as ceric ion reducing antioxidant capacity (CERAC)¹⁹ was followed in this assay. About 1 ml of the extract (100-1000mg/ml), added 1.0ml of 2mM Ce (IV) sulphate solution. After shaking for a few minutes, the solution was allowed to stand for 30 minutes at room temperature. The absorbance of the reaction mixture was measured at 320 nm against a blank composed of distilled water. The decrease in the absorbance at 320 nm was used to measure the unreacted Ce (IV) ion in the sample that indicates an increase in antioxidant power of the sample.

 

Anticancer Activity:

Topo II Inhibition Assay with the Ethanolic Fraction:

The ethanol fraction that shows positive when subjected to the relaxation assay²⁰ to check for the inhibition of the Topo II enzyme. Various concentrations of the samples were used. The reaction products were electrophoresed on a 1% agarose gel and densitometry analysis was carried out using Alpha Digidoc pro 2010 software to identify the appropriate inhibitory concentration of the test sample in comparison with the standard, Naldixic acid.

 

Decatenation Assay:

The unique property of Topoisomerase II enzyme to decatenate was checked using Kinetoplast DNA as the substrate. The total reaction volume was fixed as 20µl. Briefly to an assay buffer (50Mm Tris HCL, pH 8, 120Mm KCL, 10Mm MgCl_2, 0.5mM DTT and 30µg/ml BSA) containing 100 or 200ng of k DNA, Topo II crude enzyme extract with a protein concentration of 5-10µg was added. After an incubation of 10min at 37ºC, the reaction was stopped by the addition of 3µl of arresting solution containing the loading dye. The reaction mixture was analysed on a 1% agarose gel at 40V for 7hrs in TBE buffer. The DNA was stained with Ethidium-bromide and visualized under UV-transilluminator.²¹

 

Catalytic Activity Assay: 

To check, whether the inhibitor of Tribulus terrestris (Leaves, stem and fruits) and Bougainvillea spectabilis (Leaves) affects the catalytic activity of the enzyme, the decatenation assay²¹ was used. The inhibitors were added before the addition of kDNA (kinetoplast DNA) and incubated at 37 ̊C for 30 min. The reaction mixtures were electrophoresed in 1% agarose gel at 40V for 7 hrs in TBE buffer and observed in an UV transilluminator. 

Analysis of Chemical Components of Extract by Thin Layer Chromatography:

The general principle involved in thin layer chromatography (TLC) is adsorption chromatography. In this the solute competes with the solvent for the surface sites of the adsorbant, depending on the distribution coefficients. The compounds are distributed on the surface of the adsorbant. The adsorbant normally used contains a binding agent such as calcium sulphate which facilitates the binding of the adsorbant to the glass plate.

 

The procedure was followed as described by.²² Dry, clean glass plates were placed over a plain surface into which the silica gel was applied. Silica gel of the adsorbant was prepared in water in the ratio of 1:2 (w/v). The silica gel was thoroughly stirred for 1-2 min and poured in the applicator positioned on the neat glass plate. The slurry was coated on the glass plates at a thickness of 0.25mm for quantitative analysis by moving the applicator at a uniform speed from one end to other. The coated plates were then allowed to dry at room temperature for 15-30min. The plates were heated in an oven at 100-120 ˚C for 1-2 h to remove the moisture and to activate the adsorbant on the plate. The solvent such as n-butanol: water: acetic acid in the ratio 4:1:1 (v/v/v) was poured into the tank up to a depth of 1.5cm. It was allowed to stand for at least an hour with a cover plate at the top of the tank to ensure that the atmosphere with in the tank become saturated with the solvent vapour. The samples were applied at 2.5cm from one end of the glass plate and at equal distance from the edges by means of micropipette or capillary tubes as small spots. The standard used was rutin, quercitin and anthocyanin. The solutions were allowed to dry so that spotting could be done repeated for a more concentrated sample spot. A thin layer plate was placed vertically in the tank so that it stood in the solvent with the spotted end dipping in the solvent. Separation of the compounds occurred as the solvent moved upward. Once the solvent reached the top of the plate, the plate was removed from the tank and allowed to dry. 0.3% of ninhydrin solution was sprayed for the identification of separated compounds. The plate was then placed in hot air oven for about 10min.

 

RESULTS AND DISCUSSION:

Antiyeast activity:

The fruits of Tribulus terrestris and leaves of Bougainvillea spectabilis has potent antiyeast activity against Candida albicans at 100µl concentration showing zone of inhibition (23mm) when compared to the leaves and stem Tribulus terrestris which has minimum antiyeast activity against Candida albicans at 100µl showing zone of inhibition (17mm and 19mm). Leaves, fruits and stem of Tribulus terrestris and leaves of Bougainvillea spectabilis has lowest antiyeast activity at 50µl showing zone of inhibition (10, 11, 13 and 16mm) respectively. The standard Fluconazole antiyeast agent achieved the antiyeast activity against Candida albicans having zone of inhibition (25mm).

 

 

 

Table 1-Antiyeast activity of T. terrestris Linn and B. spectabilis Linn against candida albicans and Malassezia furfur.

Yeast	Plant Material	Concentration ( ml)	Zone of Inhibition (mm)	Control used
Candida       albicans	Tribulus terrestris Leaves Stem Fruits   Bougainvillea spectabilis Leaves	50 50 50       50	10 11 13       16	Fluconazole ( 25 mm)
Candida   albicans	Tribulus terrestris Leaves Stem Fruits   Bougainvillea spectabilis Leaves	100 100 100       100	17 19 23       23
Malassezia furfur	Tribulus terrestris Leaves Stem Fruit   Bougainvillea spectabilis Leaves	50 and100 50 and100 50 and100       50 and100	- Nil -

 

Antioxidant Activity

Graph 1:DPPH radical scavenging activity

 

DPPH radical scavenging activity of Leaves, stem and fruits of Tribulus terrestris and leaves of Bougainvillea spectabilis. The. sample A, B, C and D represents ethanolic extract of    A – Leaves of Tribulus terristris, B – leaves of Bougainvillea spectabilis, C – Fruits of Tribulus terrestris and  D – Stem of Tribulus terrestris.

 

Graph 2:Ferrous Ion Reducing Antioxidant Power

 

Reducing capacity of a compound indicates the potential antioxidant activity. The Ferrous ion reducing / antioxidant activity was observed in the ethanolic extract of Tribulus terrestris (leaves,   stem and fruits) and Bougainvillea spectabilis (leaves) was compared with rutin, quercetin and ascorbic acid. The final result was expressed as the concentration of antioxidant having a ferric reducing ability.

 

Graph 3: Ferrous Ion Chelating Ability Assay

 

In the present study ethanolic extract of T. terrestris (leaves, fruits and stem) and Bougainvillea spectabilis (leaves) compared with rutin, quercitin and ascorbic acid (vitamin C). It was observed that the ethanolic extract of T. terrestris L. (fruits) and Bougainvillea spectabilis (leaves) has potent chelating capacity.

 

Graph 4: Cerium (IV) Ion Reducing Power Assay

 

In the present study, ethanolic extract of Tribulus terrestris (leaves, fruits and stem) and Bougainvillea spectabilis (leaves) obtained in antioxidant activity. The ethanolic extracts of T. terrestris (fruits) and Bougainvillea spectabilis (leaves) have potent cerium (IV) ion reducing power.

 

Anticancer Activity :

In the assay, the kDNA (100 and 200ng) incubated along with the enzyme showed the formation of minicircles as a result of the Topo II enzyme activity. Whereas, in the control lane containing only the kDNA (lane l), there were no minicircles present and the network DNA did not migrate from the well as suggested by.²³

 

Since, Topo II is unique in its ability to catalyze the decatenation of intact double – stranded DNA, it was identified that Topo II specific enzyme activity was present in the crude enzyme extract and this extract was used for the screening of Topo II inhibitors.

 

Decatenation Assay:

                

In the presence of the inhibiting compounds in Tribulus terrestris (fruits) and Bougainvillea spectabilis (Leaves), the decatenation was not observed in lane 3 and lane 4 similar to that of the lane 1 containing only the kDNA. But, decatenation was observed by the formation of monomers in lane 2 and lane 5 containing the kDNA, Topo II enzyme and inhibitors such as Tribulus terrestris and Bougainvillea spectabills. This proved that the bioactive ethanol obtained inhibited the catalytic activity of the enzyme. In present study, the fruits of Tribulus terrestris and leaves of Bougainvillea spectabilis have an anticancer activity.

  

Catalytic Activity Assay:

 Analysis of Chemical Components of Extract by Thin Layer Chromatography:

Thin layer chromatography was performed with Silica G60 on glass plate using n-butanol: water: acetic acid in the ratio 4:1:1 (v/v/v) as mobile phase. Ninhydrin used as spraying reagent. Rutin, quercetin and leuco anthocyanin act as standard. It was observed that the fruits of Tribulus terrestris and leaves of Bougainvillea spectabilis have flavanoids and saponin compounds.

 

The antiyeast activity of plant material extracts were presented in (Table 1). These results indicate that the crude ethanol extract showed different degree of growth inhibition depending on the extract source and fungal strains used such as Candida albicans and Malassezia furfur (one of the dandruff causing yeast). The dose response curve of DPPH radical scavenging  activity (Graph 1), the ferric reducing capacity was determined by using FRAP (Ferrous ion reducing/antioxidant power assay) (Graph 2), chelating capacity (graph 3) and cerium (IV) ion reducing power (graph 4), anticancer activity (Fig 1) was observed that the Tribulus terrestris (fruits) and Bougainvillea spectabilis (leaves) has flavanoids and saponin compounds which has  potent antiyeast activity against Candida albicans, antioxidant activity and anticancer activity.

 

CONCLUSION:

As a conclusion, the fruits of T. terrestris and leaves of B. spectabilis have potent anti-yeast activity against C. albicans. This study also provide an important basis for the treatment of infection associated to the pathogens used in this study and it is useful for the development of the plants exhibited significant anti-yeast activity and properties that support folkloric use in the treatment of some diseases. The results from the antioxidant model reveal that the fruits and of T. terrestris L. and leaves of B. spectabilis L. have significant antioxidant activity, DNA binding activity and anticancer activity. Thus the scientific study on T. terrestris and B. spectabilis and their compounds such as flavanoids and saponins are of great importance.

 

REFERENCES:

1.        Hassain M, Salleem I, Khan S, Khan and Wali – ullahi, Heavy metals accumulation in medicinal plants growing in Peshawar: Pakistan, 27: 643, (2005).

2.        Sobel JD, Zervos M, Reed BC, Hooton T, Soper D, Nyirjesy P, Heine MW, Willems J, Panzer H, Antimicrob: Agents chemother, 47: 34-38,(2003).

3.        Cotran RS, Kumar V and Collins T, Robbins pathological basis of diseases, 6^th Edn, Thomas press (I) Ltd., Noida, India.

4.        Yu BP, Suescun EA, and Yand SY, Effect of age related lipid peroxidation on membrane fluidity and phospholipase A: modulation by dietary restriction Mech. Ageing Dev, 65: 17-33, (1992).

5.        Campbell IC, and Abdulla EM, Strategic approaches to in vitro neurotoxicology: In Approaches and methods: Neurotoxicology Academic Press, London, 495, (1995).

6.        Pratt DE, Hang M, Ho C, and Lee C, (Natural antioxidants from plant material, in phenolics compounds in foods and their effects on health II: antioxidants and cancer prevention I (ACS symposium series 507) (American chemical society, Washington DC), 54, (1992).

7.        Larsen AK, Escargueil AE, and Sklandanowski A,  From DNA damage to G2 arrest the many roles of Topoisomerase II: Prog Cell Cycle Re,. 5: 295, (2003).

8.        Turley H, Comley M, Houlbrook S, Nozaki N, Kuchi A, Hickson ID, Gatterk K, and Harris AL,  The distribution and expression of the 2 isoforms of DNA topoisomerase II in normal and neoplastic human tissues: Br. J. cancer, 75: 1340, (1997).

9.        Topia MO, Giordano MA, Gueper HG, An outbreak of Hepatogenous Photosensitization in sheep grazing Tribulus Terrestris in Argentina. Vet Hum Toxico, 36(4): 35-3, (1994).

10.     Bean GA,  and Abeywickrama. K, Toxigenic Aspergillus Flavus and Aflatoxins in srilankan medicinal plant material: Mycopathologia,113: 187-90, (1991).

11.     Kostova I, Dinchev D, Rentsch GH, Dimitrov V, Naturforsch Z, and Ivanova A, Two New sulfated furostanol saponins from Tribulus Terrestris linn.: Naturforsch Z,  57(1-2): 33-8, (2002).

12.     Ody P, The complete guide medicinal herbal: London, Dorling, and Kindersley, 223, (2000).

13.     Balasaraswathi. RS, Sadasivam M, Ward and Walker JM, (1998). An antiviral protein from Bougainvillea spectabilis root: Purification and Characterisation. Phytochemistry, 47(8): 1561-1565, (1998).

14.     Pun KB, Sabitha D, and Jeyarajan R, Screening of plant species for the presence of antiviral principles against okra yellow vein mosaic virus, 52(3): 221-223, (1999).

15.     Hartman HT, and Kester DI, Plant Propagation: Principles and Practices, 4^th Edn, Prentice Hall of India Private Limited, New Delhi, (1989).

16.     Oyaizu M, Antioxidative activities of browning products of glucosamine fractionated by organic - solvent and thin layer chromatography: Journal of the Japrese society for food science and technology, 35: 771-775, (1988).

17.     Decker EA, and Welch B,  Role of ferritin as a lipid oxidation catalyst in muscle food: J. Agric Food Chem, 38. 674-677 (1990).

18.     Shimada K, Fujikawa K, Yahara K, and Nakamura T, Antioxidative properties of xanthan on the antioxidation of soybean oil in cyclodextrin emulsion: J Agri Food Chem,                          40: 945–948, (1992).

19.     Resat Apak, Dilek Ozyurt, and Birsen Demirata, Determination of total antioxidant capacity by a new spectrophotometric method based on Ce(IV) reducing capacity measurement: Talanta, 71: 1155–1165, (2007).

20.     Nakamura K, Sugumi H, Yamaguchi A, Uenasea T, Kotasee Y, Okava R, Kamata J, Niijima J, Nagusu T, Koyamaji N, Yoshino H, Kito HK,  and Yoshimatsu K, Antitumor activity of – 37328, a novel herlozole topisonerest II Inhibitor vzol. Cancer, 1:169, (2002).

21.     Choi SK, Park HJ, Lee SK, Kim SW, Hog and Choo HT, Solid phase combinatorial synthesis of benothiozole and elevation of topoisomerase II inhibitory activity: Biorg. med. Chem, 14: 1229-1235, (2006).

22.     E. Stahl E, Chemiker-Ztg, 82, 323, (1958).

 

 

 

Received on 12.07.2011          Modified on 22.07.2011

Accepted on 09.08.2011         © RJPT All right reserved