KIM NGÂN HOA

Kim Ngân Hoa - Lonicera japonica Thunb

http://tuetamherbgarden.blogspot.com/2015/10/kim-ngan-hoa.html

ANTIOXIDANT ACTIVITY OF DIFFERENT EXTRACTS FROM LEAVES OF PERESKIA BLEO

Journal of Medicinal Plants Research Vol. 6(15), pp. 2932-2937, 23 April, 2012 Available online at http://www.academicjournals.org/JMPR DOI: 10.5897/JMPR11.760 ISSN 1996-0875 ©2012 Academic Journals

 Full Length Research Paper

Antioxidant activity of different extracts from leaves of Pereskia bleo (Cactaceae)

Behnaz Hassanbaglou(1) , Azizah Abdul Hamid(1,2*), A. M. Roheeyati(1), Norihan Mohd Saleh(2,3) , AhmadSahib Abdulamir(1) , Alfi Khatib(1) and Sabu M.C(4)

(1) Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400 Serdang Selangor, Malaysia.

(2) Agro-Biotechnology institutes, Ministry of Science, Technology and Innovation Malaysia.

(3) Faculty of Biotechnology and Biomolecular Science, Universiti Putra Malaysia, 43400, Serdang Selangor, Malaysia.

(4) Faculty of Pharmaceutical Science, UCSI Universiti, Cheras, Malaysia.

Accepted 9 September, 2011

The present study was conducted to assess the antioxidant properties of ethanol, ethyl acetate, methanol and hexane extracts from leaves of Pereskia bleo using 2,2-diphenyl-1-picrylhydrazyl method (DPPH assay), ferric reducing antioxidant power (FRAP assay) and β-Carotene-Linoleic acid methods. Total phenolic compounds (TPC) and flavonoids of the leaves of P. bleo were measured using FolinCiocalteu and HPLC methods respectively. Results of the present study showed that Ethyl acetate extract of P. bleo leaf exhibited significantly (p < 0.05) higher antioxidant activity as measured using DPPH free radical (IC50), FRAP and β-carotene-linoleic assays than that of hexane extract, methanol extract and ethanol extract. Pereskia bleo leaf was also found to contain high amounts of bioactive compounds including total phenolic compounds (109.43 ±0.84 mg GAE/g), epicatechin (575± 0.04 mg/100g), quercetin (110.94 ±0.12 mg/100g), catechin (918 ± 0.01 mg/100 g) and myricetin (9.49±0.55 mg/100g) while the concentrations of α- tocopherol, β-carotene and lycopene were found to be higher (69±0.25, 51.97±1.18 mg/100 g, 92.46±0.41 µg/g), respectively. 

Key words: Pereskia bleo, antioxidant activity, total phenolic compounds, total flavonoid compounds. 

INTRODUCTION 

Oxidative stress can cause a wide range of diseases including Alzheimer's and Parkinson's disease, diabetes, cardiovascular disease, rheumatoid arthritis, cancer and other diseases (Simonian, 1996). Antioxidants are compounds that are capable of slowing or preventing the oxidation of other molecules. Antioxidants can terminate chain reactions by removing free radicals and oxidation reaction blocking others by being oxidized themselves. Due to this, antioxidants such as polyphenols and flavones that are often reducing factors (Shahidi, 1998). 

Synthetic antioxidants such as butylated hydroxyanisole (BHA) and butylated hydroxyltoluene (BHT) are very effective in neutralizing free radicals and therefore they are used as inhibitors of lipid peroxidation   to stabilize food containing fat. Carcinogenicity and toxicity of these synthetic antioxidants have been reported (Shahidi, 1997; Jeong et al., 2004, Siddhuraju and Becker, 2003). Consequently, the use of synthetic antioxidants is limited. In recent years, researchers are trying to replace them with natural antioxidants such as tocopherols, flavonoids and rosemary for food preservation (Bruni et al., 2004; Frutos and HernandezHerrero, 2005; Hras et al., 2000; Williams et al., 2004). 

Fruits and vegetables are reported to contain large amount of antioxidants, so people who consume these products have a lower risk of the relevance of free radical diseases such as heart disease and neurological disorders. Moreover, medicinal plants have been shown to have antioxidant activity by virture of the presence of phenolic diterpenes, flavonoids, tannins and phenolic acids (Dawidowicz et al., 2006). Medicinal plants have been regarded as a source of bioactive natural compounds. Antioxidants have the ability to protect the body against damages caused by oxidative stress induced free radicals. There is a growing interest in natural antioxidants, for example, polyphenols present in medicinal plants and food materials, which could help to prevent oxidative damages (Silva et al., 2005). 

Plant phenolic compounds probably had multifunctional antioxidants. These compounds have been reported to quench free radicals derived from oxygen, giving a hydrogen atom or an electron to the radical (Wanasundara and Shahidi, 1998). The antioxidant properties of phenolic compounds have been demonstrated in many systems through studies in vitro and in human low-density lipoproteins and liposomes (Leanderson et al., 1997). Literature showed only one antioxidant study on DPPH radical scavenging activity reported for P. bleo (Malek et al., 2008). However, the extraction method used in their study was different from that used in the present study. Various methods have been developed to measure total antioxidant activity, but none have been shown to be ideal (Erel, 2004). These antioxidant methods detect variable characteristics in the samples. This explains why different antioxidant detection methods may lead to different observations. Consequently, usage of different antioxidant methods is necessary in antioxidant activity assessment. 

Since development of cancer can be prevented by consumption of medicinal plants with antioxidative properties, it is of interest to investigate whether P. bleo possess these properties. In the present study, the total phenolic compound of the extracts of P. bleo was assessed by the Folin-Ciocalteau’s method while the antioxidant activities were determined using three different assays, namely scavenging activity on 1, 1- diphenyl-2- picrylhydrazyl (DPPH) radicals, reducing power assay and ß-carotene method. 

In Malaysia, herbs are usually eaten fresh as a vegetable (salad), especially among the Malay communities (Frankel and Meyer, 2000). Locally known as the ‘Seven Star Needle’ (qi xing zhen), this plant is a member of the cactus family. The genus was named in honour of Nicolas Fabre de Peiresc, a French botanist of the 16th Century (Figure 1). 

Figure 1. Pereskia bleo shrub.

 However, this cactus is a leafy cactus that is not a desert-adapted plant like many other leafless cacti. The objective of this study was to investigate the antioxidant compounds and its activities of extracts from P. bleo leaves using different solvent systems.

MATERIALS AND METHODS 

Chemicals 

1,1- Diphenyl-2-picryl hydrazyl radical (DPPH), 2,4,6-tri-pyridyl-striazine (TPTZ) were obtained from Sigma-Aldrich Company(USA), β-carotene, linoleic acid, Tween 40, 2,6-ditert-butyl-4- hydroxytuloene (BHA), 2,6-di-tert-butyl-4- methylphenol (BHT) and 2,4,6-Tris(2-pyridyl)-s-triazine (TPTZ), 6-hydroxyl-2,5,7,8- tetramethylchroman- 2-carboxylic acid (Trolox), and Folin-Ciocalteu phenol reagent were also obtained from Sigma (St.Louis, MO, USA). Methanol of HPLC-grade and hexane obtained from Merck was used for extractions. All other chemicals used were of analytical grade.

 Collection of plant material 

P. bleo leaves were collected from the vicinity of Universiti Putra Malaysia (UPM) in October 2009. They were identified by Professor Dr. Norihan Saleh of Faculty of Biotechnology and Biomolecular Science, University Putra Malaysia. Voucher specimen (CA01-09) was deposited in the Department of Food Biotechnology lab, Faculty of Food Science and Technology, UPM, Malaysia.

Preparation of plant extracts 

Fresh leaves of P. bleo were harvested and all petioles were cut and removed. The leaves were washed and dried under oven (Memmert, Germany. The dried leaves were ground in a grinder (Sharp,Malaysia). The dried powder was stored in the dark glass bottles and kept in the -20°C until examinations. The finely ground samples (50 g) were extracted with ethanol, methanol, ethyl acetate and hexane using a shaking water bath (Flavil, Switzerland) for 2 h at 40ºC. After filtration with whatman No1filter paper using vacuum pump, the samples were concentrated using a rotary vacuum evaporator (EYELA, USA) at 40°C to dryness. The concentrated extracts were kept in dark bottle in t at 4ºC.

Determination of antioxidant activity assays 

1,1- Diphenyl-2-picryl hydrazyl (DPPH) radical scavenging assay 

The antioxidant activity of P. bleo was performed using the method of DPPH radical scavenging activity as described by Brand-Williams et al. (1997). This method is spectrophotometric assay using a stable radical 2, 2'-diphenylpicrylhydrazyl (DPPH) as a reagent (Bondent and Brand-Williams, 1997; Cuendet et al., 1997; Buritis and Bucar, 2000). Different concentration of sample was (0.5ml) added to 3.5 ml of methanol DPPH solution with 6× 10 -5 mol/L , concentration in test tubes and kept covered at room temperature for 30 min. After the incubation period, absorbances were read at 515 nm until the reaction reached a plateau. The percentage of radical scavenging activity was calculated as follows:

Inhibition% = (A blank – A (sample) /A blank × 100)

A blank = Absorption of blank sample (t = 0 min). Asample = Absorption of tested extract solution (t = 30 min). Extract concentration providing 50% inhibition (IC50) was calculated from the plot of percentage inhibition against concentration of the extract. Hydroxanisol (BHA) and α-tocopherol were used as positive controls. All assays were carried out in triplicate.

Ferric reducing antioxidant potential assay 

Determination of FRAP was performed using the method described by Benzie and Strain (1996) with slight modifications. The stock solutions contains 300 mM acetate buffer (3.1 g C2H3NaO2. 3H2O and 16 ml C2H4O2), pH 3.6, 10 mM TPTZ solution in 40 mM HCl and 20 mM FeCl3.6H2O solution. The new solution (FRAP reagent) was prepared by mixing 50 ml acetate buffer, 5 ml TPTZ, and 5 ml FeCl3. 6H2O solution (ratio 10:01:01). Two hundred microliters of sample was added to 3 ml of FRAP reagent. After incubation in dark for 30 min at 37°C and the absorbance was read at 593 nm (Shimadzu UV-1650 PC UV-Vis spectrophotometer). Final results were expressed as micromole Trolox equivalent (TE) per gram of extract basis (umol TE/g). The amounts of the value of FRAP show the effects of antioxidant activity in the sample.

β-carotene- linoleic acid assay 

In the assay system acid β-carotene-linoleic acid, an aqueous emulsion of β-carotene and linoleic acid under condition of heatinduced oxidation reaction is used for testing anti-oxidant activity. This test is a rapid staining of β-carotene in the absence of antioxidant (Miller, 1971). In this process, the linoleate free radicals formed during the reaction are neutralized by antioxidants.

Briefly, 200 mg linoleic acid (Sigma, 99%) and 20 mg of Tween 20 (Sigma) were placed in a flask, then 2 mg of β-carotene (Sigma, 95% purity) dissolved in 10 ml chloroform (Merck, Germany) was added. Chloroform was evaporated using a vacuum evaporator unit. Then 50 ml of distilled water saturated with oxygen by stirring for 30 min was added. Aliquots (200 ft) of each sample dissolved in methanol (2.0 mg/ml) were added to 2.5 ml of solution in test tubes. The samples were then placed in an oven at temperature of 50°C for 3 h. The absorbance was read at 470 nm using a spectrophotometer. Percent antioxidant activity was calculated from the following equation:

AA% = 100 × [1- (A0- At /A00 _ A0t)]

Where AA% is percent of antioxidant activity, Ao is the absorbance at beginning of reaction, At is the absorbance after three hour, with compound; Aoo is the absorbance at beginning of reaction, without compound (200 µl of methanol and 2.5 ml of stock solution). Aot is the absorbance after 3 h without compound. A White stock solution containing less β-carotene has been used to bring the spectrophotometer to zero. As a positive control 2, 6-di-tert-butyl-4- methylphenol (BHT, Sigma) was used with same condition and concentration (2.0 mg/ml). Each test was repeated three times and the average score and standard deviation calculated.

Determination of total phenolic compound (TPC) 

TPC was measured by colorimetry using the Folin-Ciocalteu (Sigleton et al., 1999). One ml of appropriate dilution (using 80% ethanol) ethanol extract of leaves P. bleo was added to a 25 ml volumetric flask filled with 9 ml of distilled water. Phenol reagent of Folin-Ciocalteu (0.5 ml) and 5 ml of 5% Na2CO3 were added to the sample and shake vigorously. The solution was immediately make up to 25 ml with distilled water and mix well. The incubation time was one hour at room temperature under dark condition. The absorbance was then measured at 765 nm. Quantification was based on the calibration curve of gallic acid standard, and the contents were expressed as mg gallic acid equivalents (mg)/100 g.

Statistical analysis 

Data were analyzed using statistical package for social sciences (SPSS), version 16. Difference between means was determined using ANOVA (Duncan and Tukey's test).

 RESULTS AND DISCUSSION 

1,1- Diphenyl-2-picryl hydrazyl (DPPH) radical scavenging assay 

DPPH method measured the ability of antioxidant in scavenging free radicals present. Antioxidant activity of P. bleo leaf was expressed as the concentration that inhibits 50% DPPH free radical (IC50). Results obtained in the study showed that the IC50 of ethyl acetate extract of P. bleo (168.35 ± 6.5 μg/ml) was significantly (p < 0.05) lower than that of other extracts; Hexane extract (244 ± 4.5 μg/ml), methanol extract (277.5± 6.5 μg/ml), and ethanol extract (540 88 ± 9.0 μg/ml (Figure 2). 

In this study, the ethyl acetate extract of leaves of P. bleo was found to exhibit excellent antioxidant activity compared to that of ethanol, hexane, and methanol extracts. The results are compatible with those of Wahab et al. (2008) and Sri Nurestri et al. (2009). All extracts showed low activity against BHA and-tocopherol, which act as a reference antioxidant compounds. There was broad experimental evidence suggesting that being a rich source of polyphenolic compounds, plants display strong antioxidant activity (Sabu et al., 2002). 

FRAP method 

The FRAP test measures the ability of samples to reduce ferric ion to the ferrous form of TPTZ (2,4,6- tripyridylstriazine). One FRAP unit is defined as the reduction of 1 mol of Fe3+ to Fe2+. Similarly, result of the study showed that the antioxidant capacity of ethyl acetate extract (50.48 ±1.53 μM TE/g) was significantly (p < 0.05) higher than that of hexane (41.7± 2.2 μM TE/g) methanol and ethanol extracts (45.2±1.2 μM TE/g), (40.45±1.54 μM TE/g), respectively, of leaves of P. bleo (Figure 3). 

However, there was no significant (p < 0.05) difference on the antioxidant capacity between hexane extract and methanol extract. It is interesting to note that the trend of antioxidant activity obtained from FRAP assay was similar to that obtained in DPPH assay.

β-carotene-linoleic acid assay 

β-carotene- linoleic acid assay is used to assess the ability of the extracts in protecting β-carotene and the data is expressed as percent of antioxidant activity (AA%). Results of the present study showed that AA% of ethyl acetate extract (71 ± 4.5%) was significantly (p <0.05). there was no significant (p < 0.05) difference on the antioxidant capacity between hexane extract (66 ± 1.2%), ethanol extract (65 ± 2.7%) and methanol extract (67.8 ± 4.6%) (Figure 4). In this study, methanol, ethanol, ethylacetate and hexane extracts of P. bleo leaf were evaluated for their antioxidant activities using DPPH, FRAP and β-carotene linoleic acid assays. The different methods and different percent of solvent (80% methanol, 100%ethanol) used here are crucial due to the different array of bioactive components present in the plants that maybe responsible for the antioxidant activity of medicinal plants. 

The antioxidant activity ability of the plant extracts basically depend on the composition of the extracts, hydrophobic or hydrophilic nature of the antioxidants, type of solvent used for extraction process, method of extraction, temperature and conditions of the test systems. Therefore, it is necessary to use more than one method for evaluation of antioxidant activity of plant extracts defining various mechanisms of antioxidant actions (Wong et al., 2006). Antioxidant capacity and health benefits of medicinal plants are often ascribed to their total phenolic contents (Hua-Bin, 2008; Nagai et al., 2003). High correlation was also reported between antioxidant activity as measured by DPPH method and total phenolic compound and flavonoid content of 21 selected tropical plants (Mustapha et al., 2010). Similarly, green tea exhibited higher total antioxidant activity than that of pure catechins proportionately combined based on the green tea constituent, which is mainly due to its synergistic effect (Rice-Evans et al.,1995) (Table 1).

Bioactive compounds in P. bleo leaves 

Selected bioactive compounds from P. bleo leaf were determined and showed in Table 2. Results of the study showed that TPC of P. bleo leaf were 109.43 ± 0.84 mg GAE/g of extract. The major flavonoids identified were catechin (918 ± 0.01 mg/100 g) and quercetin (110.94 ± 0.12 mg/100 g). This was followed by epicatechin (109.43 ± 0.84 mg/100 g) and myricetin (9.49 ± 0.55 mg/100 g). However, concentrations of β-carotene (51.97 ± 1.18 mg/100 g) and α- tocopherol (69 ± 0.25 mg/100 g) were quite high. 

Conclusion 

The results of experiments in vitro, including radical  scavenging DPPH, FRAP assay, determining the content of phenolic and total β-carotene bleaching showed that phytochemicals in extracts of plants studied can have a significant effect on antioxidant activity. In addition, the antioxidant activity is directly related to the total amount of phenolic compounds in plant extracts. These compounds have been reported to possess antioxidant activities. P. bleo can be used as an easily accessible source of natural antioxidants, and can be used as food supplement or in pharmaceutical and medical industries. 

ACKNOWLEDGMENTS 

The authors would like to acknowledge the Ministry of Science, Technology and Innovation (MOSTI) of Malaysia for financing the project and Faculty of Food Science and Technology, University Putra Malaysia, for the laboratory facilities. 

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Cytotoxic Components of Pereskia bleo (Kunth) DC. (Cactaceae) Leaves

Molecules 2009, 14, 1713-1724; doi:10.3390/molecules14051713

molecules

ISSN 1420-3049

www.mdpi.com/journal/molecules

 Article

Cytotoxic Components of Pereskia bleo (Kunth) DC. (Cactaceae) Leaves

Sri Nurestri Abdul Malek 1,*, Sim Kae Shin 1, Norhanom Abdul Wahab 2 and Hashim Yaacob 3

1 Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia

2 Institute of Postgraduate Studies, University of Malaya, 50603 Kuala Lumpur, Malaysia

3 International University College of Nursing, B-27-6, Block B, Jaya One, No 72A Jalan Universiti, 46000 Petaling Jaya, Selangor, Malaysia

* Author to whom correspondence should be addressed: E-mail: srimalek@um.edu.my; Tel.: +603-79677119; Fax: +603-79674178.

Received: 17 March 2009; in revised form: 7 April 2009 / Accepted: 4 May 2009 /

Published: 6 May 2009

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Abstract: Dihydroactinidiolide (1) and a mixture of sterols [campesterol (2), stigmasterol (3) and β-sitosterol (4)], together with the previously isolated individual compounds β-sitosterol (4), 2,4-di-tert-butylphenol (5), α-tocopherol (6), phytol (7) were isolated from the active ethyl acetate fraction of Pereskia bleo (Kunth) DC. (Cactaceae) leaves.

Cytotoxic activities of the above mentioned compounds against five human carcinoma cell lines, namely the human nasopharyngeal epidermoid carcinoma cell line (KB), human cervical carcinoma cell line (CasKi), human colon carcinoma cell line (HCT 116), human hormone-dependent breast carcinoma cell line (MCF7) and human lung carcinoma cell line (A549); and non-cancer human fibroblast cell line (MRC-5) were investigated. Compound 5 possessed very remarkable cytotoxic activity against KB cells, with an IC50 value of 0.81μg/mL. This is the first report on the cytotoxic activities of the compounds isolated from Pereskia bleo.

Keywords: Pereskia bleo; Cactaceae; cytotoxic activity; cell lines

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Introduction

The leaves of Pereskia bleo (Kunth) DC. (Cactaceae) are used traditionally in Malaysia for the treatment of cancer, high blood pressure, diabetes and diseases associated with heumatism and inflammation. They are also used as remedy for the relief of gastric pain, ulcers and for revitalizing the body [1]. The leaves are generally consumed by the locals either raw or taken as a concoction brewed from fresh leaves.

Chemical investigations on Pereskia bleo are rare in comparison to other Pereskia species, as there were only three phytochemical and biological studies reported for this plant. The earliest phytochemical study was by Doetsch et al. [4], who reported the isolation of four alkaloids, namely 3,4-dimethoxy-β-phenethylamine, mescaline, 3-methoxytyramine and tyramine. An investigation by Tan et al. [2] reported that the methanol extract of Pereskia bleo possessed cytotoxic effects against T-47D cells and the cell death was found to be apoptotic in nature, mainly via the activation of the caspase-3 and c-myc pathways. A more recent investigation by Er et al. [3] indicated the antiproliferative and mutagenic activities of aqueous and methanol extracts of Pereskia bleo leaves against mouse mammary cancer cells (4T1) or normal mouse fibroblast cells (NIH/3T3). In our previous cytotoxicity screenings on Pereskia bleo [5], the EtOAc fraction possessed notably high cytotoxic effects against selected human carcinoma cell lines, but exerted no damage to a non-cancer human fibroblast cell line (MRC-5). The active EtOAc fraction was found to contain β-sitosterol (4), 2,4-ditert- butylphenol (5), α-tocopherol (6) and phytol (7) [5]. As part of our ongoing research on Pereskia bleo, a pure compound and a mixture of sterols were also isolated from the leaves of Pereskia bleo.

In the present study, we report further progress in ongoing research on Pereskia bleo, which led to the isolation and identification of dihydroactinidiolide (1) and a mixture of sterols [campesterol (2), stigmasterol (3) and β-sitosterol (4)] and cytotoxic investigation on all isolated compounds against five human carcinoma cell lines, namely the human nasopharyngeal epidermoid carcinoma cell line (KB), human cervical carcinoma cell line (CasKi), human colon carcinoma cell line (HCT 116), hormonedependent breast carcinoma cell line (MCF7) and human lung carcinoma cell line (A549) and noncancer human fibroblast cell line (MRC-5).

Results and Discussion

Extraction and isolation of pure compounds and the sterol mixture

β-Sitosterol (4), 2,4-di-tert-butylphenol (5), α-tocopherol (6) and phytol (7) were obtained from Pereskia bleo as previously described by Sri Nurestri et al. [5]. On repeated chromatographic purification of the active EtOAc fraction, a red viscous oil and white colored needles were obtained and identified as dihydroactinidiolide and a mixture of sterols.

Dihydroactinidiolide (1), red viscous oil; EI-MS m/z (%): 180 [M] + (15), 137 (8), 111 (100), 109, 67. Compound 1 was identified by comparison of its mass spectral data with NIST mass-spectral library [21] and other reported spectroscopic data [6-8].

The mixture of sterols appeared as white colored needles that according to GC-MS analyses consisted of campesterol (2, 14.33%), stigmasterol (3, 4.95%) and β-sitosterol (4, 70.21%). Compound 2 (campesterol); EI-MS m/z (%): 400 (42, [M+]), 382 (34), 367 (20), 315 (30), 289 (30), 55 (100). The mass spectral data was also in agreement with reported data [9]. Stigmasterol (3) was identified by GC-MS analysis and by comparison of its mass spectral data [EI-MS m/z (%): 412 (16, [M+]), 394 (4), 369 (2), 351 (6), 271 (16), 255 (22), 229 (5), 55 (100)] with reported data [9]. Compound 4 (ß-sitosterol); EI-MS m/z (%): 414 (100, M+), 396 (57), 381 (43). ß-sitosterol (4) was identified by GC-MS analysis as well as comparison of its mass spectral data with reported data [10].

The structures of compounds 1-7 are illustrated in Figure 1.

In vitro Neutral Red cytotoxicity assay

The in vitro cytotoxicity assay was carried out using a Neutral Red cytotoxicity assay as previously described by Borenfreund and Puerner [11] with some modifications; this test determines the accumulation of the Neutral Red dye in the lysosomes of viable and uninjured cells.

The results of cytotoxicity screening of the components are summarized in Table 1. It is generally known that ethnomedical data substantially increases the chances of finding active plants relative to a random approach [2]. The consequence is that, once having found activity in the plant from the ethopharmacological observation (e.g. raw or concoction brewed from the plant leaves shows effect for cancer treatment), there is a desire to determine the chemical structures of the compounds that are responsible for the activity, as not all the compounds in the extracts have the same activity.

However, the observed activity might be due to synergism between compounds present in the plant extract. The synergism among these compounds which contribute to the cytotoxic activity, is not only dependent on the concentration of the compounds, but also on the structure and interaction(s) between the compounds [27]. This can explain the differences in the cytotoxic effect between crude extracts and isolated compounds against the same cell lines, as shown in our earlier report [5]. For example, the cytotoxic effect of the crude methanol extract on the KB cell lines showed an IC50 of 6.5 μg/mL and such impressive activity was supported by some of the isolated compounds [dihydroactinidiolide (1), 2,4-di-tert-butylphenol (5), α-tocopherol (6) and phytol (7)]. In contrast, the cytotoxic effect of the crude methanol extract on the MCF7 cell line gave IC50 of 39.0 μg/mL (mild) whilst two isolated compounds 2,4-di-tert-butylphenol (5) and α-tocopherol (6), showed good inhibitory activities with IC50 values of 5.75 and 7.5 μg/mL, respectively.

2,4-Di-tert-butylphenol (5) displayed very remarkable cytotoxic activity against KB cells with an IC50 value of 0.81 μg/mL and strong cytotoxicity against MCF7 (IC50 5.75 μg/mL), A549 (IC50 6 μg/mL) and CasKi cells (IC50 4.5 μg/mL). This in vitro data of 2,4-di-tert-butylphenol (5) support the findings that phenolic antioxidants exert cytoctoxic activity against cancer cells [14, 15]. 2,4-Di-tertbutylphenol (5) is an antioxidant widely used in the plastics industries, and its presence in plants cannot readily be explained biogenetically. It is more probable that the plant accumulated this compound from the soil it grew in, that might have contained the compound. In our experience, this compound has also been detected in other plants like Termitomyces heimi, Pleurotus sajor-caju and Hericium erinaceus collected from different locations to where the Pereskia bleo leaves were obtained (unpublished data from our group of researchers working on Termitomyces heimi, Pleurotus sajor-caju and Hericium erinaceus). The observation of 2,4-di-tert-butylphenol (5) in our study is not an isolated case, as it has also been reported to exist in natural sources by other researchers [29-31]. To support our finding that 2,4-di-tert-butylphenol (5) is not an artifact, an extraction on Pereskia bleo was repeated using redistilled methanol and ethyl acetate. GC-MS analysis on the ethyl acetate extract still showed the presence of 2,4-di-tert-butylphenol (5) representing the major component of the total ethyl acetate extract. This shows that 2,4-di-tert-butylphenol (5) is present in the extract itself and not a solvent artifact.

Other constituents in the plant also contribute to its cytotoxic activity as shown by α-tocopherol (6), phytol (7) and dihydroactinidiolide (1). In the present study, α-tocopherol (6), which is a dietary antioxidant, displayed pronounced cytotoxicity against CasKi (IC50 6 μg/mL) and A549 (IC50 6 μg/mL). The result obtained here is consistent with other reports [37-40, 47-50] on cytotoxic activities in other cell lines. Lesser number of investigations described an opposite effect [44-46]. There was no report on the cell lines that were used in this study. According to Table 1, phytol (7) demonstrated strong activity against KB cells (IC50 7.1 μg/mL). The cytotoxicity data showed in this report thus supports our hypothesis in our previous report [5] that phytol might be responsible for the remarkable cytotoxic effect of the EtOAc fraction against the KB cancer cell lines. In this study, dihydroactinidiolide (1) demonstrated strong cytotoxic effect against HCT116 with IC50 5.0 μg/mL. Dihydroactinidiolide (1) is structurally similar to the C11-terpene lactones that arise from the biological or oxidative degradation of carotenoids and has been isolated from various plants and insect sources. It has also been identified as the flavor molecule in tea and tobacco [6-8].

Sterols are important constituents of all eukaryotes and play a vital role in plant cell membranes. In addition to their cholesterol lowering effect, plant sterols may possess anti-atherosclerosis [32-33], antibacterial [36], anti-inflammation [34] and anti-oxidation activities [35]. In the present study, β-sitosterol (4) and the mixture of sterols [campesterol (2), stigmasterol (3) and β-sitosterol (4)] did not display cytotoxic effects against the tested cell lines. The results obtained here were in agreement with published data [16-20]. There have been reports that plant sterols are able to stimulate estrogen dependent cancer cells in vitro (e.g. Ju et al. [42]). The MCF7 cell line used in this study was purchased from ATCC. It was reported that MCF7 cells from ATCC were unaffected by estrogen or antiestrogen [43]. Thus, the result showed that the sterols do inhibit the growth of MCF7 cells.

Doxorubicin which is clinically used for the treatment of a great variety of cancer disease [24-26] was used as the positive control in present study. Based on the result, it can be concluded that doxorubicin is not only cytotoxic against all the human cancer cell lines tested, but also the non-cancer human cell line. This result supports the statement that doxorubicin is a potent cytostatic drug which is applied for the treatment of cancer diseases but the routine use of this drug could bring major adverse effect [24]. Although the cytotoxicity of the isolated compounds and mixture of Pereskia bleo are not as effective as doxorubicin, they however have low toxicity against normal MRC5 cell line in comparison to doxorubicin. The use of the isolated compounds as single anticancer agents would not merit consideration. However, their use in combination with cytotoxic therapeutic drugs might reduce the adverse effects of some of these drugs. Support for this suggestion is provided by Amir et al. [41], who reported that in addition to having potent antitumor properties as single agents, natural products have also demonstrated potential synergy with established cytotoxic therapeutic drugs in pre-clinical studies. At this stage, it is not possible to justify the use of isolated compounds in comparison to doxorubicin in the treatment of cancer. A more comprehensive investigation is required.

Experimental

General

GCMS analysis was performed using a Agilent Technologies 6980N gas chromatography equipped with a 5979 Mass Selective Detector (70 eV direct inlet); a HP-5ms (5% phenylmethylsiloxane) capillary column (30.0 m x 250 μm x 0.25 μm) initially set at 60C for 10 minutes, then programmed to 230C at 3C min-1and held for 1 min at 230C using helium as the carrier gas at a flow rate of 1 mL min-1. The total ion chromatogram obtained was auto integrated by ChemStation and the components were identified by comparison with an accompanying mass spectral database [21]. Thin layer chromatography (TLC) analyses were carried out using precoated TLC plates 60 F254 (20.25 mm thickness) purchased from Merck and were visualized in UV light (254 and/or 343 nm) and/or iodine vapour.

Plant sample collection and identification

The fresh leaves of Pereskia bleo were collected from Petaling Jaya, Selangor, Malaysia in September 2006. They were identified by Professor Dr. Halijah Ibrahim of Institute of Biological Sciences, Faculty of Science, University of Malaya, Malaysia and a voucher specimen (SN01-06) was deposited at the herbarium of the Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia.

Extraction and isolation of pure compound and mixture

β-Sitosterol (4), 2,4-Di-tert-butylphenol (5), α-tocopherol (6) and phytol (7) were isolated from Pereskia bleo as previously described by Sri Nurestri et al. [5]. Compound 1 and mixture of sterols were obtained according to the following procedure. Dried, ground leaves (1,050.56 g) of Pereskia bleo were extracted with MeOH (3x 1.5 L). The MeOH-containing extract obtained was initially treated with charcoal, then filtered over Celite® and the filtrate was evaporated under reduced pressure to give a crude MeOH extract (99.44 g). Treatment with charcoal was necessary to remove the high amounts of chlorophyll present in the extract, which interfered with chromatographic separation efforts. The crude MeOH extract was then further partitioned between EtOAc and H2O in a separating funnel. The EtOAc-soluble layer was concentrated in vacuo giving an 18.34 g EtOAc fraction, which was subjected to flash silica gel column chromatography (Si-gel CC) eluting with CHCl3 (10 L), and then with CHCl3-MeOH [9:1 (9 L)] and finally MeOH (7.6 L). The CHCl3 fraction was concentrated to give a dark brown residue (3.47 g). The brown residue was subjected to a Si-gel CC initially eluting with a gradient of hexane followed by hexane enriched with increasing percentages of CH2Cl2, monitoring with TLC. The volume of each fraction was 25 mL. The mixture of sterols (20.5 mg) was obtained from the fraction upon elution with CH2Cl2-hexane (3.5: 6.5). Further elution with CH2Cl2 yielded a mixture (206.7 mg) containing 1. Purification of 1 was obtained through preparative-TLC using CHCl3 as the developing solvent to yield pure compound 1 (5.4 mg).

Cell lines and culture medium 

Human nasopharyngeal epidermoid carcinoma cell line (KB), human cervical carcinoma cell line (CasKi), human colon carcinoma cell line (HCT 116), human hormone-dependent breast carcinoma cell line (MCF7), human lung carcinoma cell line (A549) and non-cancer human fibroblast cell line (MRC-5) were purchased from the American Tissue Culture Collection (ATCC, USA). KB cells were maintained in Medium 199 (Sigma), CasKi, A549 and MCF7 cells in RPMI 1640 medium (Sigma), HCT 116 in McCOY’S 5A Medium (Sigma) and MRC-5 cells in EMEM (Eagle Minimum Essential Medium) (Sigma), supplemented with 10% foetal bovine serum (FBS, PAA Lab, Austria), 100 μg/mL penicillin or streptomycin (PAA Lab, Austria) and 50 μg/mL of fungizone (PAA Lab, Austria). The cells were cultured in a 5% CO2 incubator (Shel Lab water-jacketed) kept at 37°C in a humidified atmosphere.

In vitro Neutral Red cytotoxicity assay

The Neutral Red cytotoxicity assay is based on the initial protocol described by Borenfreund and Puerner [11] with some modifications. Briefly, the cells (1x104/well) were seeded in 96-well microtiter plates (Nunc) and allowed to grow for 24 hours before treatment. After 24 hours of incubation, the cells were treated with six different concentrations (0.1-100 μg/mL) of test compounds, in three replicates. The plates were further incubated for 72 h at 37°C in a 5% CO2 incubator. A stock solution was initially obtained by dissolving the test compounds in DMSO. Further dilution to different tested concentrations were then carried out ensuring that the final concentration of DMSO in the test and control wells was not in excess of 1% (v/v). No effect due to the DMSO was observed. Doxorubicin was used as the positive control. The well containing untreated cells was the negative control. At the end of the incubation period, the media were replaced with medium containing 50 μg/mL of Neutral Red. The plates were incubated for another 3 hours to allow for uptake of the vital dye into the lysosomes of viable and injured cells. After the incubation period, the media were removed and cells were washed with the neutral red washing solution. The dye was eluted from the cells by adding 200 μL of Neutral Red resorb solution and incubated for 30 minutes at room temperature with rapid agitation on a microtiter plate shaker. Dye absorbance was measured at 540 nm using a spectrophotometer ELISA plate reader. The average data from triplicates were expressed in terms of killing percentage relative to negative control. The percentage of inhibition (%) of each of the test samples was calculated according to the following formula:

where OD control: Optical Density of negative control; OD sample: Optical Density of sample

Cytotoxicity of each sample is expressed as IC50 value. The IC50 value is the concentration of test compounds that cause 50 % inhibition or cell death, averaged from the three experiments, and was obtained by plotting the percentage inhibition versus concentration of test compounds. According to US NCI plant screening program, a plant extract is generally considered to have active cytotoxic effect if the IC50 value, following incubation between 48 to 72 hours, is 20 μg/mL or less, while it is 4 μg/mL or less for pure compounds [12, 13, 22, 23]. However, we recognized that whether an IC50 value corresponds to a significant or non-significant cytotoxicity depends on the sensitivity of the cell line.

Conclusions

In conclusion, and depending on the cell lines used, the cytotoxic activities observed for Pereskia bleo [5] are ascribable to the presence of the active compounds 1, 5, 6 and 7. Although the cytotoxicity of these compounds and mixture are not as effective as doxorubicin, in comparison to the latter they have low toxicity against normal MRC5 cell line. The cytotoxicity assay used in the present study could only provide important preliminary data to help select plant extracts or isolated compounds with potential antineoplastic properties for future work. A detailed investigation on the mechanism of cell death would provide more convincing evidence. An investigation into this phenomenon is now underway and will be reported in due course. The resulting information will certainly contribute to a better understanding of the anti-carcinogenic activity of the natural constituents in Pereskia bleo.

Pereskia bleo has been traditionally used for the treatment of cancer and the findings of the current study thus provide scientific validation on the use of the leaves of Pereskia bleo. In view of the increasing popular consumption of medicinal plants as alternative therapy, it is therefore necessary to conduct serious research to support the therapeutic claims and also to ensure that the plants are indeed safe for human consumption.

Acknowledgements

This work was supported by a research fund from the University of Malaya (Vote F PS056/2007C) and the Ministry of Science, Technology and Innovation (MOSTI) (E-sciencefund 1202032026). We are also grateful to Prof A. Hamid A Hadi for use of his laboratory space.

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