Abstract
Context: Natural products are a very productive source of leads for the development of medicines. Six Pakistani plants were chosen for study based on ethnobotanical data.
Objective: Exploration of important medicinal plants of Pakistan for cancer treatment.
Materials and methods: The crude extracts of the six plants and their fractions were tested for inhibition of nuclear factor κB (NFκB), aromatase, and nitric oxide (NO) production in lipopolysaccharide (LPS)-activated murine macrophage RAW 264.7 cells, induction of quinone reductase 1 (QR1), agonism of retinoid X receptor, and growth inhibition with MCF-7, LU-1 and MDA-MB-231 cancer cells.
Results: Two samples of Withania coagulans (Stocks) Dunal (Solanaceae) demonstrated inhibition of TNF-α induced activity of NFκB with IC50 values of 2.6 and 4.3 µg/mL, respectively. Two fractions from W. coagulans and Euphorbia wallichii Hook F. (Euphorbiaceae) aerial parts inhibited aromatase with IC50 values of 17.0 and 17.7 µg/mL, respectively. A total of 13 samples (five from E. wallichii, one from Acer oblongifolium Hort. ex Dippel (Aceraceae), one from Aster thomsonii C. B. Clarke (Asteraceae) and six from W. coagulans aerial parts with fruits) inhibited NO production with IC50 values ranging from 1.3 to 15.6 µg/mL. Fourteen samples demonstrated induction of QR1 with CD ranging from 1.0 to 20.6 µg/mL, and a total of eight extracts and fractions inhibited the proliferation of cancer cells in culture with IC50 values ranging from 1.2 to 7.8 µg/mL.
Discussion and conclusion: Selected plants can be a valuable source of chemopreventive and anticancer products. W. coagulans aerial parts showed the strongest activity.
Introduction
Carcinogenesis can be viewed a multistage process culminating in tumorigenesis. Initiation involves a change in the genetic makeup of a cell, possibly due to carcinogens or damage to a DNA repair mechanism. During promotion, the mutated cell is stimulated to grow and divide, becoming a population of highly proliferative cells. These cells can progress to expand further as tumor cells, eventually outnumbering their normal cell counterparts (CitationPezzuto et al., 2005). Any substance that can prevent, block or reverse these stages are potential candidates for cancer chemoprevention (CitationPezzuto, 1993; CitationKinghorn et al., 2004), and there is significant interest in the discovery of novel cancer chemopreventive agents.
Our work in this area has primarily focused on terrestrial plant and marine organisms as starting materials (CitationPezzuto, 1993; Park & Pezzuto, Citation2002b; CitationKinghorn et al., 2004). In most cases, the active starting materials were selected on the basis of large-scale collection followed by biological evaluation. Numerous bioassay procedures and criteria of activity have been established. In the current investigation, plants were selected after an extensive survey of different geographical areas of Pakistan and interviews with local healers prescribing folk medicines. As a result, Euphorbia wallichii, Bergenia ciliate, Acer oblongifolium, Aster thomsonii, Opuntia dillenii and Withania coagulans were selected for evaluation. Each of these plants had a history of multiple traditional uses ().
Table 1. Summary of the ethanobotanical uses and literature review of the selected plants.
E. wallichii Hook F. belongs to the family Euphorbiaceae, one of the largest families of higher plants, and is locally known as “Dodal.” The roots are traditionally used as a folk medicine for the treatment of skin diseases such as furuncle, cutaneous anthrax, exanthema and edema (CitationLal et al., 1990). This plant is mostly found in northern areas of Pakistan, Afghanistan, Tibet, Nepal and Assam. Members of the genus Euphorbia are rich in bioactive compounds and some of these plants have been used in folk medicine for hundreds of years (CitationAli et al., 2008) for the treatment of cancers, tumors, migraine, skin diseases, gonorrhea, intestinal parasites and warts (CitationSingla & Pathak 1990). Euphorbia species have constituents of different chemical classes including aromatic esters, phenolics, steroids, terpenoids, essential oils, etc. (CitationAli et al., 2008).
B. ciliata (Haw.) Sternb, a member of the family Saxifragaceae, is a very important medicinal plant locally known as “Zakhm e Hayat.” It is native to central Asia but also distributed in South and East Asia and European countries. It grows at high altitudes in the Himalaya usually in rocky areas and on cliffs. B. ciliata is known to be used ethno-botanically for dissolution of kidney and bladder stones, and for the treatment of leucorrhea, piles, and pulmonary infections (CitationAsolkar et al., 1992). It is also used for the treatment of heart diseases, hemorrhoids, ophthalmia, stomach disorders, diabetes, and boils and blisters (CitationKapur, 1993; CitationSingh, 1997). Other pharmacological activities include antiviral, antibacterial, antitussive and anti-inflammatory potential (CitationSingh et al., 2007). Bergenin is an important constituent of B. ciliata and is reported to have anti-inflammatory, antitussive, anti-HIV, anti-arrhythmic and neuroprotective activities (CitationSingh et al., 2007). Chemical investigations of B. ciliata have revealed the presence of β-sitosterol-d-glucoside, pashaano-lactone, α-afzelechin, methyl gallate, catechin leucocyanidin, and gallic acid (CitationSingh et al., 2007).
A. oblongifolium Hort. ex Dippel (Aceraceae), which grows 12–15 m tall, is an evergreen tree known locally as “Kaeen.” A. oblongifolium is reported to have phytotoxic, antitumor and cytotoxic potential (CitationInayatullah et al., 2007).
A. thomsonii C. B. Clarke is a member of the family Asteraceae. Plants of the genus Aster have been used as an expectorant for the relief of cough, and they have also shown antitumor, antibacterial, diuretic, antiulcer and antiviral activities (CitationShirota et al., 1997; CitationWang & Yu, 1998). An infusion of the aerial parts of Aster squamatus is used as an antidiarrheal (CitationMeneghetti, 1997). The genus Aster is reported to contain phenolic esters, triterpenes, simple phenols, indoles, flavonoids, quinic acid derivatives, phenylpropanoids and phenylpropanoid glycosides (CitationLin et al., 2007).
O. dillenii (Ker-Gawl) Haw (Cactaceae) is locally known as “Anar Phali.” It usually grows in semi-desert regions in the tropics and sub-tropics (CitationChang et al., 2008). O. dillenii is used in folk medicine for treatment of gastric ulcers, reducing cholesterol levels, diabetes, analgesic, inflammation (CitationLoro et al., 1999) and several other diseases. O. dillenii is also reported to have antispermatogenic effects (CitationGupta et al., 2002). It contains betalain, betacyanins, betaxanthins, ascorbic acid, betanin, isobetanin, kaempferol 3-O-arabinoside, isorhamnetin 3-O-glucoside, isorhamnetin 3-O-rutinoside (CitationAhmed et al., 2005) and steroids (CitationJiang et al., 2006).
W. coagulans (Stocks) Dunal (Solanaceae) (synonym: Puneeria coagulans [Stocks]) commonly known as Indian cheese maker, Indian rennet, vegetable rennet (English), Panir band, Panir ke phool, Punir dodi (Hindi), Paneer bandh, or Ning gu shui qie (Chinese), is distributed in the relatively drier parts of Pakistan and India (CitationKirtikar & Basu, 1995). The plant is a native of the Asia-temperate (Western Asia: Afghanistan) and Asia-tropical (Indian Subcontinent: India, Pakistan and Nepal) regions. W. coagulans is well known in the indigenous system of medicine and used as an antimicrobial, anthelmintic, antitumor, anti-inflammatory, hepatoprotective, diuretic, sedative, anti-asthmatic, blood purifier, wound healer, antirheumatic, antidyspepsia, antidropsy, antiulcer, antidiabetic and to treat consumption and sensile debility and cardiovascular problems (CitationChattopadhyay et al., 2007; CitationHemalatha et al., 2008; CitationMaurya, 2010). The smoke of the plant is inhaled for relief in toothache and twigs are chewed to clean teeth. The leaves are used as vegetable, and as fodder for camel and sheep. The seeds are useful in ophthalmia and lessen the inflammation of piles (CitationMaurya, 2010). The berries are used for milk coagulation. This plant has been reported to contain withanolides, esterases, alkaloids, lignan, essential oils, fatty oils and free amino acids (CitationRahman et al., 2003; CitationMaurya, 2010).
The goal of this work was to assess the cancer chemopreventive and growth inhibitory effect of these six naturally growing, medicinally important plants from Pakistan, through inhibition of TNF-α induced NFκB, aromatase, and of NO production in lipopolysaccharide (LPS)-activated RAW 264.7 cells, induction of quinine reductase 1 (QR1), interaction with retinoid X receptors, and cytotoxicity against MCF-7, LU-1 and MDA-MB-231 cancer cell lines.
Materials and methods
Plant collection
Euphorbia wallichii Hook F. (EW), Acer oblongifolium Hort. ex Dippel (AO) and Bergenia ciliata (Haw.) Sternb (BC) were collected from Nathia Gali, Khyber Pakhtonkhwa in July, 2008. Withania coagulans (Stocks) Dunal (WC) was collected from Karapa (Banda Daood Shah) District Karak, Khyber Pakhtonkhwa in August, 2009. Aster thomsonii C. B. Clarke (AT) was collected from Shogran, District Mansahra, Khyber Pakhtonkhwa in July, 2009. Opuntia dillenii was collected in October, 2009 from Quaid-i-Azam University, Islamabad. These plants were identified by taxonomist Dr. Rizwana Aleem Qureshi, Professor, Department of Plant Sciences, Quaid-i-Azam University, Islamabad. A voucher specimen (numbering HMP-450 to 455 respectively) of each plant was also deposited in the “Herbarium of medicinal plants,” Quaid-i-Azam University, Islamabad, Pakistan.
Extraction and fractionation
A total of 59 extracts and fractions were prepared from six plants. Taxonomic data of plants, extract name, plant part, method name and solvent used are given in . Methods for extract preparation are described in detail below.
Table 2. Detail of sample preparation scheme of selected plants for biological evaluation.
Maceration
Shade-dried plant material was ground to coarse powder and was soaked in methanol for 3 days and shaken five times daily. Methanol was used in the ratio of 4 mL for 1 g of dry powdered plant material. After 3 days, methanol was squeezed out and filtered. The filtrate was dried in a rotary evaporator (Buchi, Switzerland) and fume hood. This semisolid material is called the crude methanol extract (CME).
Solvent-solvent extraction
CME was suspended in hot distilled water in the ratio of 1 g extract in 3 mL water. This water suspension was extracted three times with the water immiscible organic solvent (as given in ) using a separating funnel. Each time, the volume of the organic solvent used was the same as that of the water layer. The organic layer was dried in the rotary evaporator, and the aqueous layer was again extracted with the other organic solvent in the same order as given in .
Flash column chromatography
CME (10 g) was dissolved in methanol and adsorbed on 10 g of silica gel 60 (70–230 mesh, Merck, Germany) and dried in a fume hood. Then a glass column was loaded with 100 g of the same silica gel and the dried sample was loaded on the top. A protective layer (2 cm) was also added after loading the sample. The column was run with the mobile phase as given in against each fraction at the pressure of 2 bar. The volume of each fraction was 100 mL.
Solid phase extraction
Extracts that could not be fractionated by solvent-solvent extraction (Sol-Sol Ext) due to the formation of a heavy emulsion layer at the interface were fractionated using solid phase extraction (SP Ext). In this method, CME was dissolved in methanol and adsorbed on the same amount of silica gel. It was dried in a fume hood and loaded to a column. Then, this was extracted with an organic solvent with the order given in .
Bioassays
Inhibition of TNF-α induced NFκB
For these experiments, 293/NFκB-Luc HEK cells were purchased from Panomics (Freemont, CA, USA). Cells were maintained in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum and antibiotic. Cells were seeded into a sterile white-walled 96-well plate at 2 × 104 cells per 200 µL. After 48 h of incubation at 37°C and 5% CO2, medium was replaced and test samples were added at 20 µg/mL. TNF-α was added to a final concentration of 10 ng/mL. After 6 h of incubation, cells were washed in PBS, 50 µL of 1X reporter lysis buffer was added, and cells were subjected to one freeze/thaw cycle (−80°C/37°C). Inhibition was measured in a luminometer using the Luciferase Assay System from Promega (Madison, WI, USA) according to the manufacturer’s instructions. Data were calculated as % inhibition. The samples which showed more than 70% inhibition at 20 µg/mL were tested in dose dependence to find the IC50 (CitationHoshino et al., 2010). N-tosyl-L-phenylalanyl chloromethyl ketone (TPCK) and (E)-3-(4-Methylphenylsulfonyl)-2-propenenenitrile (BAY-11) were used as a positive controls and exerted inhibitory activity with IC50 values of 5.1 ± 1.6 and 2.01 ± 0.37 µM, respectively. The cytotoxic effects of these samples were measured by the sulforhodamine B (SRB) procedure described below.
Aromatase inhibition assay
Aromatase inhibition was assayed as previously reported (CitationMaiti et al., 2007). Briefly, test samples were preincubated with a NADPH regenerating system for 10 min at 37°C. The enzyme and substrate mixture was added, and the plate was incubated for 30 min at 37°C before quenching with NaOH. After termination of the reaction and shaking for 5 min, the plate was further incubated for 2 h at 37°C to enhance the ratio of signal to background. Fluorescence was measured at 485 nm (excitation) and 530 nm (emission). IC50 values and dose-response curves were based on two independent experiments performed in duplicate using five concentrations of test substance. Naringenin (IC50 = 0.23 µM) was used as a positive control.
Inhibition of LPS-induced NO production (nitrite assay)
This assay was performed as previously reported (CitationPark et al., 2011). RAW 264.7 cells (10 × 104 cells per well) in 10% FBS containing DMEM were seeded in 96-well plates and incubated for 24 h. Then, the media was replaced with 190 µL of 1% FBS-containing phenol red free DMEM and cells were treated with 10 µL of test samples in 10% DMSO for 15 min followed by 1 µg/mL of LPS treatment for 20 h. The amount of nitrite, the major oxidized metabolite of NO, was measured to evaluate the effects of samples on NO biosynthesis. Incubation media (100 µL) were transferred to 96-well plates and reacted with Griess reagent [90 µL of 1% sulfanilamide in 5% phosphoric acid, and 90 µL of N-(1-naphthyl) ethylenediamine] and the absorbance was measured at 540 nm. To test the cytotoxic affect of samples a SRB assay was run simultaneously according to the procedure described below. The samples which showed more than 70% inhibition at 20 µg/mL were tested at three fold serial dilution to find the IC50 values.
Retinoid X receptor responsive element: luciferase reporter gene assay
COS-1 cells (1 × 104 cells per well) were seeded in 10% FBS containing DMEM in 96-well plates and incubated for 24 h. Cells were transiently cotransfected with RXR responsive element encoding vector (pRXRE), human RXRα protein expressing vector (phRXRα), and Renilla reniformis luciferase vector (pRL-SV40) by using Lipofctamine™ 2000 transfection reagent. After 24 h incubation, cells were treated with samples and further incubated for 24 h. Cells were lysed and the RXRE transcriptional activities were determined by measuring the reporter luciferase activities using Dual-Luciferase® Reporter Assay System (Promega, Madison, WI). Results were presented as a relative value calculated by fold induction over control after normalizing ratios of firefly luciferase/Renilla luciferase. In this assay, the cut-off was set at 4-fold induction and 9-cis-retinoic acid was used as a positive control which showed 13.1-fold induction at 100 nM.
Quinone reductase assay
Hepa 1c1c7 (murine hepatoma) cells were used in this assay. Cells were plated with 200 µL per well of solution at 0.5 × 104 cells/mL in MEM-α (minimum essential medium) without ribonucleosides or deoxyribonucleosides, supplemented with antibiotic/antimycotic and 10% FBS (Gibco). Cells were incubated for 24 h in a CO2 incubator. After 24 h, medium was replaced with 190 µL of fresh medium and 10 µL of test samples were added for a final concentration of 20 µg/mL. After incubation for 48 h, digitonin was used to permeabilize cell membranes, and enzyme activity was measured by the reduction of 3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to a blue formazan. Production was measured by absorption at 595 nm (CitationSong et al., 1999). A total protein assay using crystal violet staining was run in parallel (CitationSu et al., 2004). 4′-Bromoflavone (CD = 0.01 µM) was used as a positive control. Those samples which showed an induction ratio >2 at 20 µg/mL were tested in five-fold serial dilutions to determine the CD values.
SRB assay
The cytotoxic potential of test samples towards hormone responsive breast cancer cell line MCF-7 (ATCC number is HTB-22), human lung carcinoma cells LU-1 (established at the Department of Surgical Oncology University of Illinois at Chicago College of Medicine, Chicago, IL), and estrogen receptor negative breast cancer cell line MDA-MB-231 (ATCC number is HTB-26), was determined by using a SRB colorimetric assay as described previously (CitationYou et al., 1995). Briefly, 10 µL of various concentrations of test samples in 10% DMSO in PBS were transferred to 96-well plates along with 190 µL of cells (5 × 104 cells/mL) and incubated for 72 h at 37°C in a CO2 incubator. The incubation was stopped with the addition of 50 µL cold 20% trichloroacetic acid. The cells were washed, air-dried, and stained with 0.4% SRB in 1% acetic acid for 30 min at room temperature. Wells were then washed four times with 1% acetic acid, and the plates were dried overnight. Bound dye was solubilized with 200 µL 10 mM Tris base, pH 10, for 10 min on a gyratory shaker. Optical density was measured on a micro-plate reader (Bio-tek) at 515 nm and percent survival was determined. In each case, a zero-day control was performed by adding an equivalent number of cells to sixteen wells, incubating at 37°C for 30 min, and processing as described above. Percent of cell survival was calculated.
Results
Different plant parts were extracted separately by maceration in methanol and then the crude methanol extract was further fractionated by Sol-Sol Ext, flash column chromatography (FCC) and SP Ext (). These crude extracts and fractions, 59 in total, were subjected to a battery of cancer chemopreventive and anticancer assays ().
Table 3. Bioassay results.
In the TNF-α activated NFκB inhibition assay, a total of nine samples, including the crude extract and fractions of W. coagulans, showed more than 50% inhibition at 20 µg/mL. Out of these nine samples, two fractions (sample 51 and 52) showed more than 90% inhibition without mediating a cytotoxic effect. The IC50 values of these samples were 2.6 and 4.3 µg/mL, respectively () and 100% of cells survived at the IC50 concentration.
In the aromatase inhibition assay, a total of six samples showed more than 50% inhibition at 20 µg/mL. One fraction from E. wallichii (10) and one fraction from W. coagulans (50) showed 73.4 and 100% inhibition, respectively, at 20 µg/mL, and with corresponding IC50 values of 17.7 and 17.0 µg/mL ().
In the LPS-induced NO inhibition assay (nitrite assay), a total of 13 samples showed more than 70% inhibition at a concentration 20 µg/mL, including the crude extract and fractions of E. wallichii, A. oblongifolium, A. thomsonii, and W. coagulans. However, no considerable inhibitory activity was detected in fractions from the rhizome of B. ciliate or the fruit of O. dillenii. Notably, the ethyl acetate fraction obtained from aerial parts with the fruit of W. coagulans (sample 51) showed the most potent inhibition in nitrite production. A total of 18 samples showed more than 50% inhibition at 20 µg/mL (). The samples which showed ≥70% inhibition or cytotoxicity at 20 µg/mL were further studied to find IC50 values. Three samples (50, 51 and 52) were cytotoxic at 20 µg/mL and, in dose-dependent experiments, IC50 values of 3.1, 1.3, and 3.0 µg/mL, respectively, were observed, in the absence of cytotoxicity.
In the Quinone reductase (QR1) assay, after the initial testing of 59 samples, 14 samples were selected to determine CD values (concentration required to double the QR1 activity). These samples showed either an induction ratio (IR) >2 or cytotoxicity with cell survival ≤50% at 20 µg/mL (). Based on dose-dependent experiments, 10 samples had a CD value less than 10 µg/mL with no cytotoxicity at the CD, including the two most potent fractions (sample 51 and 52) with CD values of 1.0 and 1.1 µg/mL, respectively. The other four samples (9, 10, 35 and 48) showed cytotoxicity at the CD.
In the RXRE assay, the highest induction was observed in samples 31 and 52 (1.6-fold induction), followed by sample 13 (1.5-fold), samples 2, 5, and 51 (1.4-fold), samples 14, 15, 41 and 57 (1.3-fold), samples 32 and 34 (1.2-fold) and samples 8, 12, and 37 (1.1-fold) at concentration 20 µg/mL. Since none of the extracts/fractions showed ≥2-fold induction, they were not considered active. 9-cis-Retinoic acid, which was used as a positive control, showed 13.1-fold induction at 100 nM.
Finally, in an SRB assay, six samples (9, 10, 11, 50, 51 and 52) showed significant anti-proliferative activity against MCF-7 cancer cells with IC50 values ranging from 1.2 to 7.5 µg/mL (). Three samples (10, 11 and 51) showed significant anti-proliferative potential against LU1 cancer cells and had IC50 values ranging from 3.1 to 7.1 µg/mL. Seven extracts/fractions (samples 9, 10, 47, 50, 51, 52, and 53) showed anti-proliferative activity against MDA-MB-231 cancer cells and had IC50 values ranging from 2.5 to 7.8 µg/mL ().
Discussion
Terrestrial plants have played a leading role in the search for new therapeutics and pharmacological drugs. They have been a rich source of lead compounds for cancer treatment and chemoprevention (e.g., vinblastine, vincristine, taxol, camptothecine and its analogs, etoposide and its analogs, etc.) with diverse modes of action. TNF-α is an activator of NFκB, an inducible transcription factor that plays an important role in the regulation of apoptosis, cell differentiation, and cell migration. Its activation may promote cell proliferation and further prevent programmed cell death through transcriptional activation of genes that suppress apoptosis (CitationBaldwin, 2001; CitationKarin, 2006). As NFκB is an important regulator in cell fate decisions, such as programmed cell death, proliferation control, and cell invasion, it is critical in tumorigenesis. Inhibition of NFκB signaling has potential applications for the prevention or treatment of cancer (CitationAggarwal et al., 2004; CitationPezzuto et al., 2006; CitationSchupp et al., 2009; CitationLuqman & Pezzuto, 2010). The anti-inflammatory properties of many natural products and their abilities to inhibit the immune response upon exposure to a variety of external stresses may result from inhibition of the activation of NFκB by these external signals. Emerging evidence that an effective inhibition of NFκB signaling seems to be central to many of the observed anticancer properties of natural products supports the importance of screening of extracts/fractions for NFκB inhibition (CitationKondratyuk & Pezzuto, 2004; CitationPezzuto et al., 2006).
In this study, nine fractions from W. coagulans showed various levels of inhibition of TNF-α activated NFκB. Among them, the most potent fractions (samples 51 and 52) had IC50 values of 2.6 and 4.3 µg/mL, respectively. In the Indian subcontinent, two species of the genus Withania are found, W. somnifera and W. coagulans (CitationMaurya, 2010). These plants are a rich source of steroidal lactones called withanolides. NFκB inhibitory activity of 11 withanolides isolated from W. somnifera has been described by CitationIchikawa et al. (2006), but these compounds are not reported in W. coagulans. To our knowledge, this is the first report demonstrating the NFκB inhibitory activity of W. coagulans and the possible presence of withanolides similar to those isolated from Withania somnifera.
Aromatase is a cytochrome P450 enzyme complex responsible for the conversion of androgens to estrogens (CitationJongen et al., 2005). Estrogens are involved in many physiological processes, including the growth and maintenance of the female sexual organs, the reproductive cycle, and different neuroendocrine functions. These hormones also have critical roles in certain disease states, particularly mammary and endometrial cancers (CitationBrueggemeier et al., 2005; CitationMaiti et al., 2007). Aromatase inhibitors can block the production of estrogen, which in turn can reduce the growth of estrogen receptor positive breast cancer cells. Aromatase inhibitors are already in clinical use for the treatment of breast cancer and animal studies have demonstrated their potential as chemopreventive agents (CitationLubet et al., 1994; CitationGunson et al., 1995). Clinical aromatase inhibitors are also prescribed for chemoprevention in breast cancer survivors. Six samples from E. wallichii, A. oblongifolium, A. thomsonii and W. coagulans showed variable levels of potential as aromatase inhibitors. There is no earlier report about the aromatase inhibitory activities of any extracts or fractions of these plants.
On the basis of in vitro and in vivo studies which showed a consistent relationship between up-regulation of iNOS and cancer promotion (Park & Pezzuto, Citation2002b; CitationSharma et al., 2002; CitationCrowell et al., 2003; CitationNomelini et al., 2008), inhibition of NO production catalyzed by iNOS in LPS-activated RAW 264.7 cells was determined. A total of 18 extracts/fractions from four plants showed remarkable inhibition. Extracts/fractions of both roots and aerial parts of E. wallichii inhibited NO production significantly. A literature survey showed very few reports concerning the biological screening of root extracts of E. wallichii (CitationHaq et al., 2012), but no report about the biological activities of aerial parts or anti-inflammatory or cancer chemopreventive activity was found. There is no report about the NO inhibitory activity of A. oblongifolium or A. thomsonii, and here we found that one fraction from each plant inhibited NO production significantly. In the case of W. coagulans, the extracts of aerial parts are more potent inhibitors of NO production than the fruits, demonstrating the variation in concentration of bioactive components in different plant parts (CitationDalavayi et al., 2006) as well as possible synergistic effects. Our results indicated that some fractions showed inhibition of NFκB and NO production, which is correlated with the inhibitory pattern of withaferin A as reported by CitationOh et al. (2008). There is no earlier report about the inhibitory potential of NO production of W. coagulans, and it remains to be confirmed whether withanolides from this plant are responsible for these activities.
Strategies for protecting cells from cancer initiation events include decreasing metabolic enzymes involved in generating reactive species (phase 1 enzymes), while increasing phase II enzymes that can deactivate radicals and electrophiles known to interfere with normal cellular processes (CitationPark et al., 2001; CitationPark & Pezzuto, 2002a; CitationZhang & Gordon, 2004; CitationCuendet et al., 2006; CitationPezzuto et al., 2006; CitationYang & Liu, 2009). Quinone reductase 1 is phase II enzyme, induction of which has been demonstrated to coincide with the overall elevation of phase II enzyme levels. QR1 is a convenient biomarker because it is widely distributed in mammalian tissues, can be easily measured, and shows a large inducer response (CitationSu et al., 2004; CitationCuendet et al., 2006). Induction of QR1 is suggestive for cancer prevention potential of samples tested (CitationPark et al., 2001; CitationSu et al., 2004; CitationCuendet et al., 2006). We studied the effect of different plant extracts/fractions for the induction of QR1 enzymes with cultured Hepa 1c1c7 (murine hepatoma) cells. Results indicated that two fractions from E. wallichii aerial parts showed induction of QR1, with CD values of 18.1 and 20.6 µg/mL, although they also showed cytotoxicity at the CD. One fraction each from A. oblongifolium and A. thomsonii showed QR1 induction with CD values of 9.6 and 10.8 µg/mL, respectively. There is no report in the literature about the QR1 induction potential of these plants. A total of 10 fractions from W. coagulans showed significant induction of QR1, which may indicate the presence of potent withanolides. QR1 induction activity of withanolides from different plants has been reported (CitationKennelly et al., 1997; CitationMisico et al., 2002; CitationSu et al., 2004), but until now, evaluation of QR1 induction activity of W. coagulans has not been reported.
Retinoids are derivatives of vitamin A that affect cellular proliferation, differentiation, and apoptosis in a retinoid-specific and cell-type specific manner (CitationZhang & Jetten, 1997). Retinoids have shown efficacy as anticancer drugs that intervene in the carcinogenic process by regulating proliferation and differentiation at several stages (CitationWu et al., 2002). Retinoid receptors belong to the family of nuclear hormone receptor proteins. A cell line-based retinoid X receptor responsive element (RXRE)-luciferase reporter gene assay (CitationPark et al., 2011) was employed in this study to evaluate the interaction of extracts/fractions with RXR receptors but none of the extracts/fractions showed significant activity.
Inhibition of cell proliferation of cancer cell lines is one of the most commonly used methods to study the effectiveness of any anticancer agents. In the present study, three cancer cell lines (MCF-7, MDA-MB 231 and LU-1) were used to determine antiproliferative potential of selected samples. Three fractions from E. wallichii inhibited the proliferation of MCF-7. Of these three, two fractions also showed significant inhibition against LU-1 and MDA-MB 231 cancer cell lines. There has been no report about the inhibitory potential of the E. wallichii against cancer cell lines. These data demonstrate the potential of this plant to inhibit cancer cell growth.
In sum, we have shown that extracts/fractions of E. wallichii possess significant activity against aromatase, iNOS and cancer cell proliferation. The results of this screening showed that E. wallichii is a good choice for further isolation of cancer chemopreventive and cytotoxic compounds.
CitationInayatullah et al. (2007) reported the antitumor potential of A. oblongifolium using the brine shrimp toxicity assay (ED50 226.8 µg/mL) and potato disc tumor inhibition assay (59% inhibition at 10 µg/mL). We tested the methanol extract of A. oblongifolium and its fractions to evaluate cytotoxicity with MDA-MB 231, MCF-7, and LU-1 human cancer cell lines at 20 µg/mL, and no significant inhibition of proliferation was observed. Overall, one fraction of A. oblongifolium showed significant inhibition of aromatase and nitric oxide production, as well as induction of QR1. This suggests A. oblongifolium may have cancer chemopreventive potential.
Previously, CitationBibi et al. (2011) reported the antitumor and cytotoxic potential of the crude methanol extract of A. thomsonii and its n-hexane and aqueous fractions, using the potato disc antitumor assay and SRB cytotoxicity assay with H157 and HT144 human solid tumor cell lines. They reported that the aqueous fractions had the most potent activity among all the tested samples. Here, we tested the same samples against MDA-MB 231, MCF-7, and LU-1 human cancer cell lines, and only the n-hexane fraction showed >50% proliferation inhibition of MCF-7 at 20 µg/mL. These results suggest A. thomsonii extracts have selective inhibitory potential against different cell lines and active compounds may be isolated from the n-hexane as well as the aqueous fraction in the future.
CitationChavez-Santoscoy et al. (2009) reported cytotoxicity of nine plants of Opuntia spp. against four human cell lines: Caco-2, PC3, HepG-2, and MCF-7. In the same assays, O. dillenii demonstrated no activity. We tested the methanol extract of O. dillenii and its fractions against MCF-7, MDA-MB 231, and LU-1 and found no significant proliferation inhibition, which is in accordance with CitationChavez-Santoscoy et al. (2009). We did not find significant activity of the O. dillenii fruit extract and fractions in any of the assays performed in this study.
Withanferin A, a withanolide found in W. coagulans, is reported to possess cytotoxic activity (CitationMaurya, 2010). Many other cytotoxic withanolides have been isolated from different plants including Physalis angulata (CitationHe et al., 2007), Tubocapsicum anomalum (CitationHsieh et al., 2007), Datura metel (CitationPan et al., 2007), Acnistus arborescens (CitationMinguzzi et al., 2002), and Physalis peruviana (CitationLan et al., 2009), but W. coagulans remained relatively unexplored for cytotoxic activity. We tested W. coagulans extracts and fractions against human tumor cell lines, and many fractions showed a significant antiproliferative effects with IC50 values ranging from 1.2 to 7.8 µg/mL, much lower than some purified withanolides. This study supports a more detailed evaluation of W. coagulans for the isolation of cytotoxic compounds. In addition, extracts/fractions of W. coagulans showed significant activity in all the assays except the RXRE-luciferase reporter gene assay.
Except B. ciliata and O. dillinii, each of the plant species evaluated in this investigation showed significant activity in one or more assays, and these plants may be promising sources of cancer chemopreventive and cytotoxic compounds. These data support the notion of selecting plants on the basis of enthnomedical data and set the stage for ongoing bioactivity-guided isolation of the active compounds.
Conclusion
This study provides new information about six ethanobotanically important, naturally growing plants of Pakistan. Two fractions from W. coagulans inhibited TNF-α induced NFκB activity with IC50 values of 2.6 and 4.3 µg/mL. One fraction from E. wallichii and one fraction from W. coagulans inhibited aromatase with IC50 values of 17.7 and 17.0 µg/mL, respectively. In the nitrite assay, five fractions from E. wallichii (IC50 values ranging from 4.8 to 15.6 µg/mL), one fraction from A. oblongifolium (IC50 12.5 µg/mL), one fraction from A. thomsonii (IC50 12.5 µg/mL) and six fractions from W. coagulans (IC50 values ranging from 1.3 to 11.7 µg/mL) exhibited significant activity. Two samples from E. wallichii, one from A. oblongifolium, one from A. thomsonii, and ten from W. coagulans were found to induce QR1 expression. In an SRB assay, three samples each from E. wallichii (IC50 values ranging from 2.9 to 7.5 µg/mL) and W. coagulans (IC50 values ranging from 1.2 to 3.4 µg/mL) showed cytotoxicity with MCF-7 cells in culture. Two samples from E. wallichii (IC50 5.1 and 7.1 µg/mL) and one from W. coagulans (IC50 3.1 µg/mL) showed cytotoxicity with LU-1 cells in culture. Two samples from E. wallichii (IC50 2.5 and 3.4 µg/mL) and five from W. coagulans (IC50 values ranging from 4.2 to 7.8 µg/mL) showed cytotoxicity with MDA-MB 231 cells in culture. Among the six plants, W. coagulans showed the highest anticancer and chemopreventive potential with significant activities in multiple assays, followed by E. wallichii. These two plants are attractive candidates for further investigation.
Acknowledgements
We acknowledge the field staff and taxonomists particularly, Nazif Ullah and Prof. Dr. Rizwana Aleem Qureshi, for their help during plant collection and identification. This work was presented in part as an abstract at the 51st Annual Meeting of the American Society of Pharmacognosy and the Phytochemical Society of North America (2010), St. Petersburg Beach, Florida, USA.
Declaration of interest
The authors are grateful for funding provided by the Higher Education Commission Pakistan, under the “International Research Support Initiative program” and the National Cancer Institute, USA (P01 CA48112).
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