Extraction and sample preparation

All crude plants were weighed (0.25 g), pulverized, macerated/homogenized and extracted in 1000 μL of absolute ethanol for 7 days at 4 °C (Chakraborty et al., 2004). A stock solution for each extract was subsequently prepared by dilution to 10 mL with HBSS + 5 mM (N-[2-hydroxyethylpiperazine-N′-[2-ethanesulfonic acid]) (HEPES), pre-adjusted to a pH of 7.4 with 0.1 N NAOH. Six serial dilutions of each experimental extract were prepared from the stock solution in order to span a 1000-fold concentration gradient with the highest final plating concentration set at 5 mg/mL (w/v).

Cell culture

Neuro-2a cells were used in this study to screen for tumoricidal effects, as the cell line was established from a malignant spontaneous tumor and previously deemed an appropriate model to evaluate potential chemotherapy drugs for the treatment of cancer (Finklestein et al., 1975; Klebe and Ruddle, 1969; Mazzio et al., 2003). Briefly, N-2A cells were cultured in DMEM + phenol red supplemented with 10% FBS, 4 mM L-glutamine, 20 μM sodium pyruvate and penicillin/streptomycin (100 Units/0.1 mg, mL). The cultures were maintained at 37 °C in 5% CO₂/atmosphere and co-cultured every 2-3 days. Experimental plating media consisted of DMEM (-phenol red) supplemented with 1.8% FBS, penicillin/streptomycin (100 Units/0.1 mg/mL), 20 μM sodium pyruvate and 4 mM L-glutamine. The cells were plated in 96-well plates at a density of approximately 0.5 × 10⁶ cells/mL. A first tier screening was conducted at final plating concentrations of 0.5-5.0 mg/mL to identify extracts that exhibit an IC₅₀ less than 5.0 mg/mL. A second and third tier screening was conducted using the same procedure at lower concentrations in order to establish an accurate LC₅₀ for those demonstrating toxicity at less than 0.5 mg/mL.

Evaluation of cellular toxicity

Cell viability was assessed by resazurin-almar blue indicator dye as described previously (Mazzio et al., 2003). Experimental blanks and extract controls were run simultaneously with samples, in order to detect any interferences or reactivity with the dye or cell viability. Briefly, almar blue was dissolved in sterile PBS (0.5 mg/mL) and the cell viability was assessed by quantifying the reduction of the dye to its corresponding fluorescent intermediate - resorufin. The use of fluorescence for cell viability eliminates significant interferences introduced by experimental compounds themselves, otherwise presented during UV detection using spectrophotometric dyes. The fluorescence intensity was analysed using a microplate fluorometer - Model 7620 version 5.02 (Cambridge Technologies Inc, Watertown, Mass) with settings held at [550/580], [excitation/emission].

Evaluation of cell death

Propidium iodide (PI) was used to corroborate viability studies with the most potent extracts. PI is nucleic acid stain which represents cell death as described previously (Mazzio et al., 2003). Samples were analysed photographically using an Olympus IX-70 inverted microscope and images were captured using a MD35 Electronic Eyepiece (Zhejiang Jincheng Science and Technology Co., Ltd, China) with analysis performed using C-imaging systems confocal PCI-Simple software (Compix Inc. Cranberry Township, PA, USA).

Most potent plant extracts

Wild yam root (Dioscorea villosa) was found to be the most potent herbal extract with an LC₅₀ = 0.019 mg/mL. Although there is little or no research regarding the use of this herb in experimental investigation of malignancy, wild yam root is known to contain indigenous plant phytosterol estrogens such as diosgenin, which espouse therapeutic effects on female hormonal regulation and alleviate the symptoms of menopause. However, diosgenin is known to induce antiproliferative pro-apoptotic effects in a diverse range of tumor cells by arresting G2/M, down-regulating NF-kappa B, Akt, cyclin D, c-myc and initiating PARP cleavage/DNA fragmentation (Shishodia and Aggarwal, 2006; Leger et al., 2006; Liagre et al., 2005). This herb root is also known to contain sapogenins, phytosterols, beta-sitosterol and campesterol, which may contribute in part to the tumoricidal properties (Dr Dukes Chemical Database). Overall, the American Herbal Products Association - Botanical Safety Handbook (AHPA-BSH) has defined wild yam root as a Class 1 herb (safest herb category), further defined as `herbs which can be safely consumed when used appropriately' (McGuffin, 1997). However, caution should be advised since it has been reported that administration in animals while safe at 0.5 mg/kg, when administered at 2 g/kg can induce hypoactivity, piloerection, dyspnea and death (No authors, 2004). In this study, bloodroot extract (Sanguinaria canadensis) was the second most potent (LC<sub>50</sub> = 0.04 mg/mL). However, controversy surrounds its use as a primary ingredient in the marketed antiskin cancer salve sold under the name `black salve' or `can-x'. While there are few or no established research studies which investigate the anticancer effects of bloodroot, the inherent constituents of the plant such as sanguinarine (a toxic benzophenanthridine alkaloid) are known to incur major side effects such as tissue corrosion, scarring and possible worsening of basal cell carcinoma to an aggressive malignant form (McDaniel and Goldman, 2002). On the other hand, the therapeutic effects of sanguinarine and sanguinaria have been shown to combat gingivitis and plaque and are deemed relatively safe for the intended purpose, also having been approved by the FDA for commercial products manufactured by the Colgate-Palmolive Company (Frankos et al., 1990). While administration of sanguinarine is fairly safe at very low concentrations, at high concentrations it is deadly given its ability to inhibit Na⁺K⁺ATPase in cardiac tissue in a similar fashion to ouabain (Seifen et al., 1979). Further, bloodroot also contains the toxic alkaloid berberine, which can lead to adverse side effects on cardiac function, respiration, blood pressure and the use of this herb is contraindicated during pregnancy (McGuffin, 1997). Bloodroot is also known to contain a range of benzophenanthridine alkaloids, isoquinoline alkaloids, chelerythrine, sanguidimerine, protopine and trace elements which vary with the seasonal harvest and plant distinction (Graf et al., 2007). The dangers associated with this herb may in fact outweigh its therapeutic benefits.

Unlike wild yam and bloodroot, teasel root (Dipsacus asper) (LC₅₀ = 0.042 mg/mL) is often used by consumers at appreciably high dose without reported side effects. Teasel root is generally known to treat lime disease, fibromyalgia and serve as a general cleanser for the liver, kidney, digestive and circulatory systems, representing overall positive effects on the central organs. While there is no experimental research investigating teasel root's anticancer effects, a recent study has suggested that its antioxidant properties may contribute to a potential treatment for Alzheimer's disease (Zhang et al., 2003). Ethanol extracts of Dipsacus asper are known to contain phenolic acids including caffeic acid, 2,6-dihydroxycinnamic acid, vanillic acid, 2′-O-caffeoyl-D-glucopyranoside ester, and caffeoylquinic acid, iridoid glucosides, triterpenoids oleanic acid and akebiasaponin D (Tian et al., 2007). Teasel root is commonly sold with a suggested product dose of 6-21 g/day and categorized as an AHPA-BSH `Class 1 herb`; therefore this herb holds considerable promise as a holistic anticancer CAM (McGuffin, 1997). There is also a complete nonappearance in the scientific literature regarding investigative applications for balm of gilead bud (Populus balsamifera) (LC₅₀ = 0.078 mg/mL). Historically, this herb was used by Native Americans for the treatment of general maladies including urinary tract infection, wounds, colds, arthritis, pains and coughs and as an insect repellent. Balm of gilead bud has also been approved as `safe' by the German Commission E for topical applications only; including skin disease, hemorrhoids, frostbite and sunburn (Bundesinstitut fur Arzneimittel und Medizinprodukte, 1998). However, alcohol extracts of balm of gilead bud are sold by a number of manufacturers with instructions for internal use up to 2 mL/day, with no known reported side effects. While this is a AHPABSH `Class 1 herb' indicating safety, the German Commission E cautions use by individuals who demonstrate allergic responses to propolis or aspirin (salicylic acid), this showing a greater percentage in those who suffer from hives, asthma and sinus infections (McGuffin, 1997; Bundesinstitut fur Arzneimittel und Medizinprodukte, 1998). In addition, extracts derived from this species of plant are known to contain beta-eudesmol, alpha-eudesmol, gamma-selinene, cadinene, alpha-elemene, hemiterpenes, sesquiterpenes, alcohols, carbonyl compounds, hydroxyacids, aliphatic acids, phenolcarboxylic acids and substituted cinnamic acids (Jerkovi and Mastelic, 2003; Isidorov and Vinogorova, 2003).

Interestingly, the three biblical herbs: balm of gilead bud, frankincense and myrrh show significant tumoricidal effects as demonstrated in this study. The reported use of frankincense (Boswellia carteri) dates back to 3000 B.C. primarily used as an incense until the year 200 AD, where its use expanded to medicinal purposes for the treatment of gout, ulcers, oral health and also for the production of manufactured plasters (Hillson, 1988). The potential for frankincense as an anticancer agent in this study (LC₅₀ = 0.081 mg/mL) corroborates previous reports, where the extract is able to inhibit topoisomerase II in mouse leukemia L1210 cells in a similar fashion to etoposide and aclarubicin (Wang et al., 1991). This species of plant is known to contain plant chemical constituents including acetyl-alpha-boswellic acid, acetyl-beta-boswellic acid, lup-20(29)-ene-3 alpha-acetoxy-24-oic acid, alpha-boswellic acid, beta-boswellic acid and acetyl-11-keto-betaboswellic acid (Zhou and Cui, 2002). The boswellic acid pentacyclic triterpenes derived from the gum resin are more potent than campthothecin, amsacrine or etoposide in inhibiting human topoisomerases I and II alpha, through high-affinity binding sites (Syrovets et al., 2000). Boswellic acids also exert direct antiproliferative/pro-apoptotic effects through activation of caspase-3/8/9 and PARP cleavage in HT-29 cells (Liu et al., 2002), human leukemia cells HL-60, K 562, U937, MOLT-4, THP-1 and brain tumor cells LN-18, LN-229 (Hostanska et al., 2002). Due to the historical use of frankincense, and the AHPA-BSH classification of this herb as a Class 1 herb, this could also be potential anticancer CAM (McGuffin, 1997). Boswellia serrata extract is available and sold in capsule form with a suggested intake of approximately 400 mg (3× daily), with no known side effects having been reported.

There is also extended historical information regarding the use of myrrh (Commiphora molmol) dating back centuries for treatment of infection, pain, swelling, leprosy and halitosis. The present study shows tumoricidal effects by myrrh gum (LC₅₀ = 158 μg/mL) corroborating previous reports that it induces apoptosis in lung, pancreas, breast and prostate cancer cell lines with an LC₅₀ less than 500 μg/mL in vitro (Shoemaker et al., 2005). In animal studies, its administration induces tumoricidal effects equal to cyclophosphamide in Ehrlich-solid-tumorbearing mice (Qureshi et al., 1993). In terms of safety, it should be noted that lethal effects have been observed at high concentration, where oral intake of 1-5 g/kg day induces jaundice, ataxia, dyspnea, soft feces, hepatonephropathy, leucopenia macrocytic anemia hemorrhagic myositis and death in mammals (Omer et al., 1999). In humans, oral intake of myrrh was tolerated for 3 days (10 mg/kg) in experimental treatment of schistosomiasis without adverse affects on liver/kidney function (Sheir et al., 2001). While the Commission E has approved myrrh for topical treatment of neck and mouth inflammations, this is nonetheless a AHPA-BSH Class 2 herb where excessive use can lead to irritation of the kidneys and it is counter-indicated during pregnancy (McGuffin, 1997; Bundesinstitut fur Arzneimittel und Medizinprodukte, 1998). Extracts of myrrh contain a diverse range of constituents including selinene, furanodiene, β-selinene, lindestrene, furanoeudesma-1,3-diene, β-elemene, γ-elemene, δ-elemene, elemol, isofuranogermacrene, curzerenone, α-cubebene and β-bourbonene (Hanus et al., 2005).

Potent plant extracts with little to no reported side effects

Briefly, the data in this study show promising results for several other plants which are not associated with known side effects when used in humans some of which include: Bakuchi seed (Cyamopsis psoralioides) (LC₅₀ = 0.102 mg/mL) typically used for the treatment of leprosy, jaundice, infections, tumors, baldness, snake and scorpion bites; Dichroa root (Dichroa febrifuga) (LC₅₀ = 0.107 mg/mL), an antimalarial agent and Kanta kari (LC₅₀ = 0.157 mg/mL) commonly used for treatment of asthma and respiratory infections of which its clinical and experimental use in animals is effective for intended purpose without side effects (Govindan et al., 2004). Another popular plant extract showing a low LC₅₀ = 0.235 mg/mL was Garcinia fruit (Garcinia cambogia), effects of which has been reported in the scientific literature. Garcinia fruit contains prenylated xanthones in part responsible for inhibiting pre-neoplastic lesions in mammary/colon cancer (Nabandith et al., 2004) and induces apoptosis in mouth, leukemia, breast, gastric and lung cancer cell lines in vitro (Suksamrarn et al., 2006; Matsumoto et al., 2003). Indigenous xanthones such as gambogic acid can down-regulate c-MYC mRNA expression/telomerase reverse transcriptase gene, disrupting cell proliferation and the immortalization of human cancer cells (Liu et al., 2005; Zhang et al., 2004) also blocking tumor growth of SMMC-7721 transplanted carcinoma in vivo (Guo et al., 2004).

Another plant constituent is garcinol (a polyisoprenylated benzophenone within the plant) known to impair cell proliferation, cancer cell migration, cell adhesion and cancer cell viability, via inhibiting MAPK/ERK, PI3K/Akt, the phosphorylation of membrane focal adhesion kinase and augmenting BAX, caspase 2/3 activation, release of cytochrome C and PARP-1 cleavage in a diverse range human cancer cell lines (Liao et al., 2005; Pan et al., 2001). The recommended dosage for oral intake of Garcinia cambogia is estimated at up to 6 g/day. There is significant potential for this fruit, since it is well tolerated at significant doses also sold as a popular fruit juice under the tradename Xango®. Vitex (Agnus castus) (LC₅₀ = 0.302 mg/mL), also known as chasteberry is used traditionally for regulating homeostasis of women reproductive physiology and ameliorating the symptoms of PMS, amenorrhea, infertility and meno-pause. Like wild yam, the mechanism of action for vitex may involve its phytoestrogenic fraction where the extract (<100 μg/mL) is capable of inducing cell death in a diverse range of tumor cell lines including ovarian, cervical, breast, gastric, colon and lung via induction of pro-apoptotic caspase 3, 8 9 HO oxidase, a reduction in BCL-2 Bcl-XL and Bid protein, the increase in Bad gene expression and induction of DNA fragmentation (Ohyama et al., 2003, 2005; Liu et al., 2002). Given that low doses can yield significant tumoricidal properties, with relatively mild or reversible side effects with no reported drug interactions, this herb shows some promise.

The Commission E has approved its use for `irregularities of the menstrual cycle, premenstrual complaints and mastodynia. Unless otherwise prescribed, 30-40 mg (0.03-0.04 g) per day of crushed fruit for aqueous-alcoholic extracts in dry or fluid form is acceptable'. However, this is a AHPA-BSH Class 2b herb also known as an emmenagogue herb, where it can counteract with the efficacy of birth control pills, and should not be used during pregnancy due to its has effects on uterine contraction (McGuffin, 1997; Bundesinstitut fur Arzneimittel und Medizinprodukte, 1998). Rosemary leaf (Rosmarinus officinalis) (LC₅₀ = 0.299 mg/mL) also has a long history where its use involves application for cooking and medicinally to treat muscle pain, indigestion, arteriosclerosis, alopecia and bacterial infections. Extract of rosemary increases sensitivity and prevents efflux of chemotherapeutic agents in drug resistant MCF-7 human breast cancer cells (Plouzek et al., 1999) and inhibits 7,12-dimethylbenz[a]anthracene induced mammary tumorigenesis in female rats (Singletary et al., 1996). Inherent active constituents such as carnosol (a phenolic compound extracted from rosemary) are found to be lethal against acute lymphoblastic leukemia cells (Dorrie et al., 2001) and colon cancer in vivo (Moran et al., 2005). Due to the historical use of rosemary, and the suggested use of 4- 6 g/day, this herb could be proving significant as an anticancer CAM.

The Commission E `approved the internal use of rosemary leaf for dyspeptic complaints and external use as supportive therapy for rheumatic diseases and circulatory problems. No reported drug interactions, side effects and recommended use is 4-6 g of cut leaf for infusions, powder, dry extracts and other galenical preparations for internal and external use' (Bundesinstitut fur Arzneimittel und Medizinprodukte, 1998). AHPA-BSH has classified rosemary as a Class 2b/emmenagogue herb where it can stimulate uterine contraction and induce miscarriage, establishing counter-indication of use during pregnancy. In contrast to ethanol extracts, the oil of rosemary can have dangerous side effects including nausea, vomiting, seizure and pulmonary edema. Ginger (Zingiber officinale) (LC<sub>50</sub> = 0.477 mg/mL) has been used for centuries as a cooking spice and medicinally demonstrates a diverse range of applications having biological properties such as the ability to modulate platelet aggregation, serve as an analgesic, antiinflammatory, hypoglycemic, antimicrobial, antiparasitic activity and antioxidant, also commonly used for nausea. Ginger and its constituents induce apoptosis in human cancer cell lines (Wei et al., 2005) and display anticancer properties against spontaneous tumors in animal models (Nagasawa et al., 2002). In humans, the administration of ginger up to 6 g/day is relatively safe, yielding few side effects with the exception of a few subjects who experience nausea and drowsiness. The Commission E `approved the internal use of ginger for dyspepsia and prevention of motion sickness. Powdered rhizome, 0.25 -1.0 g, three times daily' (Bundesinstitut fur Arzneimittel und Medizinprodukte, 1998). AHPA-BSH recommends not exceeding the recommended 2-4 g/day, also warning against long term use or using during pregnancy (McGuffin, 1997).

Potent plant extracts lacking reported safety evaluation

Dryopteris Male Fern Rhizome (Dryopteris crassirhizoma) (LC₅₀ = 0.232 mg/mL) has been used historically to treat tapeworms and influenza. The plant contains kaempferol glycosides which are known to impair DNA polymerase which could possibly contribute in part to its anticancer effects. Alcohol extracts of this plant inhibit fatty acid synthase at low concentrations (50 μg/mL), an enzyme highly expressed in cancer tissue, and can antagonize the growth of cancer through down-regulation of PI3K/AKt and JNK pathways, S-phase arrest and induce apoptosis in cancer cell lines (Na et al., 2006; Zhao et al., 2006; Chiang et al., 2005). However, with little research to support safety and indication as to potential side effects of this herb it will require further research. This study also examines the resin from the fruit of Dragon's blood (Calamus draco) (LC₅₀ = 0.242 mg/mL). Dragon's blood has a potential application for cancer where an extract of Daemomorops draco contains the active constituent (dracorhodin) which induces proapoptotic tumoricidal affects in vitro (Xia et al., 2006). Although the traditional use of this resin has been as a coloring agent in varnishes, lacquers and plasters and incense, relatively few studies have been conducted to investigate potential safety issues with internal use. Red sandalwood (Pterocarpus santalinusis) (LC₅₀ = 0.326 mg/mL) has generally proven effective in experimental models of diabetes and wound healing. However, the use of sandalwood oil is known to prevent 7,12-dimethylbenz(a)anthracene-(DMBA)-initiated and 12-O-tetradecanoyl phorbol-13-acetate (TPA)-promoted skin papillomas in mice (Dwivedi and Abu-Ghazaleh, 1997). Red sandalwood is an AHPA-BSH `Class 1 herb', with little known about the side effects associated with use, independent from reported topical dermatitis in susceptible individuals (McGuffin, 1997). However, until further studies are conducted to determine safety regarding internal use, this herb warrants caution with use.

Bushy Knotweed Rhizome (Polygonum Cuspidatum) (LC₅₀ = 0.361 mg/mL) is far from an endangered species, classified as an unwanted invasive, noxious weed which grows aggressively, survives in adverse climates and dominates ever expanding habitation and vegetative life, even to the point of creating an economic threat. Yet this is a plant that may offer of superb anticancer properties. Previously reported medicinal properties include its use as a potential cholesterol lowering agent, antibacterial, antiviral/HIV and estrogenic agent. Although the extract of this plant has not yet been examined for its anticancer properties, 3,4′-dimethoxy-5-hydroxystilbene which was obtained by methylation/acid hydrolysis of resveratrol-3-O-glucoside induces apoptosis in human promyelocytic leukemic HL-60 cells (Lee et al., 2002). Likewise, another compound inherent to the herb (resveratrol) is generally known to induce pro-apoptotic/antiproliferative effects on cancer cells through inhibition of nuclear factor-kappa B, COX-2 of which its oral administration at low dose was effective against 7,12-dimethylbenz(a)anthracene (DMBA) mammary carcinogenesis in female Sprague Dawley rats (Banerjee et al., 2002).