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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Phytother Res. Author manuscript; available in PMC 2010 December 1.
Published in final edited form as:
PMCID: PMC2957567
NIHMSID: NIHMS215957

In Vitro Screening of Tumoricidal Properties of International Medicinal Herbs: Part II

Abstract

With growing use of anticancer complementary and alternative medicines (CAMs) worldwide, there is a need to assess and screen commercially available natural products for relative tumoricidal properties under standard experimental conditions. In the current study, we screened and ranked 264 traditional Chinese and Egyptian herbal medicines for tumoricidal potency against malignant neuroblastoma in vitro. The data obtained show that tumoricidal potencies of plants were randomly dispersed throughout similar orders, families and genera under the Division: Magnoliophyta, class: Magnoliopsida, subclasses: Asteridae, Caryophyllidae, Dilleniidae, Hamamelididae, Magnoliidae and Rosidae. The most potent plant extracts (LC50 < 0.08 mg/ml) were prepared from gromwell root also known as ‘Hong Tiao Zi Cao’ (Lithospermum Erythrorhizon) Family (Boraginaceae) > beth root (Trillium Pendulum), Family (Liliaceae) and galbanum (Ferula Galbaniflua), Family (Apiaceae). Gromwell root is traditionally used in the preparation of Chinese medicinal tea. In addition, galbanum was highly regarded for its sacred and medicinal value according to ancient texts and the bible. Future research will be required to isolate and identify chemical constituents within these plants which are responsible for tumoricidal effects.

Keywords: herbs, screening, cancer, bible, galbanum, beth root, Lithospermum erythrorhizon root

INTRODUCTION

There is an increase of individuals seeking self-administration of complementary and alternative medicine (CAM)s worldwide to aid in the fight against cancer. The term CAM generally refers to the use of potential holistic therapeutic practices that contribute to the integrated health of mind, body and spirit. While a number of studies provide helpful statistics on the types of individuals who use CAM modalities (Ferrucci et al., 2009; Owens et al., 2009), there continues to be a lack of established research evaluating the relative efficacy of various CAMs to treat or assist in the treatment of cancer.

With regard to chemoprevention, popular consumer choices are known to include the oral administration of antioxidant supplements, glutamine, arginine, zinc, omega-3, fatty acids, probiotics, prebiotics, garlic and phytochemical rich spices such as turmeric, red chilli, cloves, ginger, nutmeg, fennel, fenugreek and black cumin (Blot, 1997; Rosenberg et al., 2002; Conney, 2003; Kraft, 2009). Once cancer is established and diagnosed, self-administration of CAMs can occur without apprising the primary care physician (Clerici et al., 2009; Richardson et al., 2000; Ohno et al., 2009) often including oral administration of selenium, beta-carotene (van Tonder et al., 2009), herbal teas, green tea (Boon et al., 2000; Yates et al., 2005; Scott et al., 2005; Molassiotis et al., 2006), mistletoe, ginseng, cayenne, chamomile, don quai, feverfew, kava kava, milk thistle, licorice, meadowsweet, motherwort, senna leaf, shepherds purse and stinging nettle (Advance Data, CDC, 2004; Dy et al., 2004; Hu et al., 2005; Kumar et al., 2005; Gerson-Cwilich et al., 2006; Melnick, 2006; Tarhan et al., 2009). Although a number of reports suggest that the prevalence of self-administered CAMs is greatest when the disease prognosis is poor (Kristoffersen et al., 2009) or in instances of pediatric cancers (Genc et al., 2009; Clerici et al., 2009), there remains meager research on the relative potencies or efficacy of CAMs utilized in late stage cancers.

In our first report entitled ‘In Vitro Screening of Tumoricidal Properties of International Medicinal Herbs’ (Mazzio and Soliman, 2009), the tumoricidal potencies of the most popular plant based CAMs were evaluated and ranked. Hundreds of international medicines, herbs and plants which are distributed and available to the public worldwide were also tested. While the data showed significant tumoricidal effects for green tea, feverfew, senna leaf, nutmeg, ginger and clove, promising herbs with the lowest LC50s included: wild yam root, balm of gilead bud, chapparal, frankincense and bakuchi seed. In contrast, many popular CAMs used against cancer such as chamomile, milk thistle, motherwort, shepherd’s purse and fennel seed etc., showed weak, or a lack of, tumoricidal properties where the LC50 exceeded 5 mg/mL in vitro. In the current study, we continue to rank the relative efficacy of a diverse range of Chinese and Egyptian herbal medicines for tumoricidal cytotoxic properties in malignant neuroblastoma under uniform extraction and experimental conditions in vitro.

MATERIALS AND METHODS

Neuro-2A cells (N-2A) cells were purchased from American Type Culture Collection (Manassas, VA). Dulbecco’s modified Eagle medium (DMEM), l-glutamine, fetal bovine serum – heat inactivated (FBS), phosphate buffered saline (PBS), Hank’s balanced salt solution (HBSS) and penicillin/streptomycin were purchased from Fischer Scientific, Mediatech, (Pittsburgh, PA, USA). Chinese herbal medicines were purchased from Mayway Herbs (Oakland, CA) with all other herbs being obtained from Kalyx Natural Marketplace (Camden, NY, USA), Frontier Natural Brands, (Norway, Iowa, USA), Mountain Rose Herbs (Eugene, OR, USA), Scents of the Earth (Cape May, NJ) and Monterey Bay Spice Company (Santa Cruz, CA). Chemicals and research supplies were purchased from Sigma Chemical (St Louis, MO, USA).

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) in the absence of light. 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. Dilutions of each experimental extract were prepared from the stock solution in order to span a 1000-fold concentration range with the highest final plating concentration set at 5 mg/mL (w/v).

Cell culture

Neuro-2A cells (N-2A) were used to screen for tumoricidal effects, as they were originally derived from a malignant spontaneous tumor and deemed appropriate for evaluation of chemotherapy drugs (Klebe and Ruddle, 1969; Finklestein et al., 1975; Mazzio et al., 2003). Briefly, N-2A cells were cultured in DMEM containing 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 sub-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 ~0.5 × 106 cells/mL. A three tier process was established where all extracts were evaluated at 0.5–5 mg/mL (tier 1). Those inducing cell death at any level were then re-examined at (0.1–0.5 mg/mL) (tier 2), and those inducing cell death at any level of tier 2, where further evaluated at tier 3 (.05–0.1 mg/mL). Any extract that was lethal within this range was re-tested, and further assessed at lower concentrations.

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

Fluorescein diacetate (FD) was used to corroborate the loss of cell viability (Mazzio et al., 2003). FD is cleaved by viable esterases in living cells where a loss of fluorescence is indicative of cell death. 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 acquisition using C-imaging systems confocal PCI-Simple software (Compix Inc. Cranberry Township, PA, USA).

Data analysis

Statistical analysis was performed using both Origin Lab Scientific Evaluation Software (version 7.5 SR6) (Original Lab Corp., Northampton, MA, USA) and Graphpad Prism (version 3.0), (Graphpad Software Inc. San Diego, CA, USA). The lethal concentrations (LC50) were established from dose-dependent data with Origin Lab 7.5 SR6 and significance of difference between the groups was assessed using a one-way analysis of variance (ANOVA), followed by a Tukey post-hoc means comparison test using Graphpad Prism Ver 3.0 software.

RESULTS

The data in Table 1 list each natural product that was examined by the common name and respective LC50 which was calculated from dose dependent toxicity in malignant neuroblastoma across three tiers and nine concentrations ranging from 0.005–5 mg/mL (n = 4). A taxonomical cross-reference with specific Latin names, families and plant parts are presented in Table 2. The data are listed with the most potent tumoricidal properties first and separated into five classifications based on LC50 where Category 1 (Table 1A) list the strongest agents LC50 =[0.015–0.553 mg/mL]; Category 2 (Table 1B) moderate to strong LC50 =[0.554–1.504 mg/mL]; Category 3 (Table 1C), moderate LC50 =[1.509–3.026 mg/mL]; Category 4 (Table 1D), weak to moderate LC50 = [3.03–4.47 mg/mL] and Category 5 (Table 1E), weak – listing those with no tumoricidal effects and an LC50 > 5.0 mg/mL.

Table 1
The effect of natural products on cell viability in murine neuroblastoma cells originally derived from a spontaneous malignant tumor. The data represent the Common English name or Chinese name and the LC50 (mg/mL) calculated from 3–9 concentrations ...
Table 2
Taxonomy of natural products listed in Table 1A–E. in alphabetical order by Common Name, [Family]; Genus Species and Parts

The data obtained show that less than 1% of extracts screened were capable of inducing cell death at <0.1 mg/mL. The most potent plants were ‘Hong Tiao Zi Cao’ (Lithospermum erythrorhizon root) Siebold & Zucc., common name: gromwell root > (Trillium Pendulum) Willd, common name: beth root and (Ferula galbaniflua), common name: galbanum). Figure 1 (Almar blue viability test) and Fig. 2 (FD photographic validation of viability) show that the lethal effects of Lithospermum erythrorhizon root in tumor cells were observed at very low concentration. In order to assess the water soluble fraction of Lithospermum erythrorhizon root due to its general consumer use as a tea, an herbal tea was prepared by boiling powdered root in sterile water for 5 min, then brought to room temperature. The data obtained show that ethanol extracts were identical in strength to the prepared water extract where the LC50 of gromwell root tea was 0.014 mg/mL and the gromwell root extract was 0.015 mg/mL (data not shown).

Figure 1
The effects of gromwell root on the loss of cell viability in murine neuroblastoma cells derived from a malignant spontaneous tumor as determined with almar blue. The data are expressed as the mean ± SEM (n =4), and represent viability as % control. ...
Figure 2
The effect of gromwell root on the loss of cell viability in murine neuroblastoma cells derived from a malignant spontaneous tumor as determined by photographic acquisition of cells stained with FD. (A) Controls, (B) 0.008 mg/mL, (C) 0.011 mg/mL, (D) ...

DISCUSSION

The current study investigates a diverse range of plants for their tumoricidal properties. While in vitro screenings may provide valuable information regarding elucidation of potential chemotherapy agents, it should also be noted that limitations include lack of consideration as to gastrointestinal absorption, kinetics, bioavailability, tissue distribution, route of systemic circulation, catabolism and excretion, all of which contribute to efficacy in vivo (Lin, 1998). With respect to direct tumoricidal properties, the data in this study show the greatest potency for the following three herbs; gromwell root (Lithospermum erythrorhizon Siebold and Zucc.), beth root (Trillium pendulum Willd.) and galbanum (Ferula galbaniflua).

Patterns within taxonomical classifications

The results were examined to elucidate for patterns of cytotoxic potency within similar botanical categories. Similar to the results obtained from our previous work (Mazzio and Soliman, 2009), there is an inconsistent nature by which plants exert tumoricidal effects even with similar botanical categories. For example, beth root (Trillium pendulum) falls under the botanical classification: Division Magnoliophyta, Class Liliopsida, Order Liliales, Subclass Lilidae and Family Liliaceae. In this study, a total of 11 plant extracts were assessed under the Liliaceae family, with two extracts ranked in the strongest category (Category 1) including Zhi Mu (Genus Anemarrhena) Bunge., and beth root (Genus Trillium) Willd., with nine extracts falling in the weakest category >5 mg/mL (Category 5). A similar trend was noted for Ferula galbaniflua which is classified under the Division Magnoliophyta, Class Magnoliopsida, Order Apiales, Subclass Rosidae and Family Apiaceae. Of the 17 plants examined in this category, only four were identified as strong (Category 1); F. galbaniflua, F. assafoetida, S. divaricata, A sinensis root-tail, A. gracilistylus root-bark, four were ranked as moderate to strong (Category 2), P. praeruptorum root, N. incisium root, L. chuanxiong, and four as moderate (Category 3), three as weak to moderate (Category 4) and two under the weakest category <5 mg/mL (Category 5).

The data also indicate a non-systematic pattern of cytotoxicity from extracts within the same genus including those derived from Angelica (LC50 2.4–4.1 mg/kg), Citrus (LC50 2.317–>5 mg/kg), Curcuma (LC50 0.27–5 mg/kg), Dioscorea (LC50 0.89–5 mg/kg) and Gleditsia (LC50 0.287–5). Although there was a random nature by which tumoricidal effects were observed amongst botanical classifications, the data did indicate a trend amongst extracts from genus and species under the Division Coniferophyta, Class Pinopsida, Order Pinales, where 4/5 tested had an LC50 < 0.676 mg/kg, including those commonly known as arbovitae, pine and juniper.

Gromwell root

In this study, the most potent plant extract was Lithospermum erythrorhizon Siebold & Zucc. which is classified under the Boraginaceae family (Borage). Its extract yields a red-purple pigment analogous to synthetic dyes purported for use in commercial cosmetics (Lee et al., 2008). These light sensitive pigments are also attributable to the high concentration of shikonin naphthoquinones such as deoxyshikonin, shikonin, acetylshikonin, isobutylshikonin and beta-hydroxyisovalerylshikonin which vary in color according to pH (Cho et al., 1999). A large number of shikonin naphthoquinones are emerging as promising chemotherapy agents with the ability to induce apoptosis in a diverse range of cancer cells (Hou et al., 2006; Cui et al., 2008) also having the capability to inhibit DNA toperisomerase (Ahn et al., 1995) and to protect against UV damage (Ishida and Sakaguchi, 2007). Shikonins also inhibit the proliferation and migration of endothelial cells in culture and block tumor necrosis factor (TNF)-α-induced melanoma in mice (Hisa et al., 1998). It is of interest to note in this study that the tumoricidal effects of Lithospermum erythrorhizon superseded that of other traditional Chinese medicines commonly used for the treatment of cancer, including ‘chuan xin lian’ (andrographis), ‘ya dan zi’ (brucea fruit), ‘ban zhi lian’ (barbat skullcap), ‘shan dou gen’ (bush sophora), ‘shi shang bai’ (selaginella), ‘kuan dong hua’ (colts foot), ‘pai lan’ (eupatorium) and ‘Huang qi’ known as astralagus root (Bensky et al., 2004). According to the literature, the primary use for Lithospermum erythrorhizon root as a traditional Chinese homeopathic medicine is for maintaining the health of the heart and liver, to facilitate the passage of stools and urine and the treatment of skin boils, eczema and burns. The advised oral daily dose of this root is 3–9 g per day, indicating its use at high concentrations as has been used historically and is generally safe (Bensky et al., 2004). This study also examined the water soluble fraction of Lithospermum erythrorhizon root by boiling the powdered root in sterile water for 5 min, and then bringing it to room temperature. The data show that water extracts were near identical in strength yielding an LC50 of 0.014 mg/mL vs the Lithospermum erythrorhizon ethanol extract having an LC50 of 0.015 mg/mL.

Galbanum

In this study, galbanum (Ferula galbaniflua) was the third most potent extract. Galbanum is a dark brown-yellow sticky resin with a distinct pungent odor classified under the botanical family Apiaceae (carrot family). The gum is derived from cutting the stem of the plant, which upon exposure to the air forms a semi-solid substance. Galbanum has been referenced in historical literature, the bible and by ancient Egyptians as a holy anointing agent and a valuable medicine. Hippocrates described its extraordinary curative powers, and it was one of the earliest drugs known to man as a stimulant, expectorant, diuretic, antispasmodic carminative, antiseptic and antiinflammatory drug. It was commonly used to treat bronchial afflictions and arthritis. The bible in Exodus 30: 34–35 makes reference to the use of galbanum and frankincense as ingredients required in the preparation of holy incense. More recently, its medicinal use was referenced in the British Pharmacopoeia 1898, named ‘Pilula Galbani Composita’ which describes a mixture of galbanum, asafetida, myrrh and glucose. Today, the resin is used primarily as an odorant or flavoring agent associated with the fragrance of must (Bajgrowicz et al., 2003).

In this study, it was found that the extract of F. galbaniflua was ~3.5 fold more toxic to N-2A cells than F. assafoetida L. However, both Ferula species were classified in the strongest category and pre-existing reports also corroborate the substantial antitumor properties for species within this genus. Recently it was reported that the extract of F. vesceritensis Coss. & DR. contains a compound called lapiferin which is responsible for cytotoxic effects on human MCF-7 breast cancer cells (Gamal-Eldeen and Hegazy, 2010). Extracts derived from the roots of F. elaeochytris Korovin contain 6-anthraniloyljaeschkeanadiol which exerts cytotoxic properties on K562R imatinib-resistant human chronic myeloid leukemia and a dasatinib-resistant mouse leukemia cell line (Alkhatib et al., 2008). Similarly, F. szowitsiana DC (umbelliprenin) exerts tumoricidal effects on malignant melanoma, cell lung carcinoma and prostate carcinoma (Barthomeuf et al., 2008), where F. szowitziana DC contains conferone, a sesquiterpene coumarin known to inhibit protein transporter P-glycoprotein indicating potential in treating multidrug resistant carcinoma (Barthomeuf et al., 2006). The present study reports that F. assafoetida L. exerts potent tumoricidal effects. These findings have also been reported where F. assafoetida L is known to contain ferulic acid and farnesiferols, which at very low concentration can prevent vascular endothelial growth factor initiated processes, angiogenesis and the progression of mouse Lewis lung cancer in mice (Lee et al., 2010; Ghosh et al., 2009). In vitro, terpenes and other constituents within extracts of F. assafoetida L. may be responsible for cytotoxic effects which at low concentrations (<4 µg/mL) are induced against cancer cell lines such as HepG2, Hep3B and MCF-7 (Lee et al., 2009).

Beth root

There seems to be no existing research investigating the bio-therapeutic potential for beth root, which only recently was reported to contain steroidal saponins hypothesized to account for therapeutic efficacy in menopausal women (Hayes et al., 2009). It is also likely that the steroidal glycosides within the root may be accountable for cytotoxic effects on tumor cells (Yokosuka and Mimaki, 2008). While there is a lack of existing research on this plant root, historical literature suggests a benefit for the treatment of colds, hemorrhage, diarrhea and dysentery. Future research will be required to investigate constituents in this plant primarily responsible for the lethal effects on malignant cell lines as observed in this study.

In summary, the findings from this study suggest that relative to the hundreds of other plants tested, gromwell root, bethroot and galbanum are the most cytotoxic to tumor cells at low concentrations. These plants should be further explored for anticancer constituents, application to other types of tumor cells, and could be considered for future CAM strategies that apply to suppressing the growth of malignant tumors.

Acknowledgements

This work was supported by a grant from the United States of America National Institute of Health (NIH) National Center for Research Resources NCRR RCMI Program G12RR03020. The authors acknowledge the valuable technical help Ms Kathelene Park.

Footnotes

Conflict of Interest

The authors have declared that there is no conflict of interest.

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