HPLC Fractionation and Pharmacological Assessment ... - Springer Link

Biotechnology and Bioprocess Engineering 20: 1035-1043 (2015) DOI 10.1007/s12257-015-0538-6

RESEARCH PAPER

HPLC Fractionation and Pharmacological Assessment of Green Tea Seed Saponins for Antimicrobial, Anti-angiogenic and Hemolytic Activities Jong Deog Kim, Muhammad Imran Khan, Jin Hyuk Shin, Moon Geon Lee, Hyo Jin Seo, Tai Sun Shin, and Min Yong Kim

Received: 20 August 2015 / Revised: 26 August 2015 / Accepted: 5 October 2015 © The Korean Society for Biotechnology and Bioengineering and Springer 2015

Abstract Herbal medicinal products have proven to be safe, economical and effective alternatives to synthetic chemical pharmaceuticals. The green tea plant (Camellia sinensis) is of profound medicinal value due to the presence of potent bioactive constituents. The purpose of the present work is to investigate saponins from green tea seeds for potential use as anti-angiogenic, antimicrobial, and hemolytic agents. Green tea seed saponins were separated into six fractions by reverse phase HPLC. The presence of three aglycone chains in the saponins of each fraction was confirmed by acid hydrolysis. Anti-angiogenic activity was evaluated using saponin fractions at concentrations in the range of 2.5 ~ 25 µg/mL. Antimicrobial activity was evaluated by thin-layer chromatography (TLC) using E. coli; S. mutans, a zoonotic Salmonella species and the fungal strain, A. niger. Saponin fractions were more potent against E. coli (gram negative bacteria) than against S. mutans Jong Deog Kim*, Muhammad Imran Khan, Jin Hyuk Shin, Moon Geon Lee, Hyo Jin Seo Department of Biotechnology, Chonnam National University, Yeosu 550749, Korea Tel: +82-61-659-7305; Fax: +82-61-659-7305 E-mail: [email protected] Tai Sun Shin Department of Food Science and Nutrition, Chonnam National University, Yeosu 550-749, Korea Min Yong Kim Department of Refrigeration Engineering, Chonnam National University, Yeosu 550-749, Korea Jong Deog Kim, Tai Sun Shin, Min Yong Kim Research Center for Anti-Obesity and Health Care (RCAOHC), Chonnam National University, Yeosu 550-749, Korea

(gram positive bacteria) and strongly inhibited six strains of zoonotic Salmonella. Green tea saponins also showed potent anti-angiogenic effects. All saponin fractions exhibited hemolytic activity. Our results confirm that green tea saponins have antimicrobial, anti-angiogenic, and hemolytic activities; indicating their potential as natural pharmaceutical products. Keywords: tea seed saponin, anti-angiogenesis, hemolytic, antimicrobial, thin-layer chromatography

1. Introduction Since ancient times, people have utilized herbal products as folk medicines and food sources. In the Middle East, the use of herbal products goes back to 5000 BC [1,2]. China has a historical background in the research of medicinal plants and its utilization in the treatment of various diseases and ailments [3]. In developing countries, medicinal plants are particularly important sources of traditional medicines for the maintenance of good health [4]. In European countries, more than a thousand species of medicinal and aromatic plants are used as folk medicines [5]. Researchers have shifted their efforts toward bioactive phytochemical constituents as an alternative therapy for cancer, as synthetic chemical drugs are expensive and have adverse side effects [6,7]. The National Cancer Institute (U.S.A) has screened about 114,000 plants, obtained from different countries, for anticancer activities. Most of the currently marketed anticancer drugs are now fully or partially based on natural products [8,9]. Natural products

1036

Biotechnology and Bioprocess Engineering 20: 1035-1043 (2015)

from medicinal plants are now replacing unsafe drugs derived from synthetic chemicals, because of the efficacy demonstrated by herbal medicines [10]. Thus, plantderived bioactive constituents have been used to prevent or treat various disorders [11,12]. The green tea plant (Camellia sinensis) is a rich source of medicinally active biosubstances that exhibit many biological properties including anti-angiogenic, anticancer, antimicrobial, and antioxidant activities. All of these can be useful in the treatment of various diseases and complications [13,14]. Green tea is considered a medicine and a healthy beverage because of its chemical composition. For the successful application of secondary metabolites obtained from plant sources, it is essential that the secondary metabolite be available at high yield, high concentration, and with consistent activity. Research has shown that saponins from a variety of sources, including medicinal plants and foodstuffs, have considerable health benefits [15]. Saponins are present in many edible and non-edible plants, mostly as complex mixtures of lipid-soluble glycosides, consisting of water soluble glycones chemically linked to either steroids or triterpenoids [16]. Saponins can be detected based on their foam-producing and hemolytic activities. Furthermore, they are known to have many additional biological effects, including antimicrobial, anti-cancer, adjuvant, and gastroprotective properties [17-22]. The biological activities of saponins depend on their chemical structures and are affected by factors such as the saponin nucleus type, number of sugar chains, and types of functional substituents [23]. The structures of saponins from C. sinensis have been widely studied [24]. However, previous pharmacological studies are limited. Green tea seed saponins have been shown to exhibit anti-yeast activity. Saponin E1, but not saponin E2, potently kills yeast through a mechanism involving the elimination of salt tolerance. In another study, three out of seven green tea saponins exhibited potent activities against tumor cell lines [25]. Green tea saponins may have utility as pharmacological agents because natural products either have no or fewer adverse effects than their chemical counterparts [26]. Recently, antibiotic-resistant microbes have become of huge concern to human health [27,28]. Natural products from medicinal plants have been proved effective antimicrobial agents for clinical treatment of resistant pathogens [29]. The antimicrobial activities of green tea seed saponins were studied to evaluate their possible use as antimicrobial agents. In addition, the report by Ning et al. 2013 [30], prompted us to study the potential anti-angiogenic effects of green tea saponins, because uncontrolled angiogenesis is associated with the pathology of several diseases, including atherosclerosis, arthritis, diabetic retinopathy, and cancer [31].

2. Materials and Methods 2.1. Extraction and purification Green tea (Camellia sinensis) seeds were collected during the months of November and December from the Myungin Shin Gwang Su tea garden (Suncheon, Korea). The seeds were dried, de-hulled, and ground into powder. The powder (2 kg) was defatted with n-hexane (4 L) under sonication at 30°C for 5 h and then dried. The defatted seed powder was further extracted with 70% ethanol at 60°C for 4 h. The resulting extract was filtered, concentrated using a rotatory vacuum evaporator (SB-100, Eyela), freeze dried, and weighed. The extract was dissolved in methanol and fractionated on a reverse-phase column (Luna C18 (2), 250 × 21.2 mm, 15 µm; Phenomenex, Inc. Torrance, CA, USA), using a UV detector (wavelength 200 ~ 700 nm) to monitor the separation of total saponin from sugars and isoflavones. The mobile phases were methanol (A) and 0.1% formic acid in water (B). A non-linear gradient elution was used as follows: A/B (74:26) to (74.8:25.2) at 33.5 min to (100:0) at 2 min, held A/B (100:0) for 10 min. The flow rate was 7 ml/min. The HPLC-UV system consisted of a Shimadzu LC-6AD pump, CTO-20A oven, Sil-20A auto-sampler, Shimadzu DUG-20A5, FRC-10A fraction collector, CBM-20A system controller, LC Workstation software, and an SPD-M20A photodiode array detector. The separation procedure was repeated as necessary, and the six fractions were pooled based on retention time and UV spectra using an automated fraction collector controlled by the LC Workstation software program. 2.2. Thin-layer chromatography Thin-layer chromatography (TLC) was performed to analyze total saponin and saponin fractions obtained by preparative reverse-phase HPLC. 10 mg of total saponin or saponin fractions were dissolved in 1 mL of 80% methanol. 10 mL of each sample was applied to a normal-phase TLC plate (TLC Silica gel 60, glass plates 10 × 20, Merck, Germany), and developed with n-butanol, water, and acetic acid in the ratio 84:14:17, as the mobile phase. The developed plates were sprayed with 10% H2SO4, and heated in an oven at 115°C for 13 min to visualize the saponin bands. 2.3. Acid hydrolysis Total saponin or fractions (1 mg in 100 µL of 80% methanol) were refluxed with 100 µL of 10% H2SO4 in a water bath at 80°C for 4 h and then centrifuged at 12,000 × g for 15 min. The supernatant was collected in separate tubes. Fifty µL of 80% methanol was added to the pellet. Ten milliliters of resuspended pellet and supernatant from each sample were analyzed by normal-phase TLC plates using a

HPLC Fractionation and Pharmacological Assessment of Green Tea Seed Saponins for Antimicrobial …

mixture of n-butanol, water, and acetic acid (in the ratio 84:14:17) as the solvent. 2.4. Cell viability assay The human umbilical vein endothelial cell (HUVEC) line was purchased from the Korean Cell Line Bank (Seoul, Korea). HUVECs were cultured in EBM-2 media (CloneticsTM, USA) supplemented with EGM-2, Single QuotTM Kit (CloneticsTM, USA), at 37oC in a humidified 5% CO2 incubator. Cell viability was determined using the MTT assay. HUVECs (1 × 104 cells/well) were seeded in a 96well plate, incubated for 24 h, and treated with saponin samples at various concentrations for a further 24 h at 37°C in a humidified 5% CO2 incubator. Then 0.5% MTT solution (Sigma, MO, USA) was added to the medium, and incubation continued for 4 h at 37°C. MTT medium was removed by aspiration and DMSO (Sigma, MO, USA) was added for 15 min to dissolve the formazan crystals. Absorbance was measured at a wavelength of 540 nm using a microplate reader (Biochrom Ltd., Cambridge, UK). 2.5. Angiogenesis assay The formation of tubular structures by HUVECs in Matrigel was performed as previously described [32]. In brief, 24well culture plates were coated with 150 µL/well of Matrigel (BD Bioscience, MA, US), which solidified at 37°C for one hr. HUVECs suspended in medium (2.5 × 104 cells/well) were added to the Matrigel-coated wells and incubated for 4 h at 37°C. Various concentrations of total saponin and purified fractions were added to the wells and incubation continued for another 4 h. Tube formation was observed and photographed using a phase contrast inverted microscope (Nikon, Tokyo, Japan). Tube lengths in five photographs, obtained from random fields in each well, were analyzed using Scion Image software (NIH, ML, USA). 2.6. Hemolytic assay Defibrinated sheep blood (MB cell, Seoul, Korea) was centrifuged (1,000 × g for five min) to obtain erythrocytes. Erythrocytes were resuspended in phosphate-buffered saline (PBS, 1:2 v/v), and again centrifuged at 1,000 × g for five min. This procedure was repeated 3 ~ 4 times until a colorless supernatant was obtained. For the hemolytic assay using TLC bioautography, 4 mL of washed erythrocytes were added to a gelatin solution (4.5 g of gelatin (Sigma, UK) in 100 mL 0.9% sodium chloride). Ten µL of total saponin or saponin fractions were applied to a glass plate coated with silca gel. Plates were developed with a mixture of n-butanol, water, and acetic acid in the ratio 84:14:17. The plates were dried to remove solvent residue, covered with a layer of gelatin-blood solution, and incubated at 4°C for three h. The plates were photographed under white

1037

light. Hemolytic Rf (hRf) values and diameters of hemolytic rings were measured. To calculate the minimum hemolytic concentration, fresh 20% erythrocyte suspension was prepared in PBS, and the hemolytic assay was performed in 96-well plates (SPL Life Sciences, Korea). In each well, 100 µL of PBS was mixed with 100 µL of erythrocyte suspension. To determine the baseline value, one column of wells contained 200 µL of PBS without erythrocytes. Various concentrations of total saponin and saponin fractions were added to the respective wells of the 96-well plates and incubation was continued for 3 h at room temperature in the dark. Absorbance was measured at a wavelength of 650 nm using a microplate reader. 2.7. Antibacterial assay For antibacterial activity assays, saponin fractions were dissolved at a concentration of 1 mg/ml in distilled water. A disc-diffusion method was then used as a preliminary test in zoonotic bacteria including Escherichia coli (ATCC 25922), Streptococcus mutans (ATCC 25923), Salmonella typhimurium (ATCC 14028), Salmonella enteritidis (ATCC 13076), Salmonella gallinarum (ATCC 9184), Salmonella choleraesuis (ATCC 7001), Salmonella pullorum (ATCC 19945) and Salmonella dublin (ATCC 15480). Active cultures of each microorganism were spread on Muller-Hinton agar (BD biosciences) plates. Then, filter paper discs (Advantech, Japan) impregnated with saponin fractions were placed on the surfaces of the plates. Additional discs were similarly prepared with 1 mg/mL ampicillin (MP Biomedical, France) as a control. All plates were incubated at 37°C for 24 h. The minimum inhibitory concentrations (MIC) of saponin fractions were determined using a 96-well plate format in triplicate. Active cultures (10 µL) of each organism were inoculated into nutrient broth (Difco) and various concentrations of saponin fractions were added to the plate wells. Volumes were adjusted to 200 µL per well. Plates were incubated at 38°C for 24 h. After the incubation period, 20 µL of 2-(4-Iodophenyl)-3-(4-nitrophenyl)-5phenyl-2H-tetrazolium chloride (INT), at concentration 0.2 mg/mL, was added to each well, then the plates were incubated for four more h at 37°C. Absorbance was measured at a wavelength of 600 nm using a microplate reader. 2.8. Statistical analysis Data are expressed as the standard error of the mean (SEM). Statistical analyses were performed by using SPSS 2l. Data were analyzed by one-way ANOVA followed by the Tukey-Kramer test for multiple comparisons. The level of significance was set at p < 0.05 for all statistical tests. All experiments were performed in triplicate and results were similar.

1038


Fig. 1. HPLC analysis of green tea saponin fractions. A preparative HPLC system Luna C18 (2) (250 × 21.2 mm, 15 µm) column was used. Mobile Phase: (A) methanol, (B) 0.1% formic acid in water. Fractions were eluted at 21.38 min (Fr. 1), 24.49 min (Fr. 2), 26.34 min (Fr. 3), 28.79 min (Fr. 4), 31.52 min (Fr. 5), and 34.22 min (Fr. 6). Table 1. Rf values of total saponin (TS) and saponin fractions based on thin layer chromatography using different solvents. Rf values (n-butanol: water: acetic acid) Compound (84:14:17) TS Fr.1 Fr.2 Fr.3 Fr.4 Fr.5 Fr.6

Spray 0.14 0.14 0.14 0.17 0.17 0.20 0.19

Dipping 0.5 0.45 0.45 0.50 0.50 0.50 0.50

Rf values (methanol: water) (4:1) 0.88 0.88 0.86 0.88 0.88

Solvent system: n-butanol, water, and acetic acid (84:14:17), followed by spraying and dipping in 10% H2SO4. Reverse-phase solvent system: methanol, water (4:1), followed by spraying with 10% H2SO4.

3. Results 3.1. Purification of green tea seed saponins Two kg of defatted green tea seed powder was extracted using 70% ethanol, yielding 98 g of crude saponin extract. For bulk separation of saponin from isoflavones and sugars, crude extract was subjected to reverse-phase column chromatography to obtain a mixture of saponins (39 g). Saponin mixtures (referred to here as “total saponin”) were further separated and collected in six fractions based on retention time and UV spectra (Fig. 1). The weights and elution times of each fraction were as follows: Fr. 1 (1.7 g; eluted at 21.38 min), Fr. 2 (4.2 g; eluted at 24.49 min), Fr. 3 (7.5 g; eluted at 26.34 min), Fr. 4 (5.7 g; eluted at 28.79 min), Fr. 5 (2.1 g; eluted at 31.52 min) and Fr. 6 (4.8 g; eluted at 34.22 min). 3.2. Qualitative TLC analysis Total saponin and saponin fractions obtained from

Fig. 2. Thin layer chromatography (TLC) of total saponin (TS) and saponin fractions with different solvent systems. Normal-phase TLC with solvent system: chloroform, methanol, and water (65:35:10). (A) Spraying with 10% H2SO4. (B) Dipping with 10% H2SO4. Solvent system: n-butanol, water, and acetic acid (84: 14: 17). (C) Spraying with 10% H2SO4. (D) Dipping in 10% H2SO4. (E) Reverse-phase TLC with solvent system: methanol, water (4:1).

preparative HPLC. Fractions were analyzed by qualitative TLC to visualize bands corresponding to saponin. The Rf values of all fractions were similar because the bands had similar mobilities (Fig. 2, Table 1). To confirm the presence of different aglycones, the saponin fractions were hydrolyzed before separation and visualization by TLC. New compounds were then assigned to their respective types based on TLC mobility patterns. The hydrolyzed fractions exhibited triple sapogenin bands with similar Rf values (Fig. 3).


3.3. In vitro anti-angiogenic effects The effects of total saponin and saponin fractions on endothelial cell tube formation were examined by quantitative analysis of tube lengths formed on Matrigel.

1039

The anti-angiogenic effect of each fraction was found to be dose-dependent in the concentration range 2.5 ~ 25 µg/mL of each fraction (Fig. 4). The maximum safe and non-toxic doses (0.5 LD 50) were determined and considered for angiogenesis assay. The anti-angiogenic concentration of saponin fractions ranged between five and 20 µg/mL. The minimum anti-angiogenic concentrations (MAAC) are shown in (Table 2). 3.4. Hemolytic activity The TLC chromatograms of saponins and saponin fractions produced white bands against a bright red background in the blood-gelatin immersion assay. All fractions showed hemolytic activity with specific hemolytic zones. Therefore, we performed hemolytic assays in microplates to estimate the minimum hemolytic concentrations (MHC). We observed dose-dependent increases in hemolysis at concentrations in Table 2. Minimum hemolytic concentration (MHC) and minimum anti-angiogenesis concentration (MAAC) of total saponin (TS) and saponin fractions

Fig. 3. TLC analysis of hydrolyzed green tea saponins. Upper panel: Hydrolyzed sapogenin bands from total saponin and saponin fractions, each with three different Rf values. Lower panel: Sugar molecules separated on a TLC plate.

Compound TS Fr.1 Fr.2 Fr.3 Fr.4 Fr.5 Fr.6

MHC >75 >75 >75 >100 >50 >100 >100

MAAC >2.5 >15 >5 >15 >7.5 >5 >7.5

Fig. 4. Anti-angiogenic effects of total saponin (TS) and saponin fractions in a range of concentrations evaluated by quantitative analysis of tube length formed on Matrigel. (A) Total saponin (TS). (B) Fr. 1. (C) Fr. 2. (D) Fr. 3. (E) Fr. 4. (F) Fr. 5. (G) Fr. 6. Values reported are mean ±SEM, n = 3. All values are statistically significant (t-test, P < 0.05).

1040


Fig. 5. Hemolytic assay of total saponin and saponin fractions. (A) Total saponin. (B) Fr. 1. (C) Fr. 2. (D) Fr. 3. (E) Fr. 4. (F) Fr. 5. (G) Fr. 6. The amount of hemoglobin released was measured by absorbance at a wavelength of 650 nm. The hemolytic effect was dosedependent in the concentration range 50 ~ 250 µg/mL. Values reported are mean ±SEM; n = 3. All values are statistically significant (ttest, P < 0.05).

Table 3. Diameter of hemolytic zone and hRf values of total saponin (TS) and saponin fractions based on thin layer chromatography Compound TS Fr.1 Fr.2 Fr.3 Fr.4 Fr.5 Fr.6

Hemolytic zone (diameter, cm) 1.3 0.7 0.9 1.1 1.0 1.2 1.3

hRf 0.23 0.17 0.20 0.19 0.20 0.17 0.20

the range 50 ~ 250 µg/mL of each fraction (Fig. 5). The MHC of each fraction is shown in (Table 3). 3.5. Antibacterial activity Antibacterial activity depended on saponin chemical structure

and varied between microorganisms. Antibacterial activity against zoonotic bacteria including E.coli, S. mutans, S. typhimurium, S. enteritidis, S. gallinarum, S. choleraesuis, S. pullorum, and S. dublin was examined by measurement of the diameter of inhibition zones, as well as MIC values. The MIC values and inhibition diameters of green tea seed saponin fractions are given in (Table 4). All fractions showed potent antibacterial activity against both grampositive and gram-negative bacteria (Fig. 6), with the diameters of zones of inhibition ranging from 9.0 to 13.0 mm. These values were comparable to those exhibited by the antibiotic ampicillin, which showed inhibition diameters ranging from 23.05 to 33.0 mm. The saponin fractions showed MIC values in the range 0.4 ~ 0 mg/mL for gram-negative bacteria, and 0.2 ~ 0.3 mg/mL for the gram-positive bacterium, S. mutans. However, ampicillin showed higher antibacterial activity, with MIC values in the range 5 ~ 10 µg/mL (Fig. 7).


1041

Table 4. Diameters of inhibition zones, and TLC Rf values for total saponin and saponin fractions TS S. mutans E. coli S. typhimurium S. enteritidis S. gallinarum S. choleraesuis S. pullorum S. dublin A. niger

IZ 11 13 10 9.5 11 12.75 11 10 13

MIC 0.45 0.45 0.45 0.6 0.45 0.45 0.45 0.45 -

IZ 11 11.5 10 10 11 10.5 12 11 12

Fr.1 MIC 0.6 0.5 0.5 0.7 0.5 0.5 0.5 0.5 -

Fr.2 IZ MIC 11 0.45 12.25 0.45 10 0.45 9 0.5 11 0.5 11.75 0.45 10.5 0.45 10.5 0.6 13 -

IZ 10 13 10.5 9 9 11.5 10 11 12

Fr.3 MIC 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 -

IZ 10 12.5 10.5 10 11 10 11.5 10 12

Fr.4 MIC 0.6 0.75 0.75 0.75 0.75 0.75 0.75 0.75 -

Fr.5 IZ MIC 10 0.4 11 0.45 10.5 0.45 9 0.45 10 0.45 10.5 0.45 11.75 0.45 12.75 0.45 12 -

Fr.6 IZ MIC 10 0.45 11 0.45 10.5 0.45 9 0.45 11 0.45 9 0.45 12.25 0.45 13 0.45 13 -

IZ = diameter of inhibition zone (mm). MIC = minimum inhibition concentration (mg/mL).

Fig. 6. In vitro antimicrobial activity of total saponin against S. mutans, E. coli, S. typhimurium, S. enteritidis, S. gallinarum, S. choleraesuis, S. pullorum, S. dublin, and A. niger. Values are reported as mean ±SEM, n = 3. All values are statistically significant (t-test, P < 0.05). O.D., optical density.

4. Discussion

Fig. 7. Antimicrobial assay of total saponin and saponin fractions against bacteria and fungi based on thin-layer chromatography. (A) E. coli (gram–negative). (B) S. mutans (gram-positive). (C) A. niger (fungi).

Saponins are used commercially as drugs, medicines, adjuvants, foaming agents, sweeteners, taste modifiers, and cosmetics [33]. They are consumed by humans as a regular part of the diet through pulses and other vegetables. To obtain medicinal value from saponins, their concentration and biological activity should be optimal. Depending on the chemical structure, saponins may exhibit hemolytic, antimicrobial, membrane-depolarizing, cholesterol-binding, and allelopathic activities [34]. Six saponin fractions were purified based on the major HPLC peaks observed. Each fraction was then evaluated for its pharmacological potential. TLC analysis of pure saponins is inexpensive, rapid, requires only small amount of sample, and requires no specialized equipment. Various solvents such as chloroform, methanol, n-butanol, ethyl acetate, glacial acetic acid, and water were used for optimization of the mobile phase. The purist separation of

green tea saponin was achieved with the mobile phase comprising n-butanol, water, and acetic acid in the ratio 84:14:17. However, TLC bands were initially detected at similar mobilities, with no difference in Rf values. Therefore, to confirm the presence of different aglycone chains, we carried out acid hydrolysis before TLC evaluation. Aglycones, glycosides, and sugar molecules could then be separated and visualized by their different TLC mobilities. We obtained three aglycone bands in each fraction, each of which had almost the same Rf value. According to our HPLC results, all six fractions appeared to differ based on UV spectra and retention time. From the TLC analysis, we hypothesized that all fractions contained saponins with three aglycone chains, consistent with a previous report [30]. After qualitative analysis of saponin fractions by TLC, we evaluated the pharmacological potential of saponin

1042


fractions for anti-angiogenic, hemolytic, and antibacterial applications. Angiogenesis is the formation of new blood vessels by the process of sprouting from pre-existing vasculature. It is an essential component of the metastatic pathway and is homeostatically controlled under normal physiological conditions. In the healthy body, its function is to heal wounds by restoration of blood flow to tissues after an injury and to elaborate the vasculature during embryonic development and during development of the corpus luteum. Blood vessels are essential for transport of oxygen, nutrients, and hormones [31]. Angiogenesis and inflammation are prerequisites for the development of a variety of disease conditions, including proliferation and metastasis of cancer cells, rheumatoid arthritis, and diabetic blindness [35]. Therefore, anti-angiogenic agents constitute a novel therapeutic option for the prevention and treatment of human angiogenesis-associated disorders [31,34]. For example, inhibition of angiogenesis could inhibit the proliferation and metastasis of cancer cells. Since chemotherapeutic drugs produce several cytotoxic affects, nutritional and natural supplements should be considered as pharmacological agents for treatment. Natural supplements that support treatment of cancer through inhibition of angiogenesis and associated symptoms should be considered in cancer therapy, as well. Thus, we evaluated the inhibitory effects of saponin fractions on tubular network formation by HUVECs. All saponin fractions prevented tubular network formation in a dose-dependent fashion. Surprisingly, total saponin exhibited the highest anti-angiogenic activity, inhibiting by 65.5 and 79% at concentrations of 2.5 and 5 µg/mL, respectively. This may be due to a combinatorial effect among the different saponins. From our results, it is clear that all saponin fractions had anti-tumor effects. Thus, it seems possible that the anti-angiogenic and anticancer mechanisms are worth further investigation. Could green tea saponins be incorporated into diets to intervene in cancer metabolism? Tea consumption has a long history, and thus components from tea may be free of adverse effects in humans. All six saponin fractions exhibited hemolytic activity and showed differing hemolytic zone, hRf, and MHC values. When we compared the hemolytic activity of saponin fractions with their cytotoxicity, we found that these two activities were not correlated. The saponin fractions showed hemolytic activity with HD50 values >50 µg/mL, while IC50 values for cytotoxicity were in the range 25 ~ 50 µg/mL. Therefore, the compounds having stronger hemolytic activity do not have stronger cytotoxicity. Hemolytic activity depends on the interaction of saponins with cholesterol in the bilayer membrane, which increases the membrane permeability by forming pores, thereby

altering the exchange of ions and ATP activity [36]. Hemolysis can be used to identify the presence of saponins in plant extracts, however not all saponins are hemolytic. For example, Quillaja saponins exhibit hemolytic activities, while saponins from soybean do not have this property [37,38]. Therefore, hemolytic assays are semiquantitative for determination of saponin activity. The in vitro determination of hemolytic activity was based on the destruction of the erythrocyte membranes and the release of hemoglobin [39]. From the nutritional point of view, hemolytic activity does not seem to cause adverse effects because saponins neither cross the intestinal membrane barrier nor enter the blood. Antimicrobial activities are important in plants for protection against pathogens. Most antibacterial medicinal plants attack gram-positive bacteria, while few are active against gram-negative bacteria. Both E. coli (gram-negative) and S. mutans (gram-positive) were chosen for preliminary antibacterial analysis, because not all saponins inhibit the growth of E. coli, which can be resistant to antibiotics. Virulent strains of E.coli cause gastroenteritis, urinary tract infections, and meningitis. S. mutans is well known for its contribution to tooth decay and oral disease. Prompted by our finding that saponins inhibited gram-negative bacteria (E. coli), we were interested to test for activity against food borne pathogens, including Salmonella species. All saponin fractions showed potent inhibition of salmonella species. In general, the mode of action of saponin against microorganisms involves an interaction with a membrane sterol. Thus, it is unexpected that both gram-positive and gram-negative bacteria should be sensitive to saponins [33]. Gram-negative bacteria are less sensitive to antibiotics and natural products such as saponins, because the outer membrane is rich in lipopolysaccharides. This outer membrane excludes certain drugs and antibiotics from the cells. For this reason, lipopolysaccharides are often referred to as endotoxins. From our results (in which we observed greater inhibition of E. coli by green tea saponins), we assume that the growth-inhibitory activity is due to binding between saponins and lipopolysaccharide in the membrane of gram-negative bacteria. Three of seven saponins isolated from green tea seeds exhibited potent activities against tumor cell lines [30]. Our six fractions of saponin showed anti-angiogenic effects, suggesting that saponins might exert anti-cancer effects through inhibition of angiogenesis. Anti-angiogenesis is considered a promising strategy for the development of cancer chemotherapeutics. The biological activity of saponins depends upon the number and position of aglycone chains, as well as the number of sugar moieties. In relation to our previous bioassay report [32], we assume that the six fractions contain saponins that have three different


aglycone chains attached to the sugar moieties at different positions, because the biological activity of each saponin fraction is different. The results demonstrate that the six saponin fractions isolated from green tea possess diverse biological activities.

19. 20. 21.

Acknowledgement The research reported in this manuscript was funded by Korean Institute of planning and evaluation for technology in Food, Agriculture, Forestry and Fisheries (112075-3).

22. 23. 24.

References 1. Chang, J. (2000) Medicinal herbs drugs or dietary supplements? Biochem. Pharmacol. 59: 211-219. 2. Piccaglia, R., M. Marotti, E .Giovanelli, S. G. Deans, and E. Eaglesham (1993). Antibacterialband antioxidant properties of mediterranean aromatic plants. Ind. Crops Prod. 2: 47-50. 3. Huang, K., C. (1993) The Pharmacology of Chinese Herbs. Boca Raton, FL: CRC Press. pp. 3-7. 4. UNESCO (1996) Culture and Health, Orientation Texts – World Decade for Cultural 1988 – 1997, Document CLT/DEC/PRO. p.129. Paris, France. 5. Lanfranco, G. (1992) Popular Use of Medicinal Plants in the Maltese Islands. Insula 1: 34- 35. 6. Chorawala, M. R., P. M. Oza, and G. B. Shah (2012) Mechanisms of anticancer drugs resistance: An overview. Int. J. Pharma Sci. Drug Res. 1: 1-9. 7. Jung Park, E. and J. Pezzuto (2002) Botanicals in cancer chemoprevention. Cancer Metastasis Rev. 21: 231-255. 8. Newman, D. J. and G. M. Cragg (2012) Natural products as sources of new drugs over the 30 years from 1981 to 2010. J. Nat. Prod. 75: 311-335. 9. Jaspars, M. and L. A. Lawton (1998) Cyanobacteria - a novel source of pharmaceuticals. Curr. Opin. Drug. Discov. Devel. 1: 77-84. 10. Balunas, M. J. and A. D. Kinghorn (2005) Drug discovery from medicinal plants. Life Sci. 78: 431-441. 11. Sahoo, N., P. Manchikanti, and S. Dey (2010) Herbal drugs Standards and regulation. Fitoterapia. 81: 462-4 71. 12. Steenkamp, V. (2003) Phytomedicines for the prostate. Fitoterapia. 74: 545-552. 13. Neukam K, N. Pastor and F. Cortes (2008) Tea flavonols inhibit cell growth and DNA topoisomerase II activity and induce endoreduplication in cultured Chinese hamster cells. Mutat Res-Gen Toxic Environ. 654: 8-12. 14. Patel, M., D. T. Shi, T. D Bianculli, and C. Zhao (2005) Imaging and illumination engine for an optical code reader. US Patent 20050023351. 15. Abid, M. M., T. S. Naqvi, and M. S. Naqvi (2012) Identification of phytosaponins as novel biodynamic agents: An updated overview. Asian J. Exp. Biol. Sci. 3: 459-467. 16. Rao, A. V. and D. M. Gurfinkel (2000) The bioactivity of saponins: Triterpenoid and steroid glycosides. Drug Metabol. Drug Interact. 17: 211-235. 17. Rao, A. V. and M. K. Sung (1995) Saponins as anticarcinogens. J. Nutr. 125: 717-754. 18. Francis, G., Z. Kerem, H. S. Makkar, and K Becker (2002) The

25. 26.

27.

28. 29. 30.

31. 32.

33. 34. 35. 36. 37. 38. 39.

1043

biological action of saponins in animal systems: A review. Brit. J. Nutr. 8: 587-605. Guclu-Ustundag, O. and G. Mazza (2007) Saponins properties, applications and processing. Crit. Rev. Food Sci. Nutr. 47: 231258. Khalil, A. H. and T. A. Adawy (1994) Isolation, identification and toxicity of saponin from different legumes. Food Chem. 50: 197-201. Sen, S., H. P. Makkar, S. Muetzel, and K. Becker (1998) Effect of Quillaja saponaria saponins and Yucca schidigera plant extract on growth of Escherichia coli. Lett. Appl. Microbiol. 27: 35-38. Woldemichael, G. M. and M. Wink (2001) Identification and biological activities of Triterpenoid saponins from Chenopodium quinoa. J. Agr. Food Chem. 49: 2327-2332. Osbourn, A. E. (2003) Molecules of interest: Saponins in cereals. Phytochem. 62: 1-4. Mukarami, T., J. Nakrama, and T. Kageura (2000) Bioactive saponins and glycosides: Structure of assam saponins F, G, H, I and J from the seed and leaves of the tea plant. XVII. Inhibitory effect on gastric empting accelerating effect on gastrointestinal transit of tea seponins. Chem. Pharm. Bul. 48: 1720-1725 Kohata, K. Y., T. Ujihara, and H. Horie (2004) Growth inhibitory activity of tea seed saponins and glyphosate to weed seedlings. Jpn Agric. Res. Q. 38: 267- 270. Yamauchi, Y., K. Azuma, M. Tomita, H. Horie, and K. Kohata (2001) Development of a simple preparation method for tea-seed saponins and investigation on their antiyeast activity. Jpn. Agric. Res. Q. 35: 185-188. Immanuel, G., V. C. Vincybai, V. Sivaram, A. Palavesam, and M. P. Marian (2004) Effect of butanolic extracts from terrestrial herbs and seaweeds on the survival, growth and pathogen (Vibrio parahaemolyticus) load on shrimp Penaeus indicus juveniles. Aquacult. 236: 53-65. Cowan, M. M. (1999) Plant products as antimicrobial agents. Clin. Microbiol. Rev. 12: 564-582. Prabu, G. R., A. Gnanamani, and S. Sadulla (2006) Guaijaverin-a plant flavonoid as potential antiplaque agent against Streptococcus mutans. J. Appl. Microbiol. 101: 487-495. Ning, N., J. M. Zhong, C. Yang, W. Ying, and L. Xian (2013) Phytochemical analysis of the triterpenoids with cytotoxicity and QR inducing properties from the total tea seed saponin of Camellia sinensis. Fitoterapia. 84:321-325. Folkman, J. (2006) Angiogenesis. Annurev. Med. pp. 57: 1-18. Kim, J. D., N. Chaudhary, H. J. Seo, M. Y. Kim, and T. S. Shin (2014) Tea saponin E1 as effective ingredients for anti-angiogenesis and anti-obesity effects. Biosci. Biotechnol. Biochem.78: 279-287. Hostettmann, K. A. and A. Marston (1995) Saponins. Chemistry and pharmacology of natural products. Cambridge Universty Press, Cambridge, U. K. Oleszek, W. A. (2000) Saponins. pp. 295-318. In A. S. Naidu (ed.), Natural Food antimicrobial systems. Boca Raton: CRC press, FL. Zetter, B. R. (1998) Angiogenesis and metastatis. Annu. Rev. Med. 49: 407-424. Hu, M., K. Konoki, and K. Tachibana (1996) Cholesterol-independent membrane disruption caused by triterpenoidsaponins. Biochim. Biophys. Acta 1299: 252-258. Jenkins, K. J. and A. S. Atwal (1994) Effects of dietary saponins on fecal bile acids and neutral sterols, and availability of vitamins A and E in the chick. J. Nutr. Biochem. 5: 134-138. Gestetner, B., Y. Birk, and Y. Tencer (1968) Soybean saponins. Fate of ingested saponins and the physiological aspect of their hemolytic activity. J. Sci. Food Agr. 16: 1031-1035. Birk, Y. (1969) Saponins. pp 169-210. In I. E. Liener (ed.), Toxic constituents of Plant Food stuffs. Academic press Inc. NY, USA.