Medicinal plants and human health care

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CHAPTER 2 Investigation of the possible biological activities of a poisonous South African plant; Hyaenanche globosa (Euphorbiaceae)*

ABSTRACT

The present study was undertaken to explore the possible biochemical activities of Hyaenanche globosa Lamb. and its compounds. Two different extracts (ethanol and dichloromethane) of four different parts (leaves, root, stem and fruits) of H. globosa were evaluated for their possible antibacterial, anti-tyrosinase and anticancer (cytotoxicity) properties. Two pure compounds were isolated using column chromatographic techniques. Active extracts and pure compounds were investigated for their antioxidant effect on cultured HeLa cells. Antioxidant/oxidative properties of the ethanolic extract of the fruits of H. globosa and purified compounds were investigated using reactive oxygen species (ROS), ferric-reducing antioxidant power (FRAP) and lipid peroxidation thiobarbituric acid reactive substance (TBARS) assays. The ethanolic extract of the leaves and fruits of H. globosa showed the best activity, exhibiting a minimum inhibitory concentration (MIC) of 3.1 mg/ml and a minimum bactericidal concentration (MBC) of 1.56 and 6.25 mg/ml, respectively, against Mycobacterium smegmatis. The ethanolic extract of the fruits of H. globosa (F.E) showed the highest percentage of inhibitory activity of monophenolase (90.4% at 200 µg/ml).
*A modified version of this chapter was published in “Journal of Pharmacognosy Magazine”, 2010, Vol 6, Issue 21, Page 34-41.
Subsequently, F.E was fractionated using phase-partitioning with n-hexane, ethyl acetate, and n-butanol. The cytotoxicity of these fractions were determined in vitro using different cancer cell lines. The n-hexane fraction exhibited the highest activity of toxicity. Therefore, this fraction was subjected to further separation by chromatographic methods. Two pure compounds known as; ‘tutin 1’ and ‘hyenanchin 2’ were isolated and their structures were determined by NMR spectroscopic methods. Unpredictably, none of them showed significant (P< 0.01) inhibition on cell viability/proliferation at the concentrations that were used. F.E demonstrated potent inhibition of DPPH radical activity similar to vitamin C (positive control). ‘Tutin 1’ and ‘hyenanchin 2’ were found with marginal antioxidant activity of which ‘compound 1’ showed more potent activity than ‘compound 2’. F.E showed significant anti-tyrosinase, antibacterial, and cytotoxicity effects, therefore it can be considered as an effective inhibitor alone or in combination with other plant extracts.

INTRODUCTION

There is great scope for new drug discoveries based on traditional medicinal plant use throughout the world (Hasani-Ranjbar et al., 2009). Nowadays, at least 25% of the active compounds in the currently prescribed synthetic drugs were first identified in plant resources (Van Wyk et al., 1997) and 20,000 plants have been used for medicinal proposes, of which, 4,000 have been used commonly and 10% of those are commercially available. The Euphorbiaceae family is one of the largest families of plants, with about 300 genera and 7,500 species of mostly monoecious herbs, shrubs, and trees that are further frequently characterized by a milky sap or latex material. Members of Euphorbeaceae family have been investigated for providing potential treatments for cancer, tumors, and warts (Lewis and Elvin-Lewis, 1995). The chemistry of Euphorbiaceae is one of the most diverse and interesting one of the flowering plant families and is comparable to the biological diversity of the family. Of all chemical classes, the most useful for a chemotaxonomic study of the Euphorbiaceae, above the level of genus, appear to be alkaloids, cyanogenic glycosides, diterpenes, glucosinolates, tannins and triterpenes. Hyaenanche globosa Lamb. (Euphorbeaceae) is a narrow endemic plant and is restricted to a single flat-topped mountain near Van Rhynsdrop in southern Namaqualand. This plant is the single species of Hyaenanche. It is a small, rounded tree, with dark green, leathery leaves, characteristically arranged in four along the stems. Male and female flowers are both small and occur on separate trees. The fruits are large rounded capsules with several segments. Hyaenanche is a Greek word for hyena poison and was chosen because the fruits were formerly used to poison carcasses in order to destroy hyenas and other vermin. This plant contains several toxic sesquiterpene lactones, such as, tutin, mellitoxin, urushiol III and isodihydrohyaenanchine. Its main toxin, tutin, is known to cause convulsions, delirium, and coma in humans (Hasani-Ranjbar et al., 2009; Van Wyk et al., 1997).
Pigmentation has become an important phenotypical characteristic, in the pharmaceutical, medicinal as well as in the cosmetic field. Plants and their extracts are inexpensive and rich resources of active compounds that can be utilized to inhibit tyrosinase activity as well as melanin production. Natural and synthetic chemical agents can frequently modulate the metabolism of pigmentation produced. The methanolic extract of the aerial parts of H. globosa exhibited significant inhibitory effect on the monophenolase and diphenolase activated forms of tyrosinase in vitro (Momtaz et al., 2008). Therefore, it was decided to prepare different extracts from this species to investigate the possible biological activities of the plant.
Numerous physiological and biochemical processes in the human body may produce oxygen-centered free radicals and other reactive oxygen species as byproducts. Overproduction of such free radicals can cause oxidative damage to biomolecules (e.g. lipids, proteins, DNA), eventually leading to many chronic diseases, such as atherosclerosis, cancer, diabetes, aging and other degenerative diseases in humans (Halliwell, 1994; Poulson et al., 1998). Ames et al., (1995) expressed oxidative injury might induce gene mutation and promote carcinogenesis. In opposition, oxidative injury can lead to cell death (apoptosis). Oxidative stress can modulate the apoptotic programme (Bjelakovic et al., 2004; Fruehauf and Meyskens, 2007). The role of plant extracts and natural purified compounds in alteration of pro-oxidant status in cancerous cell lines has described scantily in past. This study aimed to investigate whether the experimental samples would increase the scavenging of free radicals, so suppress the growth of tumors or excess the level of oxidants and lead to cell death (apoptosis)? The pro-oxidant/antioxidant activity of samples was measured using:

  1. Measurement of radical scavenging capacity (RSC)
  2. Measurement of intracellular ferric reducing/antioxidant power (FRAP)
  3. Measurement of intracellular thiobarbituric acid reactive substances (TBARS)
  4. Measurement of intracellular reactive oxygen species (ROS)

Based on the reported ethnobotanical information about the poisonous properties of the fruits of H. globosa (Van Wyk et al., 1997), it was considered that the other parts also might have the same effects. To explore the possible bioactivities of this species, two different extracts (dichloromethane and ethanol) of fruits, leaves, roots and stem were prepared separately.

MATERIALS AND METHODS

Chemicals and reagents

Fetal bovine serum (FBS) and RPMI 1640 were purchased from Gibco (Paisley, UK). Penicillin/streptomycin was obtained from Roche (Mannheim, Germany). MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) powder, DCFH-DA (2,7-dichlorofluorescin diacetate), 1,2-diphenyl-2-picrylhydrazyl (DPPH), 2,4,6-tripyridyl-s-triazine (TPTZ) and all the other chemicals and reagents were obtained from Sigma-Aldrich (Dorset, UK). FeCl3.6H2O, sodium sulfate and FeSO4, 2-thiobarbituric acid (TBA), Mueller Hinton agar (MHA) and Sabouraud dextrose agar (SDA) were obtained from Merck (Tehran, Iran). L-Tyrosine, L-DOPA, tyrosinase, arbutin and kojic acid were obtained from Sigma-Aldrich (Kempton Park, South Africa). All chemicals and solvents were of the highest commercial grade.

Preparation of plant extracts

The H. globosa (leaves, roots, stem and fruits) materials were collected from the Botanical Garden of the University of Pretoria during May 2007. The plant was identified at the H.G.W.J. Schwelckerdt Herbarium (PRU) of the University of Pretoria (Voucher herbarium specimen number: S.M. 95499). Forty grams of each powdered part (shade dried) was soaked in 200 ml of ethanol and dichloromethane separately for four hours and after filtration the solvents were removed under vacuum (BUCHI, Rotavapor, R-200) to yield dry extracts (F.E: Fruits, ethanol extract; F.DC: Fruits, dichloromethane extract; L.E: Leaves, ethanol extract; L.DC: Leaves, dichloromethane extract; R.E: Root, ethanol extract; R.DC: Root, dichloromethane extract; S.E: Stem, ethanol extract; S.DC: Stem, dichloromethane extract).

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 Antibacterial bioassay

The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the extracts were determined against Mycobacterium smegmatis (MC2 155, American Type, USA Culture Collection) as described previously (Mativandlela et al., 2007; Mativandlela et al., 2008). The sample extracts were dissolved in 10% dimethyl sulfoxide (DMSO) in a sterile Middlebrook 7H9 broth base, to obtain a stock concentration of 50.0 mg/ml. Serial two-fold dilutions of each sample to be evaluated were made with 7H11 broth, to yield volumes of 200 µl/wells, with final concentrations ranging from 12.5 mg/ml to 0.390 mg/ml. The highest percentage of DMSO (10%), which was not toxic to bacteria, was used in this assay. Ciprofloxacin at a final concentration of 0.156 mg/ml, served as a positive drug control.
The samples were also tested against Bacillus subtilis ATCC 6633, Staphylococcus aureus ATCC 6538p, Escherichia coli ATCC 8739, Pseudomonas aeruginosa ATCC 9027, Candida albicans ATCC 10231, and Aspergillus niger ATCC 16404 (Department of Drug and Food Control, School of Pharmacy, Tehran University of Medical Sciences, Iran). The assay was performed by means of the agar-based cup–plate method (Ahmed and Beg, 2001) (Appendices C.6.1-C.6.3).

Inhibition of tyrosinase activity and DOPA auto-oxidation

This assay was performed using methods as described earlier (Curto et al., 1999; Nerya et al., 2003). The extracts were dissolved in DMSO to a final concentration of 20 mg/ml. This extract stock solution was then diluted to 600 µg/ml in a 50 mM potassium phosphate buffer (pH 6.5). The extracts were tested only at two concentrations, 20 and 200 µg/ml, for their inhibitory effect on the monophenolase and diphenolase activated forms of tyrosinase in vitro. Arbutin and kojic acid (positive controls) were also tested at the above-mentioned concentrations. In a 96-well plate, 70 µl of each extract dilution was combined with 30 µl of tyrosinase (333 units/ml in phosphate buffer) in triplicate. After incubation at room temperature for 5 minutes, 110 µl of substrate (2 mM L-tyrosine or 12 mM L-DOPA) was added to each well. Incubation commenced for 30 minutes at room temperature. The optical densities of the wells were then determined at 492 nm with the BIOTEK PowerWave XS multi-well plate reader (A.D.P., Weltevreden Park, South Africa).

Isolation of active constituents

The ethanolic extract of the fruits of H. globosa (F.E) exhibited the highest cytotoxicity effect of HeLa cells compared to the other extracts. The ethanolic extract was selected for the isolation and identification of active principle(s). One thousand two hundred (1,200) grams of air-dried fruits of the plant were milled into a fine powder using a commercial grinder. The powder was extracted thrice, each time with 3 L of ethanol at 50°C for 24 hours. The combined ethanol extract was filtered and the filtrate was concentrated to dryness under reduced pressure in a rotary evaporator.
The dried ethanolic extract of the fruits of H. globosa (70 g) was re-dissolved in 80% ethanol (ethanol/distilled water; 75:25) and partitioned with n-hexane and ethyl acetate. The organic layers were evaporated to dryness at 40°C to give 22 g, 28 g and 18 g of n-hexane, ethyl acetate and aqueous fractions, respectively (Appendices A.1). The bioassay of these fractions of H. globosa showed that the n-hexane fraction demonstrated the highest inhibition of cell growth/proliferation (82% at 100 µg/ml) in the HeLa cells. It was therefore subjected to fractionation on a Silica gel column LH-20 (7×50 cm) using a gradient of n-hexane: ethyl acetate of increasing polarity (0 to 100% ethyl acetate). Forty-two fractions were collected and those with similar thin-layer chromatography (TLC) profiles were combined. TLC plates were developed using (n-hexane: ethyl acetate; 9:1) as eluent. Acidic vanillin was used as a detecting agent. Fractions exhibiting similar TLC profiles were combined together to provide 14 major fractions (1B to 14B) (Appendices A.1).
The pure compound ‘tutin 1’ was crystallized from 12B spontaneously (white hairy crystals, yield: 456 mg; 0.038%) (Fig 2.1) (The 1H & 13C NMR spectra are presented in Appendices A.2.1-A.2.2). Fractions 13B and 14B (2.645 g) were chromatographed separately using silica gel column LH-20 (Sigma-Aldrich, Jet Park, South Africa) using n-hexane: ethyl acetate of increasing polarity (0 to 90% ethyl acetate) as an eluent, to obtain pure ‘hyenanchin 2’ from 13B (white rounded crystals, yield: 347 mg; 0.028%) (Fig 2.1) (The 1H & 13C NMR spectra are presented in Appendices A.3.1-A.3.2). The compounds were identified by mass spectrometric and NMR data, which were identical to those in the literature. The schematic presentation of the isolation steps are shown in Appendices A.4.

List of Figures
List of Tables
List of Appendices
List of Abbreviations
Summary
Abstract
TABLE OF CONTENT
CHAPTER 1: General introduction
1.1. Introduction
1.1.1. Cancer
1.1.1.1. Types of cancer
1.1.1.2. Cancer stages
1.1.2. Carcinogenesis
1.1.3. Metastasis
1.1.4. Cell death
1.1.4.1. Apoptosis
1.1.4.2. Necrosis
1.1.4.3. Oncosis
1.1.4.4. Autophagy
1.1.5. Cancer statistics
1.2. Literature review
1.2.1. Medicinal plants and human health care
1.2.2. Medicinal plants in South Africa
1.2.3. Plants-derived anticancer agents
1.2.3.1. Alkaloids and anticancer properties
1.2.3.2. Coumarins and anticancer properties
1.2.3.3. Flavonoids and anticancer properties
1.2.3.4. Saponins and anticancer properties
1.2.3.5. Terpenes and anticancer properties
1.2.3.5.1. Mechanisms of action of terpenes’cytotoxicity
1.2.3.5.2. Chemoprevention of terpenes
1.3. The genus Hyaenanche
1.3.1. Compounds isolated and identified from the genus Hyaenanche
1.3.2. Hyaenanche globosa
1.4. The genus Maytenus
1.4.1. Compounds isolated and identified from the genus Maytenus .
1.4.2. Maytenus procumbens
1.5. Rationale for this study.
1.6. Aim and hypothesis
1. 7. Objectives
1.8. References
CHAPTER 2: Hyaenanche globosa: biological activities Investigation of the possible biological activities of a poisonous South African plant; Hyaenanche g/obosa (Euphorbiaceae)
2.1. Abstract
2.2. Introduction
2.3. Materials and methods
2.3.1. Chemicals and reagents
2.3.2. Preparation of plant extracts
2.3.3. Antibacterial bioassay
2.3.4. Inhibition of tyrosinase activity and DOPA auto-oxidation
2.3.5. Isolation of active constituents
2.3.6. Cell culture
2.3. 7. Cytotoxicity
2.3.8. Measurement of radical scavenging capacity (RSC)
2.3.9. Preparation of cells for ferric-reducing antioxidant power and lipid peroxidation thiobarbituric acid reactive substance assays
2.3.1 0. Ferric-reducing antioxidant power assay (FRAP)
2.3.11. Thiobarbituric acid reactive substance assay (TBARS)
2.3.12. Measurement of intracellular reactive oxygen species
2.4. Results and discussion
2.5. Conclusion
2.6. References
CHAPTER 3: Maytenus procumbens: biological activities Growth inhibition and induction of apoptosis in human cancerous HeLa cells by Maytenus procumbens
3.1. Abstract
3.2. Introduction
3.3. Materials and methods
3.3.1. Collection, identification and extraction of plant materials
3.3.2. Isolation of bioactive compounds using bioassay-guided fractionation
3.3.3. Identification of isolated compounds
3.3.4. Cell culture
3.3.5. In vitro cytotoxicity assay
3.3.6. Determination of induced apoptosis in Hela cells by flow cytometry
3.3.7. Determination of genotoxicity in Hela cells by comet assay
3.3.8. Measurement of radical scavenging capacity (RSC)
3.3.9. Preparation of cells for ferric-reducing antioxidant power (FRAP) and lipid peroxidation thiobarbituric acid reactive substance (TBARS) assays
3.3.1 0. Ferric-reducing antioxidant power assay
3.3.11. Thiobarbituric acid reactive assay
3.3.12. Measurement of intracellular reactive oxygen species
3.3.13. Antibacterial activity
3.3.14. Statistical analysis
3.4. Results and discussions
3.4.1. Identification of compounds from L.M.P
3.4.1.1. Spectroscopic analysis of ’30-hydroxy-11 a-methoxy-1813-olean-12-en -3-one 3′
3.4.2. Cell viability
3.4.3. Apoptosis detection analysis by flow cytometry
3.4.4. Comet assay DPPH scavenging activities of experimental samples
3.4.6. Effects of plant samples on Hela cells FRAP and TBARS
3.4.7. Effects of plant samples on Hela cells ROS level
3.4.8. Antibacterial activity of plant samples
3.5. Conclusion
3.6. References
CHAPTER 4: General discussion and conclusions
4.1. Motivations for this study
4.2. Biological activities of Hyaenanche globosa
4.3. Biological activities of Maytenus procumbens
4.4. Future perspectives
4.5. References
CHAPTER 5: Acknowledgements
5.1. Acknowledgements
CHAPTER 6: Appendices
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