Volume 2 Number 1 2008

CONTENTS AND ABSTRACTS

Avinash Nagpal, Balwinder Singh, Sanjeev Sharma, Gita Rani, Gurdeep Singh Virk (India) Coleus spp.: Micropropagation and In Vitro Production of Secondary Metabolites (pp 1-17)

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Invited Review: Coleus spp. have long been prized for their colorful foliage, which may combine shades of green, yellow, pink, red and maroon. They are plants for all locations. Besides their ornamental nature, Coleus spp. are also valued in folk medicine. C. blumei Benth., a natural hybrid of several Coleus spp. is an ornamental plant growing all over the world in an enormous number of different cultivars that vary in color and shape of the leaves, and is also known for its medicinal properties, in particular rosmarinic acid, one of its most important secondary compounds. Rosmarinic acid has been shown to exhibit antioxidative, anti-inflammatory and antiallergic activities in mammalian systems. C. forskohlii Briq. is a medicinally important plant valued for the production of forskolin, a diterpenoid, present in root tubers of the plant and known to lower blood pressure and intraocular pressure. C. parviflorus Benth. (country potato) has the potential of developing into a crop similar to potato. An ample amount of work has been reported on micropropagation of different Coleus spp. from different explants like shoot tips, leaf segments, nodal and internodal segments, etc. Furthermore, large-scale production of rosmarinic acid and forskolin from cell cultures of C. blumei and C. forskohlii, respectively, has also been reported. Genetic transformation using Agrobacterium has been reported to enhance rosmarinic acid and forskolin production in transformed cultures of C. blumei and C. forskohlii.

Jian Chen, Wei-lin Li (China) Progress in Studies on Phytochemistry and Biological Activity of Folium Eriobotryae (pp 18-23)

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Invited Mini-Review: Eriobotrya japonica (Thunb.)Lindl. is widely planted as a fruit tree, specifically in China and Japan, and its dried leaves (Folium Eriobotryae) is commonly used in traditional Chinese medicine. Many compounds including essential oil, triterpenes, sesquiterpenes, flavonoids, tannins and megastigmane glycosides have been isolated from Folium Eriobotryae, and showed anti-inflammatory, hypoglycemic, anti-oxidant, anti-tumor, antiviral, cytotoxic and antimutagenic properties. In the present paper we review the research results on phytochemical constituents and biological activities of Folium Eriobotryae in the hope that it would be helpful to better understand and use traditional Chinese medicine.

Usama Aly, Moemen Hanafy (Egypt) Geranium Oil Production in Suspension Cultures of Pelargonium graveolens L. (pp 24-28)

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Original Research Paper: Studies were conducted on the morphogenetic potential and secondary metabolite production by P. graveolens L. cell culture. Leaf segments excised from field grown plants, which showed the most intensive growth of callus on B₅ medium supplemented with 0.5 mg/l BA and 2.0 mg/l NAA, were used to initiated callus and cell suspension cultures. The effect of initial concentrations of two of the major medium components, sucrose and nitrate, on the growth, biosynthetic and biochemical characteristics of essential oils was examined. Callus developed most on B₅ medium containing 4% sucrose and normal level of nitrate (2500 mg/l). Maximum essential oil was produced from plants grown on M7 medium containing 8% sucrose.

Ali Kemal Ayan, Cüneyt Çırak (Turkey) Detection of Hyperforin in Turkish Species of Hypericum (Guttiferae) (pp 29-30)

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Short Communication: In the present study, six Hypericum species from the Turkish flora were investigated for the presence of hyperforin namely H. heterophyllum Vent, H. hyssopifolium L., H. linarioides Bosse, H. orientale L., H. scabrum L. and H. triquetrifolium Turra. For this purpose, the aerial parts were collected at full flowering, dissected into floral, leaf and stem tissues, air-dried at room temperature and then assayed for hyperforin by HPLC. Hyperforin was detected only in flower tissues of H. hyssopifolium (29.2 mg/g DW) and H. linarioides (6.28 mg/g DW). This data could be useful for elucidation of the chemotaxonomical significance of hyperforin and for the phytochemical evaluation of H. hyssopifolium and H. linarioides.

Gilles Ines Fomekong Dongmo, Claudia Edwedje Noumessing Momo, Julius Enyong Oben (Cameroon) Antihyperglycemic and Hypoglycemic Effects of Aqueous and Hydroethanolic Extracts of Pentaclethra macrophylla Benth on Wistar Rats: Inhibition of α-Amylase (pp 31-34)

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Original Research Paper: This study reports on the antihyperglycemic and hypoglycemic properties of Pentaclethra macrophylla Benth on Wistar rats. The inhibition of α-amylase was also determined using 0.25, 0.5, 1, 1.5, and 2 mg/mL of plant extracts. The aqueous and hydroethanolic extract showed a pronounced antihyperglycemic activity (P<0.001) from 75 mg/kg to 300 mg/kg body weight (BW) after 60 min. The improved regulation of blood glucose was obtained at 150 mg/kg BW with 23.75% and 28.85% reduction, respectively for aqueous and hydroethanolic extracts after 2 h 30 min of treatment. In the hypoglycemic assay on normoglycemic rats, we observed a slight decrease of blood glucose from 38 mg/kg to 75 mg/kg BW during the experimental period. Between the 5^th and the 8^th hour post-treatment, we observed a significant (P<0.001) decrease of blood glucose at 150 mg/kg and 300 mg/kg BW, respectively. On the order hand, the in vitro experiments showed that amylase activity was inhibited in the presence of these plant extracts. The extent of amylase inhibition was correlated with an increase in the concentration of extracts. Also the hydroethanolic extract showed better amylase inhibition activity. We obtained a 50% inhibitory concentration (IC₅₀) of 0.292 mg/mL with the hydroethanolic extract while the aqueous extract showed an IC₅₀ of 3.19 mg/mL. The significance of plant-based amylase inhibitors for modulation of diabetes mellitus and other oxidation-linked diseases is hypothesized and discussed.

Gilles Ines Dongmo Fomekong, Jules-Roger Kuiate (Cameroon) Effects of Essential Oil of Zingiber officinalis Roscoe on Some Biochemical Parameters, Body Weight and Food Intake on Wistar Rats (pp 35-39)

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Original Research Paper: The effects of ginger (Zingiber officinalis Roscoe) essential oil were determined on some biochemical and physiological parameters on Wistar rats. The hepatic lipids appeared to decrease respectively from group 1 (0.03 g/kg body weight (BW)) to group 5 (1.00 g/kg BW). Also, the liver and serum cholesterol decrease significantly (P<0.05) from group 2 (0.15 g/kg BW) to group 4 (0.6 g/kg BW) and highly significant (P<0.01) for group 5. However, we noted harmful effects when doses >1.00 g/kg was used on animals. The main side effects were an abnormal development of hepatic cells, an increase of lipids and cholesterol as well as a decrease of protein level. 0.03 g/kg, the commonly published dose and 0.15 g/kg, which is the smallest dose without an observable side effect (P<0.05) on cholesterol, can be considered as the advisable doses. Generally, the essential oil of ginger had beneficial effects at doses £0.30 g/kg and harmful effects at doses ³0.60 g/kg.

Buyisile Mayekiso, Ntombovuyo Madlokazi, Micheal Libala Magwa (South Africa) Antimicrobial Activity of Schistostephium heptalobium Leaf Extracts (pp 40-42)

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Original Research Paper: Schistostephium heptalobium belongs to the family Asteraceae, which is a source of many aromatic and medicinal plants. The species S. heptalobium is used by healers and herbalists in the Eastern Cape Province of South Africa to treat various medical ailments. Three crude extracts of leaves were obtained by using methanol, acetone and water. Five Gram-positive species (Bacillus cereus, Staphylococcus epidermidis, Staphylococcus aureus, Micrococcus kristinae and Streptococcus pyogenes) and five Gram-negative species (Escherichia coli, Salmonella pooni, Serratia marcescens, Pseudomonas aeruginosa and Klebsiella pneumoniae) were used to determine the antibacterial activity of these three crude extracts. All three extracts exhibited positive results for all Gram-positive bacteria while both the acetone and methanol extracts were strongly effective against most Gram-negative bacteria tested. Five fungal species, Aspergillus flavus, Aspergillus niger, Penicillium notatum, Mucor heamalis and Fusarium oxysporum were used for an antimycotic assay, all of which were inhibited, showing that this plant can be used as a complementary source for traditional medicines.

Sonia Malik, Shashi Bhushan, Subash C. Verma, Naina Sharma, A.K. Sinha, Madhu Sharma, P.S. Ahuja (India) Production of Naphthoquinone Pigments in Cell Suspension Cultures of Arnebia euchroma (Royle) Johnston: Influence of pH on Growth Kinetics and Acetylshikonin (pp 43-49)

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Original Research Paper: In this paper, the effect of pH on in vitro cell growth and naphthoquinone pigment production in cell suspension cultures of Arnebia euchroma (Royle) Johnston was evaluated under a two-stage culture system. Friable callus derived from leaves of in vitro established shoots was maintained on MS medium supplemented with BAP (10 µM) and IBA (5 µM) and the same medium was used for raising cell suspension cultures. An acidic pH (i.e. 5.0-6.50) of the medium helped cell growth, while that having alkaline pH (7.25-9.50) favoured pigment production. Almost double cell biomass (198.67g, FW.L^-1) was recorded in medium at physiological pH (5.70), when compared to neutral (138.55 g FW.L^-1) or alkaline levels (130.56 g FW.L^-1). Acetylshikonin (I) was found to be the major shikonin derivative in these cultures. The maximum content i.e., 2.049% was recorded at alkaline pH (9.5) in comparison to 0.803% at acidic pH level (5.75) on a fresh weight basis, as revealed by HPLC analysis of the cell suspension cultures. Microscopic examination of the cell suspensions grown at different pH revealed that higher pH adversely affected growth and morphology of the cells. Importantly, the induction of pigment in the alkaline range of pH was independent of the growth phase and pH showed a regulatory role in its biosynthesis.

K. Padmalatha, K. Jayaram, M.N.V. Prasad (India) A Rapid Protocol for the Isolation of Polysaccharide- and Polyphenolic-Free Genomic DNA for RAPD Analysis of Threatened Medicinal Plants (pp 50-53)

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Techniques Paper: Many procedures in molecular biology require the isolation of high quality genomic DNA which is difficult in most plant species because of the presence of secondary metabolites that interfere with DNA isolation which effects downstream applications such as DNA restriction, amplification and cloning. Here we describe a modified procedure based on the hexadecyltrimethylammoniumbromide (CTAB) method to isolate DNA from leaf tissues containing high levels of polysaccharides, polyphenols, gums, tannins and secondary metabolites. This modified CTAB (2%) protocol includes the use of a combination of polyvinypyrrolidone (PVP) and polyvinylpolypyrrolidone (PVPP), 0.2% β mercaptoethanol for Pterocarpus marsupium and use of 2 M NaCl, 1% PVPP, and 1% b-mercaptoethanol for other selected medicinal plants with an initial prolonged chloroform:isoamylalcohol (24:1) step (30 min shaking) repeated twice, RNAse treatment for 1 hr and avoiding the incubation and precipitation steps in isopropanol. The yield was approximately 20 µg DNA/200 mg of initial fresh leaf tissue. This method solved the problem of DNA degradation and co-precipitation of the secondary metabolites. The isolated DNA proved amenable to restriction digestion and PCR amplification. The technique is fast, reproducible and can be applied for further molecular approaches.

Jin-ichi Sasaki, Chihiro Sutoh, Ayako Norigami, Tomomi Satoh, Nana Kumaki, Hidemitsu Uchisawa, Shinya Yamaguchi (Japan), Vachik Lazarian (USA) Emission of Odor in Plants: A Strategy to Fight Infection? (pp 54-56)

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Techniques Paper: The bacteriocidal potency of odor was studied by the Petri dish method using eight types of plant odors from Hiba tree/Japanese cypress (Thujopsis dolabrata var. hondae), garlic (Allium sativum), onion (Allium cepa), horseradish (Armoracia rusticana), cinnamon (Cinnamomum zeylanicum), dokudami (Houttuynia cordata), clove (Syzygium aromaticum) and sage (Salvia officinalis). Target bacteria in the test were Pseudomonas aeruginosa, Bacillus natto, enterohemorragic Escherichia coli O157:H7, methicillin-resistant Staphylococcus aureus (MRSA), and Candida albicans. Seven out of eight plant odors showed antibacterial activity with a different spectrum of activity against the above described bacteria. Odor released from Hiba tree, garlic, onion, horseradish and dried whole clove demonstrated the strongest antibacterial activity, followed by dried cinnamon and dokudami, but the odor of dried sage did not show any activity against these bacteria. Gas chromatography analysis clarified the presence of 15 composites in fresh garlic odor, but two of them were unidentified in an analysis 24 hours later. When viewed by scanning electron microscopy, the bacteria surface became fluffy due to exposure to Hiba odor accompanied with bleb formation on the surface, which may have been caused by protein degeneration, while formaldehyde (a representative disinfectant)-exposed bacteria maintained a smooth surface without any formation of blebs. These results suggest that anti-bacteria mechanisms of odor vary depending on the odor (i.e. chemical(s) included) emitted from plants. One of the significances of plant odor might be the protection from attacks by harmful agents such as bacteria and insectsin environmental surroundings.

Klára Szentmihályi, Mária Hajdú, Mária Then (Hungary) Inorganic Biochemistry of Medicinal Plants (pp 57-62)

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Original Research Paper: Most medicinal plants are flowering plants and they also serve as ornamental plants. Measurement of trace element content in medicinal plants, drugs and extracts may be relevant in view of e.g. human health, animal health and environmental relations. This fact has a great significance since about half of the plant drugs available in the trade originate from natural habitats and the element content of herbs may originate from soil or air pollution, or relate to the soil type on which the plant grows. Microelements in extracts may be relevant in nutritional point of view or for therapeutic purposes.

Pasupuleti Sreenivasa Rao, Majeti Narsimha Vara Prasad (India) Extraction, Purification and Characterization of Indole Alkaloids from the South Indian Strychnos wallichiana L. – an Endangered Medicinal Plant (pp 63-67)

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Original Research Paper: Strychnos wallichiana (Loganiaceae) is a critically endangered medicinal plant, commonly used in several native medical practices. The majority of members of the Strychnos genus shares a similar chemical composition and contains common indole alkaloids. Purification of strychnine and brucine from a few species of Strychnos have already been reported; however, we have purified and quantified these alkaloids for the first time from this native Indian variety by isocratic RP-HPLC from roots and seeds. The structural identity of HPLC-purified strychnine and brucine was reaffirmed by IR, ¹H-NMR, ¹³C-NMR and LC-MS spectral analysis. The present study reveals that South Indian S. wallichiana possesses indole alkaloids in a significant proportion, and that it is an alternative source for strychnine and brucine.

P. S. Sujan Ganapathy, H. V. Sudeep, S. Padmalatha Rai, Y. L. Ramachandra (India) Screening of Crude Extracts of Holarrhena antidysenterica Wall. Against Clinically Important Pathogenic Strains (pp 68-71)

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Original Research Paper: The crude ethanol, chloroform and petroleum ether extracts of Holarrhena antidysenterica (stem bark, leaf and inflorescence) were tested against 18 clinically isolated strains including identified strains using the agar-well diffusion method. Of all the extracts the activity was more pronounced against Gram-negative bacteria than against Gram-positive ones. Amongst the evaluated extracts, the ethanolic stem bark extract showed the strongest antibacterial effect. The inhibitory effect of the extracts was compared with standard antibiotic Ciprofloxacin. Our results offer a scientific basis for the traditional use of H. antidysenterica in the treatment of infectious diseases.