4^th World Congress on Medicinal Plants and Natural Products Research August 08-09, 2018 Osaka, Japan

My research interest is extraction, separation and identification of chemical composition from Traditional Tibetan Medicine, as well as activity screening of natural products and its derivatives. Current research focuses primarily on the biological activity of natural small molecule compounds (NSMC) as well as elucidation of the mechanisms underlying NSMC metabolism and the role that these compounds play in health and disease.

It is very intense and significative in the search for novel neuropharmacological activities devoid of undesirable side-effects typical of classical benzodiazepines (BZDs) in recent years, and flavonoids, as a relative new class of ligands, have been shown to possess some anxiolytic and anticonvulsant effects in vivo/vitro. The objective of the present work was to evaluated the pharmacological properties of a naturally occurring flavonoid (2’,4’,5,7-tetrahydroxy-5’6-dimethoxyflavone or DMB) at recombinant γ-aminobutyric acid-A (GABA-A) receptors, and its anti-alcohol, anxiolytic and anticonvulsant activities were tested by the models of animal behavior. DMB, isolated from traditional Tibetan herb (Arenaria kansuensis), positively modulated GABA-activated current at α1β2γ2 and α5β2γ2 GABA-A receptors expressed in HEK 293T cells. The behavioral studies showed that DMB at doses of 0.5-8.0 mg/kg (i.p.) exerted significant anti-alcohol effects in the loss of righting reflex assay (P <0.01). The anxiolytic activity was measured on an elevated plus maze, and the results indicated that DMB can alleviated significantly anxiety at the doses of 2-8 mg/kg (p.o.). Both DMB anti-alcohol and anxiolytic effects on animal behavior were antagonized by flumazenil (5 mg/kg in vivo), the BZD antagonist. DMB competitively inhibited BZD-site [3H] flunitrazepam binding (IC50, 0.10 μM), suggesting the neuropharmacological activities of DMB were mediated via the BZD-sites on GABA-A receptors. In addition, DMB (0.5-8.0 mg/kg, i.p.) exerted dose-dependently anticonvulsant activity in pentylenetetrazole induced seizure paradigms (P <0.01). Furthermore, DMB did not cause myorelaxant effects in the rotarod test and horizontal wire test. Therefore, the results of the present study suggest that the pharmacological activities (anti-alcohol, anxiolytic and anticonvulsant) of DMB are probably mediated through positive allosteric modulation of the different GABA-A receptor subtypes via interaction at the BZD-site, and these effects were not accompanied by myorelaxant side-effects typical of BZDs.

Heung-Bin Lim is a Professor at Chungbuk National University, Republic of Korea. He administers the Tobacco Smoke Analysis Center, an internationally recognized testing laboratory (KOLAS, KS Q ISQ / IEQ 17025), which analyzes the smoke components of all tobacco produced in Republic of Korea.

Pyrrolizidine alkaloids (PAs) are secondary metabolites biosynthesized in plants and are found in about 6,000 plants. Among these, the 1,2-unsaturated PAs, reported to be widely present in medicinal plants belonging to Asteraceae, Boraginaceae, and Fabaceae, cause hepatotoxicity and genotoxicity in humans and animals. Hence, there is a need for an analytical method that allows these dangerous plant toxins to be determined. In this study, we developed a method that can be used for the rapid and accurate determination of nine toxic PAs in medicinal plants using ultra-pressure liquid chromatography–electrospray ionization–quadrupole–time-of-flight mass spectrometry (UPLC-ESI-Q-TOF). The compounds were eluted onto a C18 column with 0.1% formic acid and acetonitrile, and separated with good resolution within 11 min. Each component was characterized by its precursor ions (generated by ESI-Q-TOF) and fragment ions (produced by collision-induced dissociation, CID), which were used as a reliable database. The proposed analytical method was verified with reference to the ICH guidelines. The results showed excellent linearity (R2 > 0.9951), limit of detection (2 to 12 ng/mL), limit of quantification (6 to 30 ng/mL), intra-day and inter-day precisions, and extraction recovery rates (76.9% to 103.3%) for all components. The validated UPLC-ESI-Q-TOF method was applied to medicinal plants belonging to the family Asteraceae, and senkirkine and senecionine were detected in Tussoilago farfara. In addition, with regard to the extraction efficiency of these two alkaloids, the QuEchERS method was the most efficient in comparison with methods like hot water and ethanol. The present results demonstrate that the proposed UPLC-ESI-Q-TOF method can be employed for the screening and quantification of toxic alkaloids in medicinal plants.

Gae-ddong-ssuk, Artemisia Annua L. is an annual herb belonging to Asteraceae family which is grown popularly in Asia including Korean and China. The herb is well known for the effects of fever, hemostasis, and antibiotics. When the mouse macrophage cell line, Raw264.7 was stimulated with LPS (lipopolysaccharide), the cells tremendously secreted pro-inflammatory cytokines such as IL-1β (interleukin-1 beta), IL-6, NO (nitric oxide) and TNF α (tumor necrosis factor alpha). However, the secretion of these cytokines was reduced from the cells treated with the extract of Artemisia Annua L. (EAA). In addition, EAA were able to inhibit inflammatory reaction in the acetaminophen (APAP) -stimulated human hepatocyte carcinoma cell line, HepG2 and to protect hepatocytes from damage caused by various toxins. Acute liver failure animal model was set up with injection of certain concentrations of LPS and D-galN (D-galactosamine) in C57BL/6J mice intraperitoneally, showing highly increased level of AST (aspartate transaminase) and ALT (alanine transaminase). Oral administration of EAA (100 mg/Kg) for 2 weeks reduced the level of AST and ALT up to 50% level of the negative control group treated with water vehicle. The efficacy of EAA was even more effective than the one of the comparative positive control group treated with the extract of Milk thistle. Moreover, EAA protected hepatic cells and tissues from oxidative stresses and inflammatory damages. Taken together, Artemisia Annua L. demonstrated the possibilities as a promising nutraceutical candidate for protection and recovery of liver damages as well as maintain of liver health.

Medicinal plants are good source of various secondary metabolites. Parts of these medicinal plants are being used singly or in combination with other herbs for treating various human ailments. Plant species of Indian Himalaya region (IHR) possesses rich medicinal value and uses in various systems of medicine like Ayurveda, Siddha, Unani, etc to cure numerous diseases. The reports suggest, a total of 700 species are being used by pharmaceutical companies in India. Considering the Himalayan medicinal plants, the group of medicinal plant named ‘Astavarga’ is an important ingredient of different Ayurvedic formulations. Astavarga, a composition of eight main ingredients (Habenaria intermedia, Habenaria edgeworthii, Malaxis acuminata, Malaxis muscifera, Roscoea procera, Lilium polyphyllum, Polygonatum verticillatum and Polygonatum cirrifolium), is known to strengthen the vital force of the body, cell regeneration capacity as well immunity system. Reports indicated that the Astavarga plants have been endangered since ancient time and difficult to obtain in required quantity. This is further confirmed by the recent list of CITES where complete family of the orchids have been put in the Appendix II and completely ban on collection from natural habitat for trade. Out of the eight herbs of the Astavarga group, four are orchids, of which Malaxis muscifera/ Rishbak is one important ingredient. It is useful in the cure of sterility, vitiated conditions of pitta and vata, seminal weakness, internal and external hemorrhages, dysentery, fever, emaciation, burning sensation and general debility. The species is also used to cure paralysis, body pain, fibromyalgia, myofascial pain, nervousness, sexual weakness etc. The species has sizeable market demand on account of its commercial use but the level of exploitation is high. In many cases, most of the species are regarded as the only source of important drugs, so they are heavily exploited at commercial scale. Thus, the problem not only results in the removal of plant material but also causes lack of quality plant material. However, in last few years, emphasis was laid on conservation and sustainable utilization of this plant species to reduce the pressure but no encouraging results were achieved. Therefore to protect the natural germplasm and to collaborate with rising demand there is an immense need of biotechnology-based interventions to ascertain secure conservation of elite germplasm. The conventional propagation of this species is too slow and unable to overcome the threat of extinction. In this regard, plant propagation via tissue culture has been evolved as a valuable method compared to conventional plant multiplication in last few decades. It is to be mentioned that while undertaking conservation it would be pertinent to evaluate the range of variability using various parameters to identify the elite populations/individuals on the basis of morphological, physiological, phytochemical and molecular assessment. Occurrence of genetic variation is a major limitation in these in vitro culture plants. Therefore assessment of genetic fidelity of tissue cultured plants will assure the maintenance of genetic stature of a clone throughout the procedure. Furthermore development of synthetic seeds ensures genetically identical materials with easy handling and transportation, thereby increasing the efficiency of in vitro propagation.

Dr. Vikas Vasant Patil has completed his PhD at the age of 32 years from North Maharashtra University, Jalgaon Maharashtra, India in the faculty of Medicine and Pharmacy. He is working as a Professor and PG Guide in Affiliated to North Maharashtra University. He has published more than 28 papers in reputed journals and has been serving as an editorial board member and reviewers of reputed Journals. He has guided 25 PG (M.Pharm) students. He is life Member of APTI and INPHARM Association.

In the present study attempt has made to separate terpens from crude drugs (Mangifera Indica Linn). Determination of chemical constituent in percentage by GC and GCMS analysis was done, higher content of α-terpineol (3.26%),β-selinene (3%) found in Totapuri variety other constituents also estimated. Crude drug Vitiveria Zizanoides (Linn) collected from different district region of Maharashtra vetiver oil separated by distillation method. The physical parameters of oils are determined according to ISI specifications. Percentages of constituents are estimated observed that Bharatpur variety having high % of free alcohol as vetiverol (78.10%) The odorous volatile principles of plant and animal sources are known as volatile oils, as they evaporate when exposed to air at ordinary temperature, they are also called as Ethereal oils. They represent essence or active constituents of plant, hence they are also known as Essential oils chemically they are derived from terpenes and their oxygenated compounds and they are made up of isoprene units (C5H8)

Heung-Bin Lim is a Professor at Chungbuk National University, Republic of Korea. He administers the Tobacco Smoke Analysis Center, an internationally recognized testing laboratory (KOLAS, KS Q ISQ / IEQ 17025), which analyzes the smoke components of all tobacco produced in Republic of Korea.

Statement of the Problem: Turmeric (Curcuma longa) is an herbaceous perennial plant belonging to the ginger family, Zingiberaceae, and is commonly used as a culinary spice, as well as in food coloring. Black pepper (Piper nigrum) is a flowering vine of the family Piperaceae, which is usually dried and used as a culinary spice. Co-administration of piperine and curcumin, which are the main constituents of black pepper and turmeric, respectively, has been shown to enhance the bioavailability of curcumin by 2000% in humans and 154% in rats. We evaluated the growth inhibitory effect of turmeric and black pepper ethanolic extracts on A549 and NCI-H292 cell lines. Methodology & Theoretical Orientation: The turmeric and black pepper powder was extracted with 70% ethanol, with yields of 26.3% and 14.3%, respectively. In order to confirm the specific toxicity toward lung cancer cells, the turmeric and black pepper ethanolic extracts were exposed to two lung cancer cell lines (A549 and NCI-H292), as well as to normal human lung fibroblast cell line, for 24 h. At a concentration of 100 μg/mL, viability of normal lung fibroblasts was 87.1 ± 5.7%, while those of A549 and NCI-H292 cells were 57.0 ± 3.3 and 62.7 ± 9.4%, respectively. Conclusion & Significance: Co-treatment of turmeric and black pepper ethanolic extracts showed a synergistic effect on lung cancer cell cytotoxicity. Our study confirmed the effects of turmeric and black pepper ethanolic extracts on lung cancer cell death through synergistic effects, and provides a basis for further study.

Asst. Prof. Dr Parinya Noisa is a stem cell biologist at Suranaree University of Technology in Thailand. He completed B.Sc in Biology with the first class of honors from the faculty of Science, Mahidol University, Thailand. He was then awarded an overseas scholarship from the royal Thai government to carry his PhD at Imperial College in London and completed the degree in 2010. His thesis title is “characterization of neural progenitor/stem cells derived from human embryonic stem cells” which aims to understand the molecular mechanisms underlying neural differentiation process. He scopes his current research of interest to uncovering the regulation mechanisms of neural stem cells, cancer, and natural compounds, which will be ultimately benefit both basic research and clinical applications.

Neuroblastoma is the most common cancer of the sympathetic nervous system in children. Here, the influences of curcumin on survival, apoptosis, migration, and its combined effects with doxorubicin were investigated in SH-SY5Y cells by cell survival assay, flow cytometry, migration assays, and RT-PCR. Curcumin inhibited SH-SY5Y cell growth and induced apoptosis in dose- and time-dependent manners. This apoptotic induction relied on the upregulation of p53 and p21. Moreover, the treatment of curcumin for 24 hours significantly suppressed cell migration, together with the downregulation of matrix metalloproteinase-2 (MMP-2) and upregulation of tissue inhibitor of metalloproteinases-1 (TIMP-1). The combination of curcumin augmented the anticancer activity of doxorubicin, and significantly induced apoptosis. Pretreatment with curcumin increased the fraction of doxorubicin-induced apoptotic cells from 21.76±0.50% to 57.74±2.68%. Co-treatment with doxorubicin plus curcumin further inhibited 3D tumor migration. Altogether, the results suggest that curcumin suppresses growth and migration of SH-SY5Y cells, and enhances the anticancer activity of doxorubicin. The addition of curcumin to therapeutic regimens may be promising for the treatment of neuroblastomas, if a number of problems related to its in vivo bioavailability can be resolved.

Asst. Prof. Dr Parinya Noisa is a stem cell biologist at Suranaree University of Technology in Thailand. He completed B.Sc in Biology with the first class of honors from the faculty of Science, Mahidol University, Thailand. He was then awarded an overseas scholarship from the royal Thai government to carry his PhD at Imperial College in London and completed the degree in 2010. His thesis title is “characterization of neural progenitor/stem cells derived from human embryonic stem cells” which aims to understand the molecular mechanisms underlying neural differentiation process. He scopes his current research of interest to uncovering the regulation mechanisms of neural stem cells, cancer, and natural compounds, which will be ultimately benefit both basic research and clinical applications.

Human neuroblastoma is the most common cancer of the sympathetic nervous system in children. Neuroblastoma remains a challenging therapeutic target, especially when presents with metastatic diseases. In this study, the effects of curcumin on human neuroblastoma cells were examined by using SH-SY5Y cells as a model. The influences of curcumin on cell survival, apoptosis, and migration, as well as its synergistic effect on anticancer drug were investigated. Curcumin inhibited SH-SY5Y cell growth and induced apoptosis in dose- and time-dependent manners. This apoptotic induction by curcumin relied on the upregulation of p53 and p21 levels. Moreover, the treatment of curcumin for 24 hours significantly suppressed cell migration, along with the decrease of MMP-2 and TIMP-1 gene expression. The combined treatment of 5 µg/mL doxorubicin with either 10 or 20 µM curcumin significantly augmented the anticancer activity of doxorubicin, and markedly induced apoptotic cell death. This synergistic effect of curcumin on doxorubicin was also found in the pretreating experiments, where SH-SY5Y cells were challenged with high-doses (50, 100 and 200 µM)/short-exposure times (3 hours) of curcumin, prior to the 24-hour administration of 0.05 µg/mL doxorubicin. The pretreatment of 200 µM curcumin led to the enhancement of apoptotic cell death, from 21.76±0.50% of doxorubicin alone to 57.74±2.68%. Taken together, this study suggested that curcumin suppressed the growth and migration of human neuroblastoma SH-SY5Y cells, as well as synergized the anticancer activity of doxorubicin. This study implied that curcumin could be used as a potent anticancer agent in the combination with conventional chemotherapeutic drugs, and serving a promising strategy for chemoresistant human neuroblastomas.

The Rheumatoid arthritis is a chronic, systemic inflammatory disorder that mainly attacks the joints. It also affects several tissues and organs such as lungs, pleura, pericardium, heart, and sclera. This produces an inflammatory sinovitis that often progresses to the destruction of the articular cartilage and ankylosis of the joints. About 1%of the world’s population is affected by rheumatoid arthritis. Woman are affected three times more often than men. Onset is most frequent between the ages of 40 and 50 but people of any age can be affected. In siddha medicine the same clinical symptoms are described under the Vali Azhal keel vayu. This is an observational case study and it was conducted at Rural Siddha Hospital, Kodikamam, Jaffna. The permission for this study from the MOIC, Rural Siddha Hospital, Kodikamam, Jaffna. Patient consent was taken for this study in written from to take necessary photographs. One female known Rheumatoid arthritis patient was selected and she was treated three weeks in the ward. Siddha and Ayurvedic drugs only were given internally and externally. More over the dietary regiments also were advised. Before and after the treatment the progresses of the disease were recorded as photograph and on BHT. The Rheumatoid factor was 46 IU/L after the treatment while it was 185 IU/L Before treatment. The pain and swelling of Right knee joint were markedly decreased after the treatment and the degree of swelling and pain were estimated in certain conditions. There for according this clinical observation we can recommend this treatment method as an efficient method for management of rheumatoid arthritis patients.

Quorum-sensing (QS) signaling systems of pathogens are central regulators for the expression of virulence factors and represent highly attractive targets for the development of novel therapeutics especially for antimicrobial resistant pathogens like Pseudomonas aeruginosa. P. aeruginosa is a gram-negative nosocomial pathogen that can cause serious complications in immuno-compromised patients through tissue damage by producing QS-controlled virulence factors thus, considered by the World Health Organization as one of the highest priority in terms of research and development of new antibiotics. The use of natural products as antibiotics is given attention in the recent years. Secondary metabolites have shown antimicrobial activity and some are potential QS inhibitors. The aim of this study is to determine the quorum-sensing inhibition activity of Cordyline fruticosa (L.) A. Chev. leaf extract on the virulence factors of P. aeruginosa. Extraction was carried out by percolation using methanol as solvent and gave 11.50% yield. Thin layer chromatography was used to ascertain the presence of secondary metabolites in the extract and results showed the presence of flavonoids, alkaloids, tannins, phenols, anthraquinones, and anthrones. Acute oral toxicity testing was performed following Organisation for Economic Co-operation and Development (OECD) 425 guidelines. The median lethal dose (LD50) obtained is greater than 2000mg/kg. Liquid dilution method was performed to determine the Minimum Inhibitory Concentration (MIC) and was found to be 0.55 mg/ml. Quorum-sensing inhibition activity of the extract was determined by measuring degree of inhibition of the virulence factors namely pyocyanin production, swarming motility, proteolytic activity, and biofilm formation of P. aeruginosa. Results showed that the QS inhibition activity is dose dependent. A dose as low as 0.1375 mg/ml was effective and was statistically not significant with Aspirin (6mg/ml) on its ability to inhibit pyocyanin production, swarming motility and biofilm formation at α-0.05. A higher dose of 0.275mg/mL was able to inhibit proteolytic activity in the same degree as Aspirin as statistical comparison results showed no significant difference between the two at α-0.05. Results therefore suggests that a dose of 0.275 mg/ml concentration of C. fruticosa leaf extract inhibited the four virulence factors and may be used as treatment for infections caused by P. aeruginosa. The study suggests that this action may be another way of targeting resistant infections caused by P. aeruginosa through quorum-sensing inhibition.