Mohammad Razak Hamdan

Effect of Extraction Techniques on Phenolic Content, Antioxidant and Antimicrobial Activity of Bauhinia purpurea: HPTLC Determination of Antioxidants

Bauhinia purpurea leaf was extracted by Soxhlet, ultrasonication and maceration extraction methods using ethanol (99.5%, v/v) to obtain Soxhlet (SBE), ultrasonicated (UBE) and macerated (MBE) B. purpurea leaf extract. The effects of different extracting methods on the polyphenolic content and antioxidant activities using 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), ferric-reducing antioxidant power (FRAP) and total antioxidant capacity (TAC) were investigated. Disc diffusion and broth dilution methods were also carried out to find the antibacterial activity of these extracts. Findings of this study showed that UBE possessed significant (P < 0.05) polyphenolic constituents followed by MBE and SBE. All the extracts exhibited good DPPH and ABTS radical scavenging as well as potential reducing ability in TAC and FRAP methods. UBE possessed significant (P < 0.05) radical scavenging activity and reducing ability followed by MBE and SBE. Even the results of antibacterial activity were similar to antioxidant activity, with UBE inhibiting most of the bacteria followed by MBE and SBE. All the extracts were subjected to thin layer chromatography (TLC) bioautography followed by high-performance TLC densitometric determination, and the results show that extraction using ultrasonication method yields the highest amount of antioxidant compounds among the three methods mentioned earlier. This study confirms ultrasonic extraction to be an ideal, simple and rapid method to obtain antioxidant- as well as antibacterial-enriched B. purpurea leaf extract. The HPTLC fingerprint profile can be used as a reference data for the standardisation of B. purpurea leaf.

TLC–Bioautography-Guided Isolation, HPTLC and GC–MS-Assisted Analysis of Bioactives of Piper betle Leaf Extract Obtained from Various Extraction Techniques: In vitro Evaluation of Phenolic Content, Antioxidant and Antimicrobial Activities

The polyphenolic content, antioxidant and antibacterial activities of the ethanolic extracts of Piper betle leaf obtained from soxhlet (PBSx), sonication (PBSn) and maceration (PBMn) extraction methods were studied. GC–MS analysis was carried out to determine the variation in the phytoconstituents in these extracts. Whereas, thin-layer chromatography (TLC)–bioautography was conducted to localise, separate, and identify antioxidants, and their amount was determined by the newly developed high-performance thin-layer chromatography (HPTLC) method. The results of polyphenolic content, antioxidant assays and antimicrobial assay showed that PBSn contained significant amount of polyphenolics, antioxidant and antimicrobial activity followed by PBMn and PBSx. Moreover, the obtained antioxidant activity of PBSn was significant even in comparison with butylated hydroxytoluene (BHT) and commercially available grape seed extract (GRSx). In addition, GC–MS analysis shown marked variations in the amount of the phytoconstituents among all these extracts with PBSn containing higher amount followed by PBMn and PBSx. TLC bioautography resulted in the separation of three compounds which are identified as eugenol, allylpyrocatechol, and eugenyl acetate. The HPTLC densitometric determination was also supported the results of antioxidant assays by revealing the presence of higher amount of identified antioxidants in PBSn followed by PBMn and PBSx. Since, P. betle leaf extract has been used as one of the ingredients in several herbal formulations, results of this study will not only help the herbal industries in choosing the appropriate extraction technique but also the developed HPTLC method was simple, precise, sensitive and accurate hence can be utilised for the routine quality control and standardisation of formulations containing P. betle leaf extract.

Flavonoids-Rich Orthosiphon stamineus Extract as New Candidate for Angiotensin I-Converting Enzyme Inhibition: A Molecular Docking Study

This study aims to evaluate the in vitro angiotensin-converting enzyme (ACE) inhibition activity of different extracts of Orthosiphon stamineus (OS) leaves and their main flavonoids, namely rosmarinic acid (RA), sinensetin (SIN), eupatorin (EUP) and 3′-hydroxy-5,6,7,4′-tetramethoxyflavone (TMF). Furthermore, to identify possible mechanisms of action based on structure–activity relationships and molecular docking. The in vitro ACE inhibition activity relied on determining hippuric acid (HA) formation from ACE-specific substrate (hippuryl-histidyl-leucine (HHL)) by the action of ACE enzyme. A High Performance Liquid Chromatography method combined with UV detection was developed and validated for measurement the concentration of produced HA. The chelation ability of OS extract and its reference compounds was evaluated by tetramethylmurexide reagent. Furthermore, molecular docking study was performed by LeadIT-FlexX: BioSolveIT’s LeadIT program. OS ethanolic extract (OS-E) exhibited highest inhibition and lowest IC50 value (45.77 ± 1.17 µg/mL) against ACE compared to the other extracts. Among the tested reference compounds, EUP with IC50 15.35 ± 4.49 µg/mL had highest inhibition against ACE and binding ability with Zn (II) (56.03% ± 1.26%) compared to RA, TMF and SIN. Molecular docking studies also confirmed that flavonoids inhibit ACE via interaction with the zinc ion and this interaction is stabilized by other interactions with amino acids in the active site. In this study, we have demonstrated that changes in flavonoids active core affect their capacity to inhibit ACE. Moreover, we showed that ACE inhibition activity of flavonoids compounds is directly related to their ability to bind with zinc ion in the active site of ACE enzyme. It was also revealed that OS extract contained high amount of flavonoids other than RA, TMF, SIN and EUP. As such, application of OS extract is useful as inhibitors of ACE.

Tris[3,5-bis­(trifluoro­meth­yl)phen­yl]phosphine oxide

In the title compound, C24H9F18OP, an intramolecular C—H⋯O short contact generates a five-membered ring, producing an S(5) ring motif. The dihedral angles between the benzene rings are 57.68 (10), 77.80 (11) and 79.48 (10)°. Each of the six trifluoromethyl substituents shows rotational disorder over two positions with refined site-occupany ratios of 0.64 (3)/0.36 (3), 0.649 (14)/0.351 (14), 0.52 (2)/0.48 (2), 0.545 (16)/0.455 (16), 0.774 (9)/0.226 (9) and 0.63 (5)/0.37 (5). The crystal structure is stabilized by intermolecular C—H⋯O and C—H⋯F interactions.

Evaluation of genetic diversity of Clinacanthus nutans (Acanthaceaea) using RAPD, ISSR and RAMP markers

Three polymerase chain reaction (PCR) techniques were compared to analyse the genetic diversity of Clinacanthus nutans eight populations in the northern region of Peninsular Malaysia. The PCR techniques were random amplified polymorphic deoxyribonucleic acids (RAPD), inter-simple sequence repeats (ISSR) and random amplified microsatellite polymorphisms (RAMP). Leaf genomic DNA was PCR amplified using 17 RAPD, 8 ISSR and 136 RAMP primers . However, only 10 RAPD primers, 5 ISSR primers and 37 RAMP primers produced reproducible bands. The results were evaluated for polymorphic information content (PIC), marker index (MI) and resolving power (RP). The RAMP marker was the most useful marker compared to RAPD and ISSR markers because it showed the highest average value of PIC (0.25), MI (11.36) and RP (2.86). The genetic diversity showed a high percentage of polymorphism at the species level compared to the population level. Furthermore, analysis of molecular variance revealed that the genetic diversity was higher within populations, as compared to among populations of C. nutans. From the results, the RAMP technique was recommended for the analysis of genetic diversity of C. nutans.

Abstract B73: Chemopreventive effect of Streblus asper, a bonsai plant, on osteosarcoma cells: A preliminary study

Epidemiological studies have demonstrated a positive correlation between consumption of vegetables, fruits and beverages with reduced risk of cancer. It is estimated there are around 8,100 plant species in the Malaysian rain forests, with 10% of them reported to have some medicinal value. However, to date in Malaysia, not much investigation has been done on chemopreventive activities on cancer although Malaysian plants are an exclusive source of effective chemopreventive agents and therefore, this background leads to the premise that our local plants such as Streblus asper could have greater potential for the chemoprevention activities. Streblus asper is well known as an expensive bonsai plant which is indigenous to tropical countries such as Malaysia, Thailand, Sri Lanka and India. It is used traditionally in leprosy, piles, diarrhea, dysentery, elephantiasis and cancer. It finds place in Ayurvedic Pharmacopoeia of India and also been described in some monographs, but none have reported its activity as chemopreventive agents and the underlying mechanisms remain unclear. In the present study, we try to identify its biological properties and the cytotoxicity effect on normal liver and kidney cells. Using the osteosarcoma cells as our in vitro model, the IC50 of Streblus asper root extract was determined and observed its effect on the osteosarcoma cells morphology that changes within time, the anti‐proliferative pattern and live‐death analysis under confocal microscope analysis. The results showed that the root extracts did not contained any heavy metals compound such as mercury and cadmium and with less arsenic (0.02 ppm) and plumbum (0.07 ppm) and showing non‐cytotoxic effect on vero cells (normal kidney cells) and WRL‐68 cells (normal liver cells). Our HPLC profiling analysis also revealed antioxidants compounds exist in the Streblus asper root extracts such as caffeic acid which has been shown to act as a carcinogenic inhibitor. Although the low‐level of anti‐oxidants been found from the extracts but it still can inhibit the growth of the osteosarcoma cells which also exert apoptosis features like cell shrinkage (atrophy) and vocalization in time and dose‐dependent manner. The plant extracts IC50 doses was at 0.05% of root extracts and it also demonstrated the anti‐proliferative effect by suppressed the cells growth as early as 12 hours of treatment and marked cell death till day 6. On live‐death analysis under confocal microscope using Calcein and Ethidium staining confirmed that Streblus asper root extract exert cell death to osteosarsoma cells. This study is just a preliminary study as to identify its pharmacological properties on carcinogenesis and further investigation is still on‐going to develop it as the chemopreventive agent especially to determine the signaling pathway involved. Citation Information: Cancer Prev Res 2010;3(1 Suppl):B73.

Assessment of three plastid DNA barcode markers for identification of Clinacanthus nutans (Acanthaceae)

This study was conducted to determine the feasibility of using three plastid DNA regions (matK, trnH-psbA, and rbcL) as DNA barcodes to identify the medicinal plant Clinacanthus nutans. In this study, C. nutans was collected at several different locations. Total genomic DNA was extracted, amplified by polymerase chain reaction (PCR), and sequenced using matK, trnH-psbA, and rbcL, primers. DNA sequences generated from PCR were submitted to the National Center for Biotechnology Information’s (NCBI) GenBank. Identification of C. nutans was carried out using NCBI’s Basic Local Alignment Search Tool (BLAST). The rbcL and trnH-psbA regions successfully identified C. nutans with sequencing rates of 100% through BLAST identification. Molecular Evolutionary Genetics Analysis (MEGA) 6.0 was used to analyze interspecific and intraspecific divergence of plastid DNA sequences. rbcL and matK exhibited the lowest average interspecific distance (0.0487 and 0.0963, respectively), whereas trnH-psbA exhibited the highest average interspecific distance (0.2029). The R package Spider revealed that trnH-psbA correctly identified Barcode of Life Data System (BOLD) 96%, best close match 79%, and near neighbor 100% of the species, compared to matK (BOLD 72%; best close match 64%; near neighbor 78%) and rbcL (BOLD 77%; best close match 62%; near neighbor 88%). These results indicate that trnH-psbA is very effective at identifying C. nutans, as it performed well in discriminating species in Acanthaceae.