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PROFESSOR GERALD BLUNDEN The School of Pharmacy & Biomedical Sciences,

University of Portsmouth, Portsmouth, PO1 2DT U.K.

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Identification and in vitro Evaluation of Lipids from Sclerotia of Lignosus rhinocerotis for Antioxidant and Anti-neuroinflammatory Activities Neeranjini Nallathambya,b, Lee Guan Serma,b, Jegadeesh Raman,a Sri Nurestri Abd Maleka,b, Sharmili Vidyadarana,c, Murali Naidua,e, Umah Rani Kuppusamya,d and Vikineswary Sabaratnama,b* aMushroom Research Centre, University of Malaya, 50603 Kuala Lumpur, Malaysia

bInstitute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia

cImmunology Laboratory, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 Serdang, Malaysia

dDepartment of Biomedical Science, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia

eDepartment of Anatomy, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia viki@um.edu.my

Received: February 5th, 2016; Accepted: May 18th, 2016

Lignosus rhinocerotis (Cooke) Ryvarden (Tiger milk mushroom) is traditionally used to treat inflammation triggered symptoms and illnesses such as cough, fever and asthma. The present study evaluated the in vitro antioxidant, cytotoxic and anti-neuroinflammatory activities of the extract and fractions of sclerotia powder of L. rhinocerotis on brain microglial (BV2) cells. The ethyl acetate fraction had a total phenolic content of 0.30 ± 0.11 mg GAE/g. This fraction had ferric reducing capacity of 61.8 ± 1.8 mg FSE/g, ABTS•+ scavenging activity of 36.8 ± 1.8 mg TE/g and DPPH free radical scavenging activity of 21.8% ± 0.7. At doses ranging from 0.1 µg/mL – 100 µg/mL, the extract and fractions were not cytotoxic to BV2 cells. At 100 µg/mL, the crude hydroethanolic extract and the ethyl acetate fraction elicited the highest nitric oxide reduction activities of 68.7% and 58.2%, respectively. Linoleic and oleic acids were the major lipid constituents in the ethyl acetate fraction based on FID and GC-MS analysis. Linoleic acid reduced nitric oxide production and down regulated the expression of neuroinflammatory iNOS and COX2 genes in BV2 cells. Keywords: Anti-neuroinflammation, Lignosus rhinocerotis, BV2 Cells, Lipid component, Linoleic acid, Oleic acid, Antioxidant. Mushrooms are consumed globally and are valued not only for their unique taste and flavor but also for their high medicinal and nutritional properties [1]. In recent years, the search for mushrooms is focused on ethnomedicinal knowledge. In Malaysia, the indigenous communities use many species of mushrooms such as Amauroderma sp., Lignosus rhinocerotis (Cooke) Ryvarden, Pycnoporus sanguineus (L.) Murrill and Termitomyces clypeatus (R.) Heim as food and/or medicine [2]. Many of these species are used to treat a number of ailments related to inflammation such as fever, cough, cold, epilepsy and asthma [2]. L. rhinocerotis can be found in small geographic regions encompassing South China, Thailand, Malaysia, Indonesia, Philippines, Papua New Guinea, New Zealand, and Australia [3]. In Malaysia, this mushroom is also known as “cendawan susu rimau”, which translates to tiger milk mushroom. L. rhinocerotis has more than 15 traditional uses including treatment or prevention of cancer, fever, cough, asthma, hunger, food poisoning, wounds and it is also used as a general tonic [4]. Asthma, fever and cough are attributes of inflammation. Recent in vitro and in vivo studies supported the medicinal properties including the anti-inflammatory activities of the aqueous extract L. rhinocerotis (Table 1). To date, however, there are no reports on the anti-neuroinflammatory activities of solvent extracts. Therefore, this study aimed to investigate the hydroethanolic extract of the sclerotia of L. rhinocerotis and its fractions. The extracts and fractions were examined to determine their chemical composition, antioxidant activities, cytotoxicity and effect on nitric oxide production in microglial cells.

Table 1: Medicinal properties of sclerotia of L. rhinocerotis

Hydroethanolic extraction yielded 234.2 g of dark brown extract from an initial 2.5 kg freeze dried mushroom powder (that is 93.7 mg/g freeze dried powder). The n-hexane fraction yield was 16.8 g (6.7 mg/g freeze dried powder) whilst the ethyl acetate fraction was 9.9 g (4.0 mg/ g freeze dried powder). Both n-hexane and ethyl acetate fractions of L. rhinocerotis were dark brown coloured resinous oil. The highest TPC was observed in the crude hydroethanolic extract (0.4 ± 0.1 mg GAE/g extract). However, the n-hexane and ethyl acetate fractions contained low TPC of 0.1 ± 0.0 and 0.3 ± 0.1 mg GAE/g extract, respectively. The FRAP assay showed that the hydroethanolic extract had the highest ferric reducing ability of 122.6 ± 4.8 mmol FSE/g extract. The ABTS•+ scavenging activity was expressed in terms of TEAC. The higher the TEAC value, the more potent the radical scavenging effect. The most potent radical

Medicinal properties Active extracts Cell line References Anti-cancer cold aqueous

aqueous

breast (MCF 7) and lung (A549) cancer leukemia (HL-60, K562 and THP-1)

[5]

[6]

Immunomodulating Polysaccharides Immune cells [7] Neurite outgrowth hot aqueous

hot aqueous

PC 12 N2a and BALB/3T3

[8,9] [10]

Antioxidant cold aqueous, hot aqueous

and methanol

[11]

Anti-inflammatory cold aqueous, hot aqueous

and methanol

carrageen induced paw edema in Sprague

Dawley rats

[12]

NPC Natural Product Communications 2016 Vol. 11 No. 10

1485 - 1490

1486 Natural Product Communications Vol. 11 (10) 2016 Nallathamby et al.

scavenger was the hydroethanolic extract with 86.5 ± 4 mg TE /g and the least potent extract was the n-hexane fraction with only 30.9 ± 5.3 mg TE /g. At a concentration of 5 mg/mL, the hydroethanolic extract showed the highest DPPH radical scavenging activity of 29.4 ±1.7% followed by the ethyl acetate fraction (21.8±0.7%) and n-hexane fraction (17.2 ±1.2%). This study demonstrated that the hydroethanolic extract and its fractions (n-hexane and ethyl acetate fractions) possessed free radical reduction and scavenging activities. However, each extract showed different in vitro assay patterns, probably due to the different mechanisms involved in the steps of the oxidation process. Some studies found a correlation between the phenolic content and the antioxidant activities, while others did not [13-15]. In this study the hydroethanolic extract and ethyl acetate fraction showed high ABTS+ (36-86 mg TE/g extract) and DPPH (21-30%) scavenging activities and free radical reduction (61-122 mg FE/g extract), but the antioxidant activities were not correlated with its total phenolic content. Hassimotto et al. [13] also reported that the antioxidant activity of vegetable and fruit extracts did not correlate with either phenolics or vitamin C content. Thus, other non-phenolic compounds such as fatty acids may also be responsible for the antioxidant activity observed in the hydroethanolic extract and ethyl acetate fraction. Li et al. [16] also reported that the ethanolic extract of Corpinus comatus mushroom possessed higher antioxidant activity compared with its hot water extract. The effects of various concentrations of the hydroethanolic extract and fractions of L. rhinocerotis on the viability of BV2 cells determined by the MTS assay are given in Figure 1. The cell viability of the positive control (untreated BV2 cells) was denoted as 100%. The crude extract/fractions were not cytotoxic to BV2 cells at concentrations up to 100 µg/mL. However, at 1000 µg/mL all extracts tested were cytotoxic to the BV2 cells. A dose-dependent increase in the viability of cells treated with the extracts was observed at concentrations ranging from 0.1 to 100 μg/mL followed by a dose-dependent decrease from 100 to 1000 μg/mL. An increase of 4-10% in viable cell number was seen in BV2 cells treated with 10 μg/mL of each extract tested. However, there was no significant (p<0.05) difference in cytotoxic effects at concentrations 0.1 μg/mL – 100 μg/mL compared with the positive control. Hence, in all subsequent assays, 1000 µg/mL concentration of extract/fractions was omitted. Lee et al. [5] demonstrated that L. rhinocerotis cold water extract (LR-CW) did not show significant cytotoxic effect on human normal breast and lung cell lines (184B5 and NL 20) at concentrations ranging from 15.6 to 1000 μg/mL. However, anti-proliferative activity against both MCF-7 and A549 cancer cell lines was exhibited. The concentrations used were similar to the range of concentration used in the present study. Further, 0.1-100 μg/mL concentrations were used in this study to determine the anti-inflammatory effects on BV2 cells. The effect of L. rhinocerotis crude extract/fractions (0.1 to 100 µg/mL) on NO production by LPS stimulated BV2 cells is presented in Figure 2. The LPS stimulation of the cells resulted in an increase in NO production (39.1 ± 0.8 µM) compared with the unstimulated cells basal levels (0.7 ± 0.2 µM). L-NAME, a commercial nitric oxide suppressant, was used as a positive control at 200 µM. It was able to suppress 50% of NO production in the LPS induced BV2 cells. A dose dependent inhibition of NO production from 14% to 69% in BV2 cells occurred when cells were treated with the hydroethanolic extract at concentrations from

Figure 1: Effects of L. rhinocerotis extract and fractions on the number of viable BV2 cells. Data represent the mean ± SD from triplicate values in three independent experiments. * denotes p < 0.05 compared with the corresponding value of the control. Control (BV2) = 100%.

1 – 100 µg/mL. At 100 µg/mL, the hydroethanolic extract inhibited the production of NO by 50% in BV2 cells. At 0.1 µg/mL, there was an increase of NO production by BV2 cells for both fractions (n-hexane and ethyl acetate). A dose dependent inhibition of NO production ranging from 12% to 60% by BV2 cells was obtained when the cells were treated with n-hexane and ethyl acetate fractions at concentrations of 1 - 100 µg/mL. The ethyl acetate fraction showed the highest NO inhibition (58.2%) at 100 µg/mL compared with the n-hexane fraction (28.7%).

Figure 2: Effects of L. rhinocerotis extracts on LPS-induced NO production in BV2 microglial cells. Data represent the mean ± SD from triplicate values in three independent experiments. * denotes significant difference at p < 0.05 when compared with BV2 cells treated with LPS.

In general, inflammation is a naturally occurring reaction in the body in response to trauma, infection and tissue injury [17]. Although activated microglia scavenge dead cells from the CNS and secrete different neurotrophic factors for neuronal survival, overproductions of activated microglia may lead to neuronal death and brain injuries [18]. The activated cells also increase the secretion of various pro-inflammatory mediators such as nitric oxide (NO), prostaglandin E2 (PGE2), and cytokines. Nitric oxide, a short-lived free radical produced from L-arginine by nitric oxide synthase (NOS), mediates [19] a variety of pathophysiological actions ranging from vasodilatation, neurotransmission, inhibition of platelet adherence and aggregation, as well as the macrophage- and neutrophil-mediated killing of pathogens [20]. Overproduction of these mediators is responsible for inflammation. Therefore, inhibition of proinflammatory mediator(s) is beneficial in attenuating an inflammatory disorder. In the last few decades, evidence suggests that excessive NO production may play a role in neurodegenerative diseases.

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The hydroethanolic extract and its ethyl acetate fraction significantly (p < 0.05) inhibited (> 60%) NO production compared with the control. These extracts also showed significant (p < 0.05) antioxidant properties. A study with Tricholoma matsutake Sing (pine mushrooms) showed that the ethyl acetate fraction exhibited the highest inhibition (61.6%) of NO by BV2 cells [21], similar to the findings in this study. Further, Houttuynia cordata, a traditional plant used as folk medicine for treating several ailments including allergic inflammation and anaphylaxis showed that HC-EA (ethyl acetate fraction) inhibited the LPS-stimulated increase of NO release by BV2 cells in a concentration-dependent manner [22]. Eight major lipid components, comprising 41.7% of the total components detected in the ethyl acetate fraction, were identified using GC-MS (Table 2). Two other components amounting to 38.2% of the total oil were not identified. The three major components were identified as linoleic acid (23.3%), ethyl linoleate (8.1%) and oleic acid (2.1%). About 25% of the total identified components in the ethyl acetate fraction were linoleic acid and oleic acid. The presence of these fatty acids as major components instead of phenolic compounds may have contributed to the inhibition of NO production. This correlates with the antioxidant findings in the present study in that the ethyl acetate fraction has a high antioxidant activity but relatively low TPC levels. The dominant components; linoleic acid, ethyl linoleate and oleic acid were tested individually to measure their effect on viability and nitrite production in BV2 cells. The results are shown in Figure 3. The positive control (untreated BV2 cells) for cell viability was denoted as 100%. The unstimulated cell basal levels (0.2 µM ± 0.6) of NO were detected while LPS stimulation of the cells resulted in an increase in NO production (81.2 µM ± 2.1). The major compounds were not cytotoxic to BV2 cells at concentrations up to 100 µg/mL. Increase in the concentrations of linoleic acid led to a decrease in NO production. There was a significant (p < 0.05) reduction of 57% in NO production when compared with the control at 100 µg/mL concentration. The ethyl linoleate and oleic acid treatments did not show any significant (p < 0.05) reduction of NO in BV2 cells. The major fatty acids present in the EA fraction were linoleic acid, ethyl linoleic and oleic acid. These essential unsaturated fatty acids cannot be synthesised in mammalian tissue, therefore, must be obtained from the diet [23]. The identified major compound, linoleic acid, was not obtained in the findings of Lau et al. [24]. However, in this study, only linoleic acid demonstrated anti- inflammatory activity via reduction of nitric oxide production without exerting any cytotoxic effect on the cells. There have been many studies on the biological effects of these fatty acids and growing evidence that they modify neuronal membrane structure and functions [25]. Low concentrations of several unsaturated fatty

Table 2: Chemical constituents of the ethyl acetate fraction of L. rhinocerotis. RT (min)

Chemical constituent

Molecular formula

Molecular weight

Area Area (%)

24.717 Palmitic acid C16H32O2 256.4 172.0 4.8 25.393 Ethyl palmitate C18H36O2 284.5 81.2 2.2 27.471 Methyl linoleate C19H34O2 294.5 15.9 0.4 27.573 Methyl oleate C19H36O2 296.5 7.0 0.2 28.305 Linoleic acid C18H32O2 280.5 842.9 23.3 28.788 Ethyl linoleate C20H36O2 308.5 293.1 8.1 28.873 Oleic acid C18H34O2 282.5 74.2 2.1 29.316 Ethyl stearate C20H40O2 312.5 20.9 0.6 37.927 Unidentified 293.7 8.1 48.370 Unidentified 1091.2 30.1

2892.1 79.9

acids such as oleic, linoleic and linolenic acid may cross the blood brain barrier (BBB) [26]. Real time PCR was performed to investigate the effect of linoleic acid on LPS stimulated iNOS and COX 2 genes which are involved in the proinflammatory response. After LPS stimulation for 24 h, the expression of iNOS and COX 2 increased by 1.5 fold and 2-fold, respectively, when compared with the control (cDNA of unstimulated BV2 cells), as shown in Figure 4. Treatment with linoleic acid significantly (p < 0.05) decreased both iNOS and COX2 expression by 1.2 fold compared with the LPS (negative control). Unsaturated fatty acid components are able to penetrate into the cells to reduce NO production of glial cells by down regulating the expression of iNOS. The major lipid component, linoleic acid, caused a significant (p<0.05) reduction of iNOS and COX2 gene expression. Linoleic acid down regulated the expression of the proinflammatory genes, iNOS (30%) and COX2 (15%), lower than in aspirin treated cells. Aspirin is a non-steroidal anti-inflammatory drug (NSAID) known to exert its effects through inhibition of COX2. Pharmacological inhibition of COX2 can provide relief from the symptoms of inflammation and pain. In this study, linoleic acid was demonstrated to mimic aspirin in reducing inflammation via the COX2 mediated pathway. Yu et al. [27] had shown that linoleic acid reduced NO production by LPS activated cells and decreased the IFNγ-dependent expression of inducible NOS (iNOS) and iNOS promoter activity. Linoleic acid is also one of the fatty acids responsible for the inhibition of COX2 catalysed prostaglandin biosynthesis, as shown in Plantago major L. (Plantaginaceae) [28]. Excess NO may be produced by a higher promoter activity of iNOS and to induce also COX2 in various in vitro and in vivo models causing chronic inflammation [29]. Inflammatory cytokines, iNOS and COX2 are known downstream genes of nuclear factor-κB (NF-κB) and signal transducers and activators of transcription 3 (STAT3) pathways. These are the two most important transcription factors in the inflammation pathway. Thus linoleic acid may inhibit inflammation via either the NF-κB pathway, STAT3 pathway or both pathways [30].

Figure 3: Effect of treatment with major lipid components on normalised cell number and nitrite production in BV2 cells. The bar represents nitrite production (µM) and the line represents normalised cell number. The * symbol denotes a significant difference (p < 0.05) from the corresponding value of the control.

1488 Natural Product Communications Vol. 11 (10) 2016 Nallathamby et al.

Figure 4: Effects of linoleic acid on iNOS and COX2 expression in LPS stimulated BV2 cells. Fold increase values are calculated relative to the ACTB gene. BV2= untreated cells, LPS= LPS stimulated cells, ASP= Aspirin, LA= linoleic acid treatment on LPS stimulated BV2 cells. Data represent the mean ± SE and from a representative experiment carried out in triplicates. * denotes p < 0.05 compared with the negative control (LPS).

In conclusion, the EA fraction has the potential to suppress inflammation by reducing NO/iNOS and COX2 proinflammatory genes and to supress inflammation via NF-κB and the STAT3 pathway. The presence of linoleic acid as the major bioactive lipid component may have contributed to the anti-oxidant and anti neuroinflammatory activities. However, the synergistic effect of the components in the ethyl acetate fraction may have also played a role in inhibiting neuroinflammation. To our knowledge this is the first report on the chemical constituents of the sclerotia of L. rhinocerotis and its in vitro anti-neuroinflammatory activity on BV2 cells. Experimental

Materials: Freeze dried powdered sclerotia of L. rhinocerotis (TM02; commercial cultivar) were purchased from Ligno Biotech, Selangor, Malaysia, and linoleic (L1367) and oleic acids (O1008) from Sigma, USA. Extraction and fractionation of sample: The powdered sclerotia were extracted and fractionated for biological screening according to a method described earlier [31]. Freeze dried powder (2.5 kg) was soaked in hydroethanol 80% and kept at room temperature for 2 days. The extract was filtered using a vacuum filter and the filtrate was concentrated on a rotary evaporator at 45°C (Buchi, Switzerland) under reduced pressure. This process was repeated 5 times; the filtrate from each extraction was concentrated and combined to obtain the crude hydroethanolic extract. This was further fractionized with n-hexane to yield a hexane soluble and hexane insoluble fractions, which were further partitioned with ethyl acetate: water mixture (1:1) by a counter current technique. The ethyl acetate soluble fraction was separated from the aqueous layer. Total phenolic content (TPC) estimation: The TPC assay was conducted using the method outlined by Cheung et al. [32]. The absorbance of the sample was measured at 750 nm in a microplate reader (BioTek Instruments, USA). Gallic acid, up to 100 µg/mL, was used as a standard. The TPC results are mean values of

triplicate assays and are expressed as gallic acid equivalents (GAE) per g mushroom (mg GAE/g mushroom extract). Antioxidant activity: The antioxidant potential of the hydro-ethanolic extract of L. rhinocerotis and its n-hexane and ethyl acetate fractions was investigated using the following standard assays. Ferric reducing antioxidant power (FRAP) assay: The FRAP assay was performed using the method described by Benzie and Strain [33]. FRAP reagent was prepared by mixing 50 mL of 300 mM acetate buffer, 5 mL of 10 mM 2,4,6-tripyridyl-s-triazine solution (TPTZ) in 40 mM of hydrochloric acid (HCl) and 5 mL of 20 mM ferric chloride (FeCl3•6H2O) in the ratio of 10:1:1. FRAP reagent (300 µL) was added to 10 μL of mushroom extract plated in a 96 well plate and absorbance was measured at 593 nm after 4 min in microplate reader. The standard used was iron sulfate (FeSO4). FRAP results are mean values of triplicate assays and are expressed in mM FeSO4 equivalent (FSE) per g mushroom (mmol FSE/g mushroom extract). Trolox equivalent antioxidant capacity (TEAC) assay: TEAC was determined by using the method outlined by Re et al. [34]. The absorbance of the reaction mixture was measured at 734 nm in a microplate reader. Trolox was used as the standard. TEAC values are mean values of triplicates assay and expressed as mg Trolox equivalent (TE) per g mushroom extract (mg TE/g mushroom extract). Diphenyl-1-picryl-hydrazyl (DPPH) radical scavenging assay: DPPH radical activity was determined using the method of Brand-Williams et al. [35]. Ascorbic acid at different concentrations was used as standard. Mushroom extract (5 µL) was mixed with 195 µL of a methanolic solution of DPPH radical in a 96 well plate. The mixture was shaken vigorously and left to stand for 3 h in the dark, and the absorbance was measured at 515 nm. The assay was carried out in triplicate. DPPH activity was expressed in DPPH inhibition percentage (%). BV2 cell culture: BV2 cells were maintained in Dulbecco Modified Eagle’s medium (DMEM) supplemented with 5% heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin, 100 µg/mL streptomycin, 1 mL/L gentamycin, 250 µg/mL fungizone, 1X non-essential amino acids, 2 mg/mL insulin and 1.5 g/L sodium bicarbonate. Cultures were maintained at 37C in 95% humidified air and 5% CO2. Cells were harvested by treating with 0.25% trypsin in 1 mM ethylenediaminetetraacetic acid (EDTA) for 5 min at 37C. Cell viability assay: The cytotoxic effects were determined by using a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. This assay was carried out according to the method of Tan et al. [36]. In a 96 well flat-bottomed microplate, 5×104 cells were seeded per well and incubated at 37°C overnight for attachment. Different concentrations of the extract were then added. After 24 h incubation, MTS solution was added and further incubated for 2 h. The absorbance was measured at 490 nm with a microplate reader (Dynex MRX II microplate reader, USA). Each assay was performed in triplicate. Absorbance of all wells was deducted from the absorbance of complete growth medium which served as background reading. Cell number was calculated in comparison to untreated cells.

Lipids from Lignosus rhinocerotis Natural Product Communications Vol. 11 (10) 2016 1489

Nitric oxide determination assay: Nitrite that accumulated in the culture medium was measured as an indicator of NO production based on the Griess reaction. The BV2 cells were plated in a 96 well plate at a density of 5 × 104 cells/well and incubated overnight. Cells were then incubated with different concentrations of the extract (0.01-1000 μg/mL) and 1µg/mL of Escherichia coli (O55:B5) lipopolysaccharide (LPS) (Sigma, US). After 24 h, the culture supernatant was collected for nitrite measurement. Fifty μL of the spent medium were plated in a 96 well plate and 50 μL of Griess reagent (0.1% N-1-[naphthyl]ethylenediamine-diHCl and 1% sulfanilamide and 2.5% H3PO4) was added. The plate was incubated for 15 min, and the absorbance measured at 530 nm using a microplate reader (Dynex MRX II microplate reader, USA). The amount of NO was calculated using a sodium nitrite standard curve [36]. GC-FID and GC-MS analysis: The sample was analyzed using Agilent Technologies gas chromatography (GC) 7890A with FID equipped with a fused silica capillary column HP-5ms, 5% phenylethylsiloxane (30.0 m by 0.25 mm ID by 0.25-lm film thickness). Purified nitrogen was used as carrier gas at a flow rate of 1 mL per min, a split ratio of 1:10 and 2 µL injection volume. The column temperature was programmed initially at 100°C, then increased by 5°C per min to 300°C and was kept isothermally for 20 min. The temperature of the injector port and FID jet was 250°C and 300°C, respectively. Identification of constituents was performed on an Agilent GC 7890A equipped with a 5975C inert mass selective detector (70 eV direct inlet) using the same capillary column as the GC-FID, HP-5ms. The carrier gas was purified helium at a flow rate of 1 mL per min, a split ratio of 1:10 and 1 µL injection volume. The column temperature was programmed as for the GC-FID. The temperatures of the injector port and interface of the MS were 250°C and 270°C, respectively. The constituents were identified by comparison with constituents identified in the mass spectral database (Wiley Registry 9th Edition / NIST 2011 Library, 2011).

RNA extraction and quantitative real time PCR (qPCR): The BV2 cells were lysed and total RNA was extracted as recommended by the manufacturer’s manual using an Ambion-RNAqueous Micro® kit (Applied Biosystems, USA). Complementary DNA (cDNA) was synthesized from the purified RNA using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, USA). Reaction setup for all qualitative real time PCR reactions was performed according to the reaction setup instructions generated by the StepOne software (Ver 2.0, Applied Biosystems). The reactions were carried out with TaqMan® probes (Applied Biosystems, USA), the reaction volume being 20 µL. Briefly, a reaction which consisted of the TaqMan® Gene Expression Master Mix and the assay mix was prepared separately, as each assay mix contained corresponding primers and probe for each gene targeted. Each reaction was assayed in triplicate. The reaction mix was mixed with either sterile ultra-pure water for no template control reactions (NTC) or isolated cDNA. The strips were centrifuged and loaded into the real time PCR thermal cycler (StepOnePlusTM Real Time PCR System). The relative expression of the investigated genes was normalized with the endogenous control (β actin rRNA). Statistical analysis: All data triplicates were recorded as mean ± standard deviation (SD) and from a representative experiment carried out from 3 independent experiments and analysed by SPSS for Windows (ver. 18). One-way analysis of variance (ANOVA) and Dunnett comparisons were carried out to test any significant differences. p < 0.05 was considered statistically significant. Acknowledgments - This research is supported by University Malaya High Impact Research Grant UM-MOHE UM.C/625/1/HIR/MOHE/SC/02 and UM-MOHE UM.C / 625 / 1 / HIR/MOHE/ASG/01 from the Ministry of Education Malaysia; University of Malaya for grants PG110-2012B, J-21001-76536. Authors acknowledge Dr Daniel L. Thomas for the English editing.

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Natural Product Communications Vol. 11 (10) 2016 Published online (www.naturalproduct.us)

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Chemical Profiles and Anti-inflammatory Activity of the Essential Oils from Seseli gummiferum and Seseli corymbosum subsp. corymbosum Alev Tosun, Jaemoo Chun, Igor Jerković, Zvonimir Marijanović, Maurizio A. Fenu, Sena S. Aslan, Carlo I. G. Tuberoso and Yeong S. Kim 1523

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Biological Properties of Some Volatile Phenylpropanoids Radmila Ilijeva and Gerhard Buchbauer 1619

Natural Product Communications 2016

Volume 11, Number 10

Contents

Editorial i

Greeting Message Herbert Ipser and Erich Leitner iiiWolfgang Kubelka and Helmut Viernstein v

Preface Ernst Urban viiNurhayat Tabanca, Iris Stappen and Gerhard Buchbauer x

Original Paper

Analgesic Activity of Novel GABA Esters after Transdermal Delivery Mariia Nesterkina and Iryna Kravchenko 1419

11-Hydroxy-2,4-cycloeudesmane from the Leaf Oil of Juglans regia and Evaluation of its Larvicidal ActivityAyşegül Köroğlu, Ayşe Baldemir, Gülmira Özek, Erdal Bedir, Nurhayat Tabanca, Abbas Ali, Ikhlas A. Khan, Kemal Hüsnü Can Başer and Temel Özek 1421

Insecticidal Pregnane Glycosides from the Root Barks of Periploca sepium Renfeng Li, Ximei Zhao, Baojun Shi, Shaopeng Wei, Jiwen Zhang, Wenjun Wu and Zhaonong Hu 1425

Synthesis and Antimicrobial Activity of Calycanthaceous Alkaloid Analogues Shaojun Zheng, Longbo Li, Yu Wang, Rui Zhu, Hogjin Bai and Jiwen Zhang 1429

Apigenin Suppresses Angiogenesis by Inhibiting Tube Formation and Inducing Apoptosis Hyun Ju Kim and Mok-Ryeon Ahn 1433

A Novel Genistein Prodrug: Design, Synthesis and Bioactivity on Mouse RAW264.7 Macrophages Burkhard Kloesch, Silvia Loebsch, Jenny Breitenbach, Katrin Goldhahn, Norbert Handler, Philipp Schreppel and Thomas Erker 1437

Cytotoxic Effects of Resveratrol, Rutin and Rosmarinic Acid on ARH–77 Human (Multiple Myeloma) Cell Line Zerrin Canturk, Miris Dikmen, Oge Artagan, Mustafa Guclu Ozarda and Nilgun Ozturk 1441

Evaluation of Antioxidant Interactions of Combined Model Systems of Phenolics in the Presence of Sugars Mirela Kopjar, Ante Lončarić, Mateja Mikulinjak, Žaklina Šrajbek, Mihaela Šrajbek and Anita Pichler 1445

HPLC Fingerprint Combined with Quantitation of Phenolic Compounds and Chemometrics as an Efficient Strategy for Quality Consistency Evaluation of Sambucus nigra Berries Agnieszka Viapiana and Marek Wesolowski 1449

In vitro Antioxidant and Antimicrobial Effects of Ceratostigma plumbaginoides Hosam O. Elansary, Kowiyou Yessoufou, Eman A. Mahmoud and Krystyna Skalicka-Woźniak 1455

Enzyme-hydrolyzed Fruit of Jurinea mollis: a Rich Source of (-)-(8R,8’R)-Arctigenin Rita Könye, Ágnes Evelin Ress, Anna Sólyomváry, Gergő Tóth, András Darcsi, Balázs Komjáti, Péter Horváth, Béla Noszál, Ibolya Molnár-Perl, Szabolcs Béni and Imre Boldizsár 1459

Aroma Profile of Galangal Composed of Cinnamic Acid Derivatives and Their Structure-Odor Relationships Toshio Hasegawa, Momohiro Hashimoto, Takashi Fujihara and Hideo Yamada 1463

Natural and Synthetic Furanones with Anticancer Activity Alessio Cimmino, Patrizia Scafato, Veronique Mathieu, Aude Ingels, Wanda D’Amico, Laura Pisani, Lucia Maddau, Stefano Superchi, Robert Kiss and Antonio Evidente 1471

Structural Features for Furan-Derived Fruity and Meaty Aroma Impressions Bettina Wailzer, Johanna Klocker, Peter Wolschann and Gerhard Buchbauer 1475

Phytotoxic Fungal Exopolysaccharides Produced by Fungi Involved in Grapevine Trunk Diseases Alessio Cimmino, Tamara Cinelli, Marco Evidente, Marco Masi, Laura Mugnai, Marcondes A. Silva, Sami J. Michereff, Giuseppe Surico and Antonio Evidente 1481

Identification and in vitro Evaluation of Lipids from Sclerotia of Lignosus rhinocerotis for Antioxidant and Anti-neuroinflammatory Activities Neeranjini Nallathamby, Lee Guan Serm, Jegadeesh Raman, Sri Nurestri Abd Malek, Sharmili Vidyadaran, Murali Naidu, Umah Rani Kuppusamy and Vikineswary Sabaratnam 1485

Plant Genomic DNA Extraction for Selected Herbs and Sequencing their Internal Transcribed Spacer Regions Amplified by Specific Primers Farah Izana Abdullah, Lee Suan Chua, Zaidah Rahmat, Azman Abd Samad and Alina Wagiran 1491

Effect of Angelica acutiloba Extract on Blood flow Regulation in Stroke-prone Spontaneously Hypertensive Rats Hiroko Negishi, Sari Sugahama, Ayaka Kawakami, Junna Kondo, Yuriko Nishigaki, Masato Yoshikawa, Taketeru Ueyama and Katsumi Ikeda 1497

Pharmacophore Models Derived From Molecular Dynamics Simulations of Protein-Ligand Complexes: A Case Study Marcus Wieder, Ugo Perricone, Thomas Seidel and Thierry Langer 1499

Continued inside backcover