Research Article Inhibitory Effect on -Hexosaminidase Release …downloads.hindawi.com/journals/ecam/2014/828760.pdf · 2019-07-31 · Research Article Inhibitory Effect on -Hexosaminidase

Research ArticleInhibitory Effect on 120573-Hexosaminidase Release fromRBL-2H3 Cells of Extracts and Some Pure Constituents ofBenchalokawichian a Thai Herbal RemedyUsed for Allergic Disorders

Thana Juckmeta1 Pakakrong Thongdeeying2 and Arunporn Itharat23

1Faculty of Medicine Thammasat University Rangsit Campus Khlong Luang PathumThani 12120 Thailand2Department of Applied Thai Traditional Medicine Faculty of Medicine Thammasat University Rangsit Campus Khlong LuangPathumThani 12120 Thailand3Center of Excellence on Applied Thai Traditional Medicine Research (CEATMR) Faculty of Medicine Thammasat UniversityRangsit Campus Khlong Luang PathumThani 12120 Thailand

Correspondence should be addressed to Arunporn Itharat iarunpornyahoocom

Received 3 November 2014 Revised 20 November 2014 Accepted 22 November 2014 Published 16 December 2014

Academic Editor Il-Moo Chang

Copyright copy 2014 Thana Juckmeta et alThis is an open access article distributed under theCreative CommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Introduction Benchalokawichian (BCW) aThai traditional herbal formulation has long been used as antipyretic and to treat skindisorders It comprises roots from five herbs Ficus racemosa Capparis micracantha Clerodendrum petasites Harrisonia perforataand Tiliacora triandra This polyherbal remedy has recently been included in the Thailand National List of Essential Medicines(Herbal Products list)Methodology A Bioassay-guided fractionation technique was used to evaluate antiallergy activities of crudeextracts and those obtained by themultistep column chromatography isolation of pure compounds Inhibitory effect on the releaseof 120573-hexosaminidase from RBL-2H3 cells was used to determine antiallergic activity Results Two pure compounds from BCWformulation showed higher antiallergic activity than crude or semipure extracts Pectolinarigenin showed the highest antiallergicactivity followed by O-methylalloptaeroxylin with IC

50values of 63 120583gmL and 1416 120583gmL respectively Moreover the highest

activities of pure compounds were significantly higher than chlorpheniramine (162120583gmL)ConclusionsThis study provides somesupport for the use of BCWin reducing itching and treatment of other skin allergic disordersThe two isolated constituents exhibitedhigh antiallergic activity and it is necessary to determine their mechanism of action Further phytochemical and safety studies ofpure compounds are required before development of these as antiallergy commercial remedies

1 Introduction

Allergic diseases are manifested as hyperresponsiveness toallergenic environmental substances in the various targetorgans of the body (skin nose lung gastrointestinal tractetc) and involve both IgE-mediated and non-IgE-mediatedcomponents [1 2] Exposure to allergenic materials results inproduction by B cells of a multitude of antibodies collec-tively called immunoglobulins (Ig) that are antigen-specificThe allergic reaction starts when immunoglobulin E bindsto specific receptors (FcRI) on the surface of mast cells andbasophils [3 4] which in turn induces degranulation of thecells and release ofmediators such as histamine leukotrienes

serotonin and platelet activating factors [5ndash7] Histamine isthe main cause of many of the symptoms of allergies such asrunnynose sneezing and itchingHistamine also contributesto the progression of allergic-inflammatory responses byenhancement of the secretion of proinflammatory cytokines[8] Although antihistamines are the first drugs of choicefor treatment of many types of allergic disorders they dohave certain side effects A large proportion (70ndash95) ofthe worldrsquos population still relies on herbal medicines forprimary health care [9] Therefore there is a continuoussearch for newer and better drugs for allergy treatmentincluding evaluation of traditional herbal remedies [10] SinceIgEs play an important role in the allergic reaction it has been

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2014 Article ID 828760 8 pageshttpdxdoiorg1011552014828760

2 Evidence-Based Complementary and Alternative Medicine

suggested that the way to treat and prevent allergic diseases isto block the activity of IgE response [11ndash13]

Benchalokawichian (BCW) is aThai traditional medicineformulation containing parts from roots of five plants inequal amounts Ficus racemosa Linn (Moraceae) CapparismicracanthaDC (Capparidaceae) Clerodendrum petasites SMoore (Lamiaceae)Harrisonia perforataMerr (Simarouba-ceae) and Tiliacora triandra Diels (Menispermaceae) It haslong been used for relief of fever and to treat skin rashThis formulation is included in the Thailand National List ofEssential Medicines [14] It has also been used for preventionof influenza H1N1 infections and in recent years this remedyhas been used to treat acne skin rashes and other similar skindisorders

Thepolyherbal formulationBCWhas not been systemati-cally studied before but there are numerous previous reportson the chemical constituents of some individual plants in thisherbal remedy Harrisonia perforata leaves fruits brachesand roots were shown to contain several chromones limon-oids triterpenoids and prenylated polyketides including har-risotone AndashE haperforine A haperforine E 12-desacetylhap-erforine A haperforine C2 haperforine F haperforine GForitin harrisonol A peucenin-7-methylether O-methyla-lloptaeroxylin perforatic acid eugenin saikochromone Agreveichromenol and perforamoneAndashD [15] Other reportedconstituents are 120573-sitosterol obacunone herteropeucenin-7-methyl ether perforatic acid and harrisonin [16ndash19] harper-foratin harperfolide and harperamone [20] Tiliacora trian-dra has been reported to contain alkaloids especially bisben-zylisoquinoline alkaloids including tiliacorinine tiliacorinenortiliacorinine and others [21ndash23] Ficus racemosa has beenreported to contain tannins flavonoids coumarins phenoliccompounds glycosides and phytosterols [24 25]

Currently the antipyretic and anti-inflammatory activi-ties of BCWhave only been studied in vivo in rats [26 27]Theantimicrobial activity of ethanolic and water extracts of BCWhas recently been reported [28 29] There are no other invitro studies on antioxidant antiallergy or anti-inflammatoryactivities on BCW However a recent study on fruits of Hperforata has demonstrated that organic extracts exhibitedhigh antioxidant activity by the DPPH method but failed toshow any cytotoxicity against human myelogenous leukemia(K562) and human cancer (SGC-7901) cell lines in vitro bytheMTTmethod [30] Another report has also described theantioxidant activity in extracts of fruits ofH perforata by theDPPH method [31] The results of in vivo rats suggest thatH perforata bark aqueous extracts does not cause acute andsubchronic toxicities [32]

Benchalokawichian remedy has yet not been thoroughlystudied either in vitro or in vivo Its recent inclusion in theThailandNational List of EssentialMedicines has encouragedus to carry out systematic phytochemical and bioactivityassessment of crude extracts Singharachai et al studiedmor-phological characters including macroscopic microscopicexamination and pharmacognostic parameters and investi-gated 3D-HPLC fingerprint profile [33] We also report theuse of bioactivity-guided isolation of semipure and some pureconstituents from this polyherbal remedy In this preliminarystudy we have concentrated on antiallergic activity and

on limited number of pure compounds It is hoped thatfurthermore detailed studies of this type along with safetystudies in animals will provide data that may allow theinvestigation of its clinical efficacy in controlled clinical trialsfor some of the conditions it is currently used for inThailandbyThai Traditional medicine (TTM) practitioners

2 Materials and Methods

21 Chemicals Reagents and Instrumentation RBL-2H3 Ratbasophilic leukemia cell line was from American Type Cul-ture Collection (ATCC CRL-2256 VA USA) fetal bovineserum (FBS) trypsin-EDTA and trypan blue were purchasedfromGibco (OKUSA)Minimumessentialmedium (MEM)penicillin-streptomycin (PS) and phosphate buffer saline(PBS) were purchased from Biochrom (MA Germany) 4-NitrophenylN-acetyl-120573-D-glucosaminide (PNAG) antidini-trophenylated bovine albumin (DNP-BSA) anti-DNP IgE(monoclonal anti-DNP) ketotifen fumarate and chlorpheni-ramine were purchased from Sigma (MO USA) Dimethylsulfoxide (DMSO) was purchased from Fluka (MunichGermany) Calcium chloride dehydrate magnesium chlo-ride 6H

2O potassium chloride and sodium carbonate were

purchased from Merck (Darmstadt Germany) Piperazine-NN1015840-bis(2-ethanesulfonic acid) (PIPES)was purchased fromAmresco (OH USA) Sodium chloride and sodium hydrox-ide (analytical grade) were purchased from Univar (AjaxFinechem Australia) Sodium bicarbonate was purchasedfrom BDH (Poole UK) The solvents for analysis hexanechloroform ethyl acetate and methanol (analytical grades)were purchased from RCI Labscan (Bangkok Thailand)Sterile water was obtained by purification using a Milli Qsystem from Millipore (Bedford MA USA) Silica gel 60grade numbers 107734 and 109385 (70ndash230mesh and 230ndash400mesh) and TLC silica gel 60 F

254were purchased from

Merck (Darmstadt Germany) Chromatographic column(45 times 54 cm glass) was purchased from Becthai (BangkokThailand) CO

2humidified incubator was purchased from

Shellab (OR USA) Laminar air flow cabinet was purchasedfrom Boss tech (Bangkok Thailand) Microplate reader waspurchased from BioTek (VT USA)

22 Plant Materials Roots of five plants were collected fromDan-Chang Suphan Buri Province in Thailand in March2012 Authentication of plant materials was by comparisonagainst specimens deposited at the herbarium of SouthernCenter of Thai Medicinal Plants Faculty of PharmaceuticalScience Prince of Songkla University Songkla Thailand

23 Preparation of the Extracts The roots from each of thesefive plants were cleaned cut in small pieces and dried at50∘C 24 h Each dried plant material was powdered using anelectric grinder (40mesh particle size) Five plant powders inthe same ratio weremixed to provide the BCW remedy BCWand each of the five plants were macerated with 95 ethanolfiltered with whatman number 1 and solvent removed usinga rotary evaporator under reduced pressure (40∘C) to obtainthe dry ethanolic extracts These extracts were further dried

Evidence-Based Complementary and Alternative Medicine 3

to constant weight in a vacuum desiccator All extracts werekept at minus20∘C until required for further use

24 Preparation of Semipure Extracts and Isolation ofPure Compounds

241 Vacuum Liquid Chromatography (VLC) Bioassay-guided fractionation was used to isolate the pure com-pounds by the following modified method of Tewtrakuland Itharat [34] The BCW ethanolic extract (5046 g) wassubjected to silica vacuum liquid chromatography (VLC)using five solvent systems of increasing polarity hex-ane (1500mL) hexane chloroform (1 1 2000mL) chloro-form (2500mL) chloroform methanol (1 1 2500mL) andmethanol (2000mL)TheVLC columnwas packedwith silicagel 60 (mesh 230ndash400) the crude extract was applied ontop of the column then they were eluded with these fivedifferent solvents The solvent in each fraction (fractions 1ndash5) was removed by rotary evaporator (40∘C) and the drysemipure extract further dried to constant weight

242 Further Purification by Gravity Feed CC to Isolate PureCompound The fraction 3 (chloroform 502 g) which exhib-ited the highest antiallergy activity was rechromatographedby column chromatography (450 cm diameter and 54 cmlength) using silica gel G (70ndash230mesh) with gravity feed ofthe following solvents in sequence hexane chloroform (1 1500mL) chloroform (300mL) chloroform methanol (9 1500mL) chloroform methanol (1 1 200mL) methanol(300mL) Fractions (12mL) were collected during elutionwith each solvent (the first and the last fractions 500mLper each fraction were not collected) Each of total fractionscollected was examined by TLC

The fraction 12 (2826mg) was rechromatographed bycolumn chromatograph (375 cm times 54 cm) of silica gel gradenumbers 70ndash230mesh by eluting sequentially with sol-vents of increasing polarity hexane EtOAc (7 3 1000mL)hexane EtOAc (1 1 400mL) hexane EtOAc (3 7 300mL)EtOAc (700mL) EtOAc MeOH (1 1 100mL) and finallyMeOH (200mL) Fractions (5mL) were collected for eacheluting solvent and all fractions were examined by TLC(GF254

)

243 Identification of Compounds 1 and 2 TLC (silica gel 60GF254

aluminium sheets Merck) were used to demonstratethe purity of compounds with three different solvent systemsof varying polarity and detection with anisaldehyde reagentThe structures of the isolated compounds were determinedby their NMR data [1H and 13C on a Varian Unity Inova500 spectrometer (500MHz for 1H 125MHz for 13C)] UVspectra [SPECORD S 100 (Analytikjena) spectrometer] andESImass spectra bothHRMSandLRMSwere obtained froma Agilent Technologies 1200 Binary LC System coupled to aBruker microtof mass spectrometer

25 Determination of Antiallergic Activity Inhibitory effectson the release of 120573-hexosaminidase from Rat BasophilicLeukemia cell line (RBL-2H3) were evaluated by the fol-lowing modified method [35] RBL-2H3 cells were cultured

in MEM medium supplemented with 15 fetal bovineserum (FBS) penicillin (100 unitsmL) and streptomycin(100 120583gmL) The cells were seeded in 24-wells plate (5 times105 cellsmL) and incubated to adhere at 37∘C in 5 CO

2for

15 hour RBL-2H3 cells were sensitized with anti-DNP IgE(antidinitrophenyl-immunoglobulin E) (045 120583gmL) andincubated at 37∘C in 5 CO

2for 24 h The cells were washed

with 400 120583L of Siraganian buffer (buffer A) [119mM NaCl5mM KCl 56mM glucose 04mM MgCl

2 1 mM CaCl

2

25mM piperazine-NN1015840-bis(2-ethanesulfonic acid) (PIPES)01 bovine serum albumin (BSA) and 40mM NaOHpH 72] An aliquot (160 120583L) of buffer A was added andincubation was continued for an additional 10min at 37∘CThe test sample (20120583L) solution was added to each welland incubated for 10min followed by addition of 20120583L ofantigen (DNP-BSA final concentration 10 120583gmL) at 37∘C for20min to stimulate cell degranulationThe supernatants weretransferred into 96-well plate in 50120583Lwells and incubatedwith 50120583L of substrate PNAG (1mM p-nitrophenyl-N-acetyl-b-D-glucosaminide) in 01M citrate buffer (pH 45) at 37∘Cfor 3 h The reaction was stopped by adding 200120583L of stopsolution (01M Na

2CO3NaHCO

3 pH 100) The absorbance

was measured with a microplate reader at 405 nm Thetest samples were dissolved in dimethyl sulfoxide (DMSO)and Siraganian buffer was added for dilution (final DMSOconcentration was 01) The positive controls showed clearyellow color whereas the negative control was colorless Thesamples were pale yellow to colorless representing the per-centage of inhibition antiallergic activity Chlorpheniraminewas used by positive controls The percentage of inhibitionwas calculated according to the following formula

Inhibition = [1 minus (119879 minus 119861 minus 119873)(119862 minus 119873)

] times 100 (1)

Control (119862) DNP-BSA (+) Test sample (minus) Test (119879) DNP-BSA (+) Test sample (+) Blank (119861) DNP-BSA (minus) Testsample (+) Normal (119873) DNP-BSA (minus) Test sample (minus)

26 Statistical Analysis The results are based on three sepa-rate experiments Each sample was analyzed in triplicate inany experiment The activity values are expressed as mean plusmnSD IC

50values were calculated using the Prism Program

3 Results

31 Yield of Crude Extracts and Fractions (F1ndashF5) Yields (ww) of 95 ethanolic crude extracts of BCW remedy and itsconstituent were low in range of 2ndash4 (see in Table 1)

The ethanolic extract of BCW (5046 g) was subjectedto vacuum liquid chromatography (VLC) to obtained fivesemipure extracts (F1ndashF5) Fraction 4 (chloroform methanolelution) showed the highest yield followed by fraction 3 andfraction 5 with yields being 6540 1320 and 1052respectively The yields are shown in Table 2 All extractsand fractions were test inhibitory effects on release of 120573-hexosaminidase The fractions which showed highest antial-lergy activity were further subjected to purification to isolatepure compounds


Table 1 Yields (ww) and voucher specimen number of Benchalokawichian formulation and constituent plants

Sample Family name Voucher specimens Yieldlowast

Ficus racemosa Linn Moraceae SKP 117 06 18 01 418Capparis micracantha DC Capparidaceae SKP 391 03 13 01 189Tiliacora triandra Diels Menispermaceae SKP 114 20 20 01 392Harrisonia perforataMerr Simaroubaceae SKP 178 08 16 01 278Clerodendrum petasites S Moore Lamiaceae SKP 202 03 09 01 220Benchalokawichian remedy (Ha-Rak) mdash 359lowastExtraction by maceration with 95 ethanol Yields are ww of starting dry weight of powered roots or BCW formulation

Table 2 Yields (ww) of fractions after VLC of the crude ethanolicextract of Benchalokawichian formulation

Fraction solvent system Yieldlowast

1 hexane 006 (0002)2 hexane chloroform 598 (0214)3 chloroform 1326 (047)4 chloroform methanol 6540 (235)5 methanol 1052 (038)lowastYield is ww of crude ethanolic extract (values in brackets are wwexpressed of DPW)

32 Isolation and Identification of Isolated Pure Compounds1 and 2 The semipure extract from fraction F3 of VLC waspurified using silica gel chromatogram (70ndash230mesh) usingsolvents of increasing polarity for elution with gravity feed ofsolvents This step of purification resulted in the isolation oftwo pure compounds All collected fractions were examinedby TLC using anisaldehyde (in H

2SO4) as detection spray

Compound 1 appeared as a yellow spot on TLC plates onheating and compound 2 appeared as yellow spot with noheating required Compounds 1 and 2 were further identifiedby 1H and 13C NMR and the structure identity confirmedby mass spectroscopy compound 1 was pectolinarigenin andcompound 2 was O-methylalloptaeroxylin

On recrystallization of material from methanol affordeda crystalline pale yellow solid (5mg) as compound 1 pec-tolinarigenin or 57-dihydroxy-6-methoxy-2-(4-methoxy-phenyl)-4H-chromen-4-one (PubChem CID 5320438) Themolecular formula of compound 1 was proposed to beC17H14O6 as deduced from ESI mass spectra [mz 3150863

(M+H)+] 1H NMR (500MHz CDCl3) and the 13C NMR

(125MHz CDCl3) 17 carbon signals observed 10 of which

correspond with 14 protons as observed from HMBCspectrum (Figure 1) NMR data is shown in Table 3

The fraction 6 was a yellow solid (108mg) and was iden-tified as compound 2 O-methylalloptaeroxylin (PubChemCID 441968) The molecular formula of compound 2 wasproposed to be C

16H16O4 as deduced from ESI mass spectra

[mz 2731121 (M+H+)] 1H NMR (500MHz CDCl3) and

the 13CNMR (125MHz CDCl3) in Table 4 16 carbon signals

observed 7 of which correspond with 16 protons as observedfrom HMBC spectrum (Figure 2)

H

H

HH

HH

O

O

HO

35

OH

3998400

6998400

1998400

OCH3

H3CO

Figure 1 HMBC correlation of compound pectolinarigenin

Table 3 NMR spectrum data (500MHz for 1H and 125MHz for13C) of compound 1

Position 1H (mult 119869 in Hz)a 13C HMBC (1Hrarr 13C)2 mdash 16423 657 (s) 1038 C-2 C-4 C-4a C-11015840

4 mdash 18304a mdash 10585 mdash 15216 mdash 13037 mdash 15498 659 (s) 933 C-6 C-7 C-8a8a mdash 153211015840 mdash 123621015840 783 (dd 89 19) 1281 C-2 C-41015840 C-61015840

31015840 701 (dd 89 19) 1145 C-11015840 C-41015840 C-51015840

41015840 mdash 162651015840 701 (dd 89 19) 1145 C-11015840 C-31015840 C-41015840

61015840 783 (dd 89 19) 1281 C-2 C-21015840 C-41015840

5-OH 1310 (s) C-4a C-5 C-67-OH 653 (s) C-6 C-7 C-841015840-OCH3 390 (3H s) 555 C-41015840

6-OCH3 404 (3H s) 608 C-6NoteCompound 1 in CDCl3

aif not indicated the integration of each protonsignal equal to one proton

33 Inhibitory Effects on Release of 120573-Hexosaminidase IC50

values of antiallergic activity against release of120573-hexosamini-dase in RBL-2H3 cell lines are shown in Table 5The ethanolic


H

HH

H

O

O

O 8

7

5

1

3

3998400

2998400

1998400

OCH3

CH3

4a

8a

Figure 2 HMBC correlation of compound O-methylalloptaeroxylin


Position 1H (mult 119869 in Hz)a 13C HMBC (1Hrarr 13C)2 16253 599 (s) 1118 C-2 C-2 C-4 C-4a4 17754a 10855 16066 629 (s) 964 C-4a C-5 C-7 C-87 15768 10238a 154311015840 times 2 147 (s) 282 C-11015840 C-21015840 C-31015840

21015840 77931015840 555 (d 100) 1273 C-21015840 C-841015840 670 (d 100) 1153 C-7 C-8 C-8a C-21015840

2-CH3 228 (3H s) 196 C-2 C-35-OCH3 391 (s) 564 C-5NoteCompound 2 inCDCl3


crude extract from H perforata exhibited the most potentantiallergic activity followed by F racemosa and the poly-herbal BCW formulation (IC

50= 145 277 and 398 120583gmL

resp) C petasites showed only moderate activity (IC50

=578120583gmL) whereas C micracantha and T triandra wereinactive (IC

50gt 100 120583gmL)

The semipure extracts from fraction F3 and fraction F4after VLC showed high inhibitory activity (IC

50values 179

and 196 120583gmL resp) Other semipure extracts (F2 and F5)had moderate activity whereas the hexane extract (F1) wasnot tested

Pectolinarigenin and O-methylalloptaeroxylin exhibitedthe highest antiallergic activity with IC

50values of 63 and

142 120583gmL (201 and 518120583M) respectively These two com-pounds showhigher inhibitory effect than chlorpheniraminepositive control (IC

50= 162 120583gmL 588120583M)

4 Discussion and Conclusions

This study has isolated for the first time two pure com-pounds from the ethanolic extract of BCW formulation and

identified them as pectolinarigenin (compound 1) and O-methylalloptaeroxylin (compound 2) which have not pre-sented on 3D-HPLC chromatogram in previous study [33]Compound 2 has previously been isolated from branches ofH perforata [36 37] In a recent studyChoodej et al [20] haveinvestigated the effects of compound 2 on NO productionin LPS- stimulated macrophages and showed it had potentactivity with IC

50value of 6641 plusmn 521120583M Although this

structure was described more than ten years ago researchon inhibitory effect against release of 120573-hexosaminidase inRBL-2H3 cell lines has not been described to date This istherefore the first report on antiallergic effect of compound 2

Pectolinarigenin (compound 1) had never been reportedin any of the constituent plants of BCW formulationHowever this compound has been isolated from severalother plant families Scrophulariaceae [36ndash38] Compositae[39ndash42] Bignoniaceae [43] Fabaceae [44] Verbenaceae orLamiaceae [45ndash49] Interestingly pectolinarigenin has beenisolated fromClerodendrum spp [46 47 49] related toC pet-asites a constituent plant of the BCW formulation In anotherstudy the ethyl acetate fraction of Cirsium chanroenicum(Compositae) also showed strong inhibition of COX-2-mediated PGE2 and 5-LOX-mediated LT production in vitroIt showed the same inhibitory effect in several animal modelsof inflammationallergy such as arachidonic acid-inducedmouse ear edema carrageenan-induced mouse paw edemaand passive cutaneous anaphylaxis [39] Pectolinarigenin hasalso been shown to have hepatoprotective activity in a ratmodel of hepatic injury caused by D-galactosamine (GalN)mainly via SOD antioxidant mechanism [40] Inflammationmediators and reactive free radicals do have a role inthe pathophysiology of allergic disorders and thereforeanti-inflammatory and antioxidant compounds often exhibitantiallergic activities

The study on TLC chromatogram of two compounds(Figure 3) we found that compound 1 (pectolinarigenin) ispresented onH perforata whereas compound 2 (O-methyla-lloptaeroxylin) is presented on C petasites in the same asprevious reviews Both of pure compounds show exhibitedhigher antiallergic activity than all fractions BLWextract andpositive control represented in Figure 4 This preliminarystudy provides some supports for the use of BCW fortreatment of allergic skin rash in Thai traditional medicineThis is the first report on the two isolated compounds fromBCW ethanolic extract and their antiallergic activities Theethanolic extract of BCW could be further developed into


Table 5 Anti-allergic activity of the ethanolic extract fractions and pure constituents from Benchalokawichian remedy

Samples Inhibition at various concentrations IC50 of Antiallergic activityCrude semipure extracts and purecompounds 01 1 5 10 50 100 IC50 plusmn SEM 120583gmL

(120583M data for pure compound)Ficus racemosa Linn mdash 150 plusmn 105 mdash 289 plusmn 91 636 plusmn 16 758 plusmn 43 271 plusmn 16Capparis micracantha DC mdash 42 plusmn 27 mdash 95 plusmn 71 229 plusmn 89 323 plusmn 63 gt100Tiliacora triandra Diels minus2448 plusmn 57 minus00 plusmn 18 117 plusmn 48 442 plusmn 46 gt100Harrisonia perforataMerr mdash 216 plusmn 53 mdash 424 plusmn 13 628 plusmn 00 785 plusmn 08 145 plusmn 01Clerodendrum petasites S Moore minus63 plusmn 51 79 plusmn 22 440 plusmn 14 850 plusmn 19 578 plusmn 14Benchalokawichian (Ha-Rak) mdash 97 plusmn 55 mdash 242 plusmn 36 521 plusmn 15 452 plusmn 51 398 plusmn 13Fraction 1 hexane (F1) mdash mdash mdash mdash mdash mdash NTFraction 2 hexane chloroform (F2) mdash 225 plusmn 04 mdash 304 plusmn 21 436 plusmn 08 530 plusmn 07 861 plusmn 11Fraction 3 chloroform (F3) mdash 231 plusmn 45 mdash 403 plusmn 34 595 plusmn 44 712 plusmn 17 179 plusmn 30Fraction 4 chloroform methanol (F4) mdash 209 plusmn 77 mdash 389 plusmn 39 582 plusmn 27 705 plusmn 37 196 plusmn 34Fraction 5 methanol (F5) mdash 105 plusmn 76 mdash 260 plusmn 62 521 plusmn 15 639 plusmn 29 399 plusmn 19Compound 1 pectolinarigenin 171 plusmn 17 251 plusmn 39 386 plusmn 72 1011 plusmn 95 mdash mdash 63 plusmn 07 (201 120583M)Compound 2 O-methylalloptaeroxylin mdash minus158 plusmn 23 189 plusmn 09 405 plusmn 00 mdash 919 plusmn 16 142 plusmn 09 (518 120583M)Chlorpheniraminea mdash 40 plusmn 19 mdash 351 plusmn 08 444 plusmn 18 811 plusmn 32 162 plusmn 25 (588 120583M)aPositive controlNT (mdash) means not tested

HP CM CP TT FR BCW MIX

Hexane EtOAc (7 3)

cpd1

(a)


Hexane EtOAc (2 8)

cpd2

(b)

Figure 3 TLC chromatogram of five plants BCW extract mix of all extracts and pure compounds compound 1 (a) and compound 2 (b)Note CM = Capparis micracanthaDC CP = Clerodendrum petasites S Moore FR = Ficus racemosa Linn HP =Harrisonia perforataMerrand TT = Tiliacora triandra Diels

commercial formulations for the treatment of allergic dermaldiseases whereas the two pure compounds can serve asbioactive markers for the analysis and standardization of anynew formulated products

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

Thiswork was supported by theNational ResearchUniversityProject of Thailand Office of Higher Education Commission(NRU) The authors also thank the Center of Excellence inApplied Thai Traditional Medicine Research and Faculty ofMedicine Thammasat University for support and providelaboratory facilities and Professor L A Damani visitingProfessor atThammasat University for his critical commentsand careful scientific editing of this paper


0

20

40

60

80

100

120

0 50 100 150

Inhi

bitio

n (

)

Various concentrations

BCWF3 chloroformCompound 1

Compound 2

minus50

minus20

minus40

Chlorpheniraminelowast

(a)

05

1015202530354045

Chlo

rofo

rm(F

3)fr

actio

n

Com

poun

d 2

Com

poun

d 1

BCW

extr

act

Chlo

rphe

nira

min

elowast

IC50plusmn

SEM

(120583g

mL)

(b)

Figure 4 Effect of antiallergic activity against release of 120573-hexosaminidase in RBL-2H3 cell lines comparison of the BLW crude extractfraction 3 pure compounds and positive controls (a) is inhibition at various concentration (b) Data are present as the IC

50(Mean plusmn SEM

120583gmL) and lowast = positive control

References

[1] S T Holgate M K Church and L M Lichtenstein AllergyMosby London UK 3rd edition 2006

[2] B Alberts A Johnson J Lewis M Raff K Roberts and PWalter Molecular Biology of the Cell Garland Science NewYork NY USA 2007

[3] H Metzger G Alcaraz R Hohman J P Kinet V Pribluda andR Quarto ldquoThe receptor with high affinity for immunoglobulinErdquo Annual Review of Immunology vol 4 pp 419ndash470 1986

[4] I B Haydik and W S Ma ldquoBasophil histamine releasemdashassaysand interpretationrdquo Clinical Reviews in Allergy vol 6 no 2 pp141ndash162 1988

[5] K Ishizaka T Ishizaka and E H Lee ldquoBiologic function of theFc fragments of E myeloma proteinrdquo Immunochemistry vol 7no 8 pp 687ndash694 1970

[6] T Ishizaka K Ishizaka D H Conrad and A Froese ldquoA newconcept of triggering mechanisms of IgE-mediated histaminereleaserdquoThe Journal of Allergy and Clinical Immunology vol 61no 5 pp 320ndash330 1978

[7] T Ishizaka D H Conrad E S Schulman A R Sterk and KIshizaka ldquoBiochemical analysis of initial triggering events ofIgE-mediated histamine release from human lung mast cellsrdquoThe Journal of Immunology vol 130 no 5 pp 2357ndash2362 1983

[8] H Bayram J L Devalia O A Khair et al ldquoEffect of lorata-dine on nitrogen dioxide-induced changes in electrical resis-tance and release of inflammatory mediators from culturedhuman bronchial epithelial cellsrdquo Journal of Allergy and ClinicalImmunology vol 104 no 1 pp 93ndash99 1999

[9] M M Robinson and X Zhang The World Medicines Situation2011mdashTraditional Medicines Global Situation Issues and Chal-lenges WHO Press Geneva Switzerland 3rd edition 2011

[10] K Ohmori K Hasegawa T Tamura et al ldquoProperties ofolopatadine hydrochloride a new antiallergicantihistaminicdrugrdquoArzneimittel-ForschungDrug Research vol 54 no 12 pp809ndash829 2004

[11] L M Lichtenstein P S Norman and W L WinkenwerderldquoClinical and in vitro studies on the role of immunotherapy inragweed hay feverrdquo The American Journal of Medicine vol 44no 4 pp 514ndash524 1968

[12] D A Levy ldquoManipulation of the immune response to antigensin the management of atopic disease in manrdquo in Proceedingsof the Conference on the Biological Role of Immunoglobulin ESystem K Ishizaka and D H Dayton Jr Eds pp 239ndash245 USGovernment Printing Office Washington DC USA 1972

[13] F M Davis L A Gossett K L Pinkston et al ldquoCan anti-IgE beused to treat allergyrdquo Springer Seminars in Immunopathologyvol 15 no 1 pp 51ndash73 1993

[14] Bureau of Drug Control (Thailand) ldquoThe Ministry of PublicHealthNational List of EssentialMedicinesrdquo httpdrugfdamophgoth81nleminthsitesdefaultfilesattachmentsherbal book56pdf

[15] P Tuntiwachwuttikul P Phansa Y Pootaeng-on and W CTaylor ldquoChromones from the branches ofHarrisonia perforatardquoChemical and Pharmaceutical Bulletin vol 54 no 1 pp 44ndash472006

[16] Q Khuong-Huu A Chiaroni C Riche H Nguyen-Ngoc KNguyen-Viet and F Khuong-Huu ldquoNew rearranged limonoidsfromHarrisonia perforatardquo Journal of Natural Products vol 63no 7 pp 1015ndash1018 2000

[17] Q Khuong-Huu A Chiaroni C Riche H Nguyen-Ngoc KNguyen-Viet and F Khuong-Huu ldquoNew rearranged limonoidsfromHarrisonia perforata IIIrdquo Journal of Natural Products vol64 no 5 pp 634ndash637 2001


[18] H D TThu M V Tri N B Ngoc T Sevenet M Paıs and M-T Martin ldquoForitin a new limonoid from Harrisonia perforatardquoNatural Product Letters vol 14 no 3 pp 191ndash195 2000

[19] S Yin X Chen Z-S Su et al ldquoHarrisotones A-E five novelprenylated polyketides with a rare spirocyclic skeleton fromHarrisonia perforatardquo Tetrahedron vol 65 no 6 pp 1147ndash11522009

[20] S Choodej D Sommit and K Pudhom ldquoRearranged limon-oids and chromones from Harrisonia perforata and their anti-inflammatory activityrdquo Bioorganic and Medicinal ChemistryLetters vol 23 no 13 pp 3896ndash3900 2013

[21] D Thaweephol K Panida and N Kazumitsu ldquoIsolation ofactive principle from Yanangrdquo The Bulletin of the Departmentof Medical Sciences vol 16 pp 75ndash81 1974

[22] C Mahidol P Sahakitpichan and S Ruchirawat ldquoBioactivenatural products fromThai plantsrdquo International Journal of Pureand Applied Chemistry vol 66 pp 2353ndash2356 1994

[23] P Wiriyachitra and B Phuriyakorn ldquoAlkaloids of Tiliacoratriandrardquo Australian Journal of Chemistry vol 34 pp 2001ndash2004 1981

[24] K K Baruah and A K Gohain ldquoChemical composition andnutritive value of Dimaru (Ficus glomerata Roxb) leavesrdquoIndian Journal of Animal Nutrition vol 9 pp 107ndash108 1992

[25] N Khan and S Sultana ldquoChemomodulatory effect of Ficus race-mosa extract against chemically induced renal carcinogenesisand oxidative damage response inWistar ratsrdquo Life Sciences vol77 no 11 pp 1194ndash1210 2005

[26] A Konsue J Sattayasai P Puapairoj et al ldquoAntipyretic effectsof Bencha-Loga-Wichien herbal drug in ratsrdquo Thai Journal ofPharmacology vol 29 pp 79ndash82 2008

[27] A Jongchanapong C Singharachai C Palanuvej N Ruan-grungsi and P Towiwat ldquoAntipyretic and antinociceptiveeffects of Ben-Cha-Lo-Ka-Wi-Chian remedyrdquo Journal of HealthResearch vol 24 no 1 pp 15ndash22 2010

[28] C Sangrapee S Kantham S Panthong et al ldquoAntibacterialactivity of Benchalokawichian formulardquo Journal of Thai Tradi-tional amp Alternative Medicine vol 7 p 121 2009

[29] S Nuaeissara S Kondo and A Itharat ldquoAntimicrobial activ-ity of the extracts from Benchalokawichian remedy and itscomponentsrdquo Journal of the Medical Association of Thailand =Chotmaihet thangphaet vol 94 pp S172ndashS177 2011

[30] Susan ldquoStudy on the bioactive constitutents from Harrisoniaperforatardquo 2013 httpwwwresearch-degree-thesiscomshow-info-42-398304-0htmlsthashjPz1WmaLdpuf

[31] JWangMWang Z Liang et al ldquoLiposoluble components andtheir antioxidant activities from Harrisonia perforatardquo Journalof Tropical Agriculture 2013 httpencnkicomcnArticle enCJFDTOTAL-RDNK201109011htm

[32] S Sireeratawong N Lertprasertsuke U Srisawat et al ldquoAcuteand subchronic toxicity study of the water extract fromHarriso-nia perforata Merr in ratsrdquo Songklanakarin Journal of Scienceand Technology vol 31 no 1 pp 63ndash71 2009

[33] C Singharachai C Palanuvej H Kiyohara H Yamada andN Ruangrungsi ldquoPharmacognostic specification of five rootspecies in Thai traditional medicine remedy ben-Cha-Lo-Ka-Wi-Chianrdquo Pharmacognosy Journal vol 3 no 21 pp 1ndash11 2011

[34] S Tewtrakul and A Itharat ldquoAnti-allergic substances from therhizomes of Dioscorea membranaceardquo Bioorganic amp MedicinalChemistry vol 14 no 24 pp 8707ndash8711 2006

[35] S Tewtrakul and S Subhadhirasakul ldquoAnti-allergic activity ofsome selected plants in the Zingiberaceae familyrdquo Journal ofEthnopharmacology vol 109 no 3 pp 535ndash538 2007

[36] S Thadaniti W Archakunakorn P Tuntiwachwuttikul et alldquoChromones fromHarrisonia perforata (Blanco)Merrrdquo Journalof the Scientific Society vol 20 pp 183ndash187 1994

[37] R Tundis B Deguin M R Loizzo et al ldquoPotential antitumoragents flavones and their derivatives from Linaria reflexaDesfrdquoBioorganic and Medicinal Chemistry Letters vol 15 no 21 pp4757ndash4760 2005

[38] M Bonesi R Tundis B Deguin M R Loizzo F Meni-chini and F Tillequin ldquoIn vitro biological evaluation of novel7-119874-dialkylaminoalkyl cytotoxic pectolinarigenin derivativesagainst a panel of human cancer cell linesrdquo Bioorganic andMedicinal Chemistry Letters vol 18 no 20 pp 5431ndash5434 2008

[39] H Lim K H Son H W Chang K Bae S S Kang andH P Kim ldquoAnti-inflammatory activity of pectolinarigenin andpectolinarin isolated from Cirsium chanroenicumrdquo Biologicaland Pharmaceutical Bulletin vol 31 no 11 pp 2063ndash2067 2008

[40] Y-M Yoo J-H Nam M-Y Kim J Choi and H-J ParkldquoPectolinarin and pectolinarigenin of Cirsium setidens preventthe hepatic injury in rats caused by D-galactosamine via anantioxidant mechanismrdquo Biological and Pharmaceutical Bul-letin vol 31 no 4 pp 760ndash764 2008

[41] Y Yin F-Y Gong X-X Wu et al ldquoAnti-inflammatory andimmunosuppressive effect of flavones isolated from Artemisiavestitardquo Journal of Ethnopharmacology vol 120 no 1 pp 1ndash62008

[42] A Nugroho S-C Lim J S Byeon J S Choi and H-J ParkldquoSimultaneous quantification and validation of caffeoylquinicacids and flavonoids in Hemistepta lyrata and peroxynitrite-scavenging activityrdquo Journal of Pharmaceutical and BiomedicalAnalysis vol 76 pp 139ndash144 2013

[43] T Hase K Ohtani R Kasai K Yamasaki and C Pichean-soonthon ldquoRevised structure for hortensin a flavonoid fromMillingtonia hortensisrdquo Phytochemistry vol 40 no 1 pp 287ndash290 1995

[44] X-G He L-Z Lin and L-Z Lian ldquoAnalysis of flavonoidsfrom red clover by liquid chromatographymdashelectrospray massspectrometryrdquo Journal of Chromatography A vol 755 no 1 pp127ndash132 1996

[45] K K Seth V B Pandey and B Dasgupta ldquoFlavonoids ofClerodendron phlomidis flowersrdquo Pharmazie vol 37 no 1 pp74ndash75 1982

[46] S Pal A Chowdhury and N Adityachaudhury ldquoIsolation ofrice weevil feeding inhibitors uncinatone and pectolinarigeninfrom Clerodendron siphonenthusrdquo Journal of Agricultural andFood Chemistry vol 37 no 1 pp 234ndash236 1989

[47] R J Grayer N C Veitch G C Kite A M Price and TKokubun ldquoDistribution of 8-oxygenated leaf-surface flavonesin the genus Ocimumrdquo Phytochemistry vol 56 no 6 pp 559ndash567 2001

[48] R F Vieira R J Grayer and A J Paton ldquoChemical profilingofOcimum americanum using external flavonoidsrdquo Phytochem-istry vol 63 no 5 pp 555ndash567 2003

[49] N P Babu P Pandikumar and S Ignacimuthu ldquoLysoso-mal membrane stabilization and anti-inflammatory activity ofClerodendrum phlomidis Lf A traditional medicinal plantrdquoJournal of Ethnopharmacology vol 135 no 3 pp 779ndash785 2011

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


suggested that the way to treat and prevent allergic diseases isto block the activity of IgE response [11ndash13]

Benchalokawichian (BCW) is aThai traditional medicineformulation containing parts from roots of five plants inequal amounts Ficus racemosa Linn (Moraceae) CapparismicracanthaDC (Capparidaceae) Clerodendrum petasites SMoore (Lamiaceae)Harrisonia perforataMerr (Simarouba-ceae) and Tiliacora triandra Diels (Menispermaceae) It haslong been used for relief of fever and to treat skin rashThis formulation is included in the Thailand National List ofEssential Medicines [14] It has also been used for preventionof influenza H1N1 infections and in recent years this remedyhas been used to treat acne skin rashes and other similar skindisorders

Thepolyherbal formulationBCWhas not been systemati-cally studied before but there are numerous previous reportson the chemical constituents of some individual plants in thisherbal remedy Harrisonia perforata leaves fruits brachesand roots were shown to contain several chromones limon-oids triterpenoids and prenylated polyketides including har-risotone AndashE haperforine A haperforine E 12-desacetylhap-erforine A haperforine C2 haperforine F haperforine GForitin harrisonol A peucenin-7-methylether O-methyla-lloptaeroxylin perforatic acid eugenin saikochromone Agreveichromenol and perforamoneAndashD [15] Other reportedconstituents are 120573-sitosterol obacunone herteropeucenin-7-methyl ether perforatic acid and harrisonin [16ndash19] harper-foratin harperfolide and harperamone [20] Tiliacora trian-dra has been reported to contain alkaloids especially bisben-zylisoquinoline alkaloids including tiliacorinine tiliacorinenortiliacorinine and others [21ndash23] Ficus racemosa has beenreported to contain tannins flavonoids coumarins phenoliccompounds glycosides and phytosterols [24 25]

Currently the antipyretic and anti-inflammatory activi-ties of BCWhave only been studied in vivo in rats [26 27]Theantimicrobial activity of ethanolic and water extracts of BCWhas recently been reported [28 29] There are no other invitro studies on antioxidant antiallergy or anti-inflammatoryactivities on BCW However a recent study on fruits of Hperforata has demonstrated that organic extracts exhibitedhigh antioxidant activity by the DPPH method but failed toshow any cytotoxicity against human myelogenous leukemia(K562) and human cancer (SGC-7901) cell lines in vitro bytheMTTmethod [30] Another report has also described theantioxidant activity in extracts of fruits ofH perforata by theDPPH method [31] The results of in vivo rats suggest thatH perforata bark aqueous extracts does not cause acute andsubchronic toxicities [32]

Benchalokawichian remedy has yet not been thoroughlystudied either in vitro or in vivo Its recent inclusion in theThailandNational List of EssentialMedicines has encouragedus to carry out systematic phytochemical and bioactivityassessment of crude extracts Singharachai et al studiedmor-phological characters including macroscopic microscopicexamination and pharmacognostic parameters and investi-gated 3D-HPLC fingerprint profile [33] We also report theuse of bioactivity-guided isolation of semipure and some pureconstituents from this polyherbal remedy In this preliminarystudy we have concentrated on antiallergic activity and

on limited number of pure compounds It is hoped thatfurthermore detailed studies of this type along with safetystudies in animals will provide data that may allow theinvestigation of its clinical efficacy in controlled clinical trialsfor some of the conditions it is currently used for inThailandbyThai Traditional medicine (TTM) practitioners

2 Materials and Methods

21 Chemicals Reagents and Instrumentation RBL-2H3 Ratbasophilic leukemia cell line was from American Type Cul-ture Collection (ATCC CRL-2256 VA USA) fetal bovineserum (FBS) trypsin-EDTA and trypan blue were purchasedfromGibco (OKUSA)Minimumessentialmedium (MEM)penicillin-streptomycin (PS) and phosphate buffer saline(PBS) were purchased from Biochrom (MA Germany) 4-NitrophenylN-acetyl-120573-D-glucosaminide (PNAG) antidini-trophenylated bovine albumin (DNP-BSA) anti-DNP IgE(monoclonal anti-DNP) ketotifen fumarate and chlorpheni-ramine were purchased from Sigma (MO USA) Dimethylsulfoxide (DMSO) was purchased from Fluka (MunichGermany) Calcium chloride dehydrate magnesium chlo-ride 6H

2O potassium chloride and sodium carbonate were

purchased from Merck (Darmstadt Germany) Piperazine-NN1015840-bis(2-ethanesulfonic acid) (PIPES)was purchased fromAmresco (OH USA) Sodium chloride and sodium hydrox-ide (analytical grade) were purchased from Univar (AjaxFinechem Australia) Sodium bicarbonate was purchasedfrom BDH (Poole UK) The solvents for analysis hexanechloroform ethyl acetate and methanol (analytical grades)were purchased from RCI Labscan (Bangkok Thailand)Sterile water was obtained by purification using a Milli Qsystem from Millipore (Bedford MA USA) Silica gel 60grade numbers 107734 and 109385 (70ndash230mesh and 230ndash400mesh) and TLC silica gel 60 F

254were purchased from

Merck (Darmstadt Germany) Chromatographic column(45 times 54 cm glass) was purchased from Becthai (BangkokThailand) CO

2humidified incubator was purchased from

Shellab (OR USA) Laminar air flow cabinet was purchasedfrom Boss tech (Bangkok Thailand) Microplate reader waspurchased from BioTek (VT USA)

22 Plant Materials Roots of five plants were collected fromDan-Chang Suphan Buri Province in Thailand in March2012 Authentication of plant materials was by comparisonagainst specimens deposited at the herbarium of SouthernCenter of Thai Medicinal Plants Faculty of PharmaceuticalScience Prince of Songkla University Songkla Thailand

23 Preparation of the Extracts The roots from each of thesefive plants were cleaned cut in small pieces and dried at50∘C 24 h Each dried plant material was powdered using anelectric grinder (40mesh particle size) Five plant powders inthe same ratio weremixed to provide the BCW remedy BCWand each of the five plants were macerated with 95 ethanolfiltered with whatman number 1 and solvent removed usinga rotary evaporator under reduced pressure (40∘C) to obtainthe dry ethanolic extracts These extracts were further dried







)





2for




2 1 mM CaCl

2


2CO3NaHCO




] times 100 (1)




3 Results






















H

H

HH

HH

O

O

HO

35

OH

3998400

6998400

1998400

OCH3

H3CO





31015840 701 (dd 89 19) 1145 C-11015840 C-41015840 C-51015840

41015840 mdash 162651015840 701 (dd 89 19) 1145 C-11015840 C-31015840 C-41015840

61015840 783 (dd 89 19) 1281 C-2 C-21015840 C-41015840







H

HH

H

O

O

O 8

7

5

1

3

3998400

2998400

1998400

OCH3

CH3

4a

8a




21015840 77931015840 555 (d 100) 1273 C-21015840 C-841015840 670 (d 100) 1153 C-7 C-8 C-8a C-21015840




50= 145 277 and 398 120583gmL



50gt 100 120583gmL)


50values 179



50values of 63 and


50= 162 120583gmL 588120583M)













Hexane EtOAc (7 3)

cpd1

(a)


Hexane EtOAc (2 8)

cpd2

(b)





Acknowledgments



0

20

40

60

80

100

120

0 50 100 150

Inhi

bitio

n (

)



Compound 2

minus50

minus20

minus40


(a)

05

1015202530354045

Chlo

rofo

rm(F

3)fr

actio

n

Com

poun

d 2

Com

poun

d 1

BCW

extr

act

Chlo

rphe

nira

min

elowast

IC50plusmn

SEM

(120583g

mL)

(b)


50(Mean plusmn SEM


References
























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















)





2for




2 1 mM CaCl

2


2CO3NaHCO




] times 100 (1)




3 Results






















H

H

HH

HH

O

O

HO

35

OH

3998400

6998400

1998400

OCH3

H3CO





31015840 701 (dd 89 19) 1145 C-11015840 C-41015840 C-51015840

41015840 mdash 162651015840 701 (dd 89 19) 1145 C-11015840 C-31015840 C-41015840

61015840 783 (dd 89 19) 1281 C-2 C-21015840 C-41015840







H

HH

H

O

O

O 8

7

5

1

3

3998400

2998400

1998400

OCH3

CH3

4a

8a




21015840 77931015840 555 (d 100) 1273 C-21015840 C-841015840 670 (d 100) 1153 C-7 C-8 C-8a C-21015840




50= 145 277 and 398 120583gmL



50gt 100 120583gmL)


50values 179



50values of 63 and


50= 162 120583gmL 588120583M)













Hexane EtOAc (7 3)

cpd1

(a)


Hexane EtOAc (2 8)

cpd2

(b)





Acknowledgments



0

20

40

60

80

100

120

0 50 100 150

Inhi

bitio

n (

)



Compound 2

minus50

minus20

minus40


(a)

05

1015202530354045

Chlo

rofo

rm(F

3)fr

actio

n

Com

poun

d 2

Com

poun

d 1

BCW

extr

act

Chlo

rphe

nira

min

elowast

IC50plusmn

SEM

(120583g

mL)

(b)


50(Mean plusmn SEM


References
























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



































H

H

HH

HH

O

O

HO

35

OH

3998400

6998400

1998400

OCH3

H3CO





31015840 701 (dd 89 19) 1145 C-11015840 C-41015840 C-51015840

41015840 mdash 162651015840 701 (dd 89 19) 1145 C-11015840 C-31015840 C-41015840

61015840 783 (dd 89 19) 1281 C-2 C-21015840 C-41015840







H

HH

H

O

O

O 8

7

5

1

3

3998400

2998400

1998400

OCH3

CH3

4a

8a




21015840 77931015840 555 (d 100) 1273 C-21015840 C-841015840 670 (d 100) 1153 C-7 C-8 C-8a C-21015840




50= 145 277 and 398 120583gmL



50gt 100 120583gmL)


50values 179



50values of 63 and


50= 162 120583gmL 588120583M)













Hexane EtOAc (7 3)

cpd1

(a)


Hexane EtOAc (2 8)

cpd2

(b)





Acknowledgments



0

20

40

60

80

100

120

0 50 100 150

Inhi

bitio

n (

)



Compound 2

minus50

minus20

minus40


(a)

05

1015202530354045

Chlo

rofo

rm(F

3)fr

actio

n

Com

poun

d 2

Com

poun

d 1

BCW

extr

act

Chlo

rphe

nira

min

elowast

IC50plusmn

SEM

(120583g

mL)

(b)


50(Mean plusmn SEM


References
























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















H

HH

H

O

O

O 8

7

5

1

3

3998400

2998400

1998400

OCH3

CH3

4a

8a




21015840 77931015840 555 (d 100) 1273 C-21015840 C-841015840 670 (d 100) 1153 C-7 C-8 C-8a C-21015840




50= 145 277 and 398 120583gmL



50gt 100 120583gmL)


50values 179



50values of 63 and


50= 162 120583gmL 588120583M)













Hexane EtOAc (7 3)

cpd1

(a)


Hexane EtOAc (2 8)

cpd2

(b)





Acknowledgments



0

20

40

60

80

100

120

0 50 100 150

Inhi

bitio

n (

)



Compound 2

minus50

minus20

minus40


(a)

05

1015202530354045

Chlo

rofo

rm(F

3)fr

actio

n

Com

poun

d 2

Com

poun

d 1

BCW

extr

act

Chlo

rphe

nira

min

elowast

IC50plusmn

SEM

(120583g

mL)

(b)


50(Mean plusmn SEM


References
























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















Hexane EtOAc (7 3)

cpd1

(a)


Hexane EtOAc (2 8)

cpd2

(b)





Acknowledgments



0

20

40

60

80

100

120

0 50 100 150

Inhi

bitio

n (

)



Compound 2

minus50

minus20

minus40


(a)

05

1015202530354045

Chlo

rofo

rm(F

3)fr

actio

n

Com

poun

d 2

Com

poun

d 1

BCW

extr

act

Chlo

rphe

nira

min

elowast

IC50plusmn

SEM

(120583g

mL)

(b)


50(Mean plusmn SEM


References
























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

20

40

60

80

100

120

0 50 100 150

Inhi

bitio

n (

)



Compound 2

minus50

minus20

minus40


(a)

05

1015202530354045

Chlo

rofo

rm(F

3)fr

actio

n

Com

poun

d 2

Com

poun

d 1

BCW

extr

act

Chlo

rphe

nira

min

elowast

IC50plusmn

SEM

(120583g

mL)

(b)


50(Mean plusmn SEM


References
























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Documents

Research Article Inhibitory Effect on -Hexosaminidase Release …downloads.hindawi.com/journals/ecam/2014/828760.pdf · 2019-07-31 · Research Article Inhibitory Effect on -Hexosaminidase