research article antiherpetic effects of gynura...

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2013 Article ID 394865 10 pageshttpdxdoiorg1011552013394865

Research ArticleAntiherpetic Effects of Gynura procumbens

Siripen Jarikasem12 Somyot Charuwichitratana3 Sontana Siritantikorn4 Wasan Chantratita5 Magdy Iskander6 August Wilhelm Frahm7 and Weena Jiratchariyakul1

1 Department of Pharmacognosy Faculty of Pharmacy Mahidol University 447 Sri Ayudhya Road Rajathevi DistrictBangkok 10400 Thailand

2 Pharmaceutical and Natural Products Department Thailand Institute of Scientific and Technological ResearchPathumThani 12120 Thailand

3Division of Dermatology Department of Medicine Faculty of Medicine Ramathibodi Hospital Mahidol UniversityRama VI Road Bangkok 10400 Thailand

4Department of Microbiology Faculty of Medicine Siriraj Hospital Prannok Road Bangkok Noi District Bangkok 10700 Thailand5 Department of Pathology Faculty of Medicine Ramathibodi Hospital Mahidol University Rama VI Road Bangkok 10400 Thailand6Department of Medicinal Chemistry Victorian College of Pharmacy Monash University Melbourne VIC 3052 Australia7 Department of Pharmaceutical and Medicinal Chemistry Institute of Pharmaceutical Sciences Freiburg University79104 Freiburg Germany

Correspondence should be addressed to Weena Jiratchariyakul weenajirmahidolacth

Received 1 March 2013 Revised 18 July 2013 Accepted 23 July 2013

Academic Editor Ludger Beerhues

Copyright copy 2013 Siripen Jarikasem et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The ethanol extract of Gynura procumbens showed virucidal and antireplicative actions against herpes simplex virus HSV-1 andHSV-2 It was further chromatographed onMCI gel CHP20P column giving the extract fractions F1 (water) F2 (water-methanol) F3(methanol) and F4 (ethyl acetate) All but F1 had virucidal action against both viral types We reported here the active compoundsfrom F2 and F3 The antiherpetic compounds of F2 was a mixture of dicaffeoylquinic acids with virucidal and antireplicativeactions against HSV-2 (IC

50960 and 610 120583gmL resp) Virucidal compounds of F3 were a mixture of 120573-sitosterol and stigmasterol

(IC502500 120583gmL against HSV-1) a mixture of 120573-sitosteryl and stigmasteryl glucosides (IC

50500 120583gmL against HSV-2) and 1

2-bis-dodecanoyl-3-120572-D-glucopyranosyl-sn-glycerol (IC50

of 400 120583gmL against HSV-2) Herbal products containing 1 and 2of standardized ethanol extract were prepared Double-blind randomized controlled clinical trial of the products was performedin patients with recurrent herpes labialis Results showed that the number of patients whose lesions healed within 7 days and theaverage healing time of both groups differed insignificantly Viral culture onD7 indicated a decrease of infected patients from 487to 769 in treated group whereas in placebo group the infected patients decreased from 3125 to 2000 The viral reduction intreated group indicated the benefit of the product Insignificant result might arise from a low number of participated patients andinsufficient concentration of plant extract in herbal product

1 Introduction

Gynura procumbens (Lour) Merr is mentioned in tra-ditional Chinese medicine as a topical anti-inflammatoryremedy [1] In Southeast Asia Gynura plants are widelydistributed In Thailand G pseudochina var hispida (Thainame Wan Mahakaan) is externally used as anti-itching

anti-inflammatory and antiherpes virus [2] In SingaporeMalaysia and Indonesia the plant has been traditionallyused as remedies for eruptive fever rash kidney diseasemigraine constipation hypertension diabetes mellitus andcancer [3] The ethanol extract from the leaf reduced themouse ear oedema induced by croton oil [4] Phytochem-ical studies of Gynura plants resulted in the discovery of

2 Evidence-Based Complementary and Alternative Medicine

MPLC RP-18 (8 2 5 5) gradiently

LPLC RP-18 WM (55 45)

flow rate

5

inactive LPLC RP-8 WM 6 4 flow rate

Recryst from MeOH

F221 F222

3

inactive 4

Sephadex LH 20 WM (3 7)

per fraction


per fraction

MCI gel CHP20P WM (1 1)

F21 F22 F23

F2

HPLC-RP18 WM (96 4)

flow rate

HPLC-RP18 WM (8 2)

flow rate

F211

F2111

1

LPLC RP-18 WM (8 2)

flow rate

F212

21 22

2

active

W water

(123 g)

per fraction100mL

(87 g) (075g) (08 g)

20mL 5mL

1mLmin

1mLmin

R 190ndash240mL

R 150ndash165mLR 175ndash280mL

R 470ndash600mL(250mg) (80mg)

(60mg)(100mg)

(24mg)(30mg)

(90mg) (37mg)

(4mg)

(3mg) (4mg)

(8mg)

1mLmin

4mLmin

4mLmin

tR 10ndash80min

tR 20ndash40min tR 51ndash65min

NDlowast

NDlowast

not determined according to the insufficient amount of the isolated substance NDlowast

NDlowast

NDlowast

tR 80min

tR 75min

rarr

M methanol

Figure 1 Isolation of components 1ndash5 from F2

pyrrolizidine alkaloids [5ndash7] spirostanol [8] coumarins [7ndash9] and anthocyanins [10] SinceG procumbens so far has notbeen explored towards antiherpes viral activity on the otherhand according to traditional Thai medicine the utilizationof the plant may be associated with herpes viral infectionthe investigation of the plant components for the antiherpeticactivity is performed

2 Materials and Methods

PlantMaterialGynura procumbenswas collected fromChan-thaburi province Thailand in October 1994 The plant wasidentified by Ms Leena Phupatpong an expert botanist atthe Forest Herbarium (BKF) Royal Forest Department Min-istry of Agriculture and Cooperatives Bangkok ThailandA voucher specimen (BKF no 127362) was deposited at thesame placeExtraction and Isolation The aerial parts of the plant (25 kgfresh) were washed cut into pieces dried in a hot-air oven(60∘C) and ground yielding 17 kg of the coarse-powdereddrug It was successively extracted in a Soxhlet appara-tus using petroleum ether (40ndash60∘C) dichloromethane

and ethanol The solvents were removed under reducedpressure The dry ethanol extract (114 g) was further sepa-rated using chromatographic columns with different packingmaterials MCI gel CHP20P column fractionated the extractinto the fractions F1 (water) F2 (water-methanol 1 1) F3(methanol) and F4 (ethyl acetate) F2 F3 and F4 were activeagainst herpes virus

F2 (123 g) and F3 (64 g) were progressively chro-matographed on MCI gel CHP20P using aqueous methanol(1 1) for F2 and methanol for F3 as solvent systems resultingin F21 (87 g) F22 (075 g) and F23 (08 g) and F31 (20 g)F32 (30 g) and F33 (08 g) respectively Compounds 1 2 34 and 5 were isolated from F2 (Figure 1) while compounds6 7 8 and 9 were isolated from F3 (Figure 2) F4 was notfurther studied

Cell Line The Vero cell line (African green monkey kidneycells) was grown and maintained in Eaglersquos minimum essen-tial medium (MEM) supplemented with 10 fetal calf serumand antibiotics (5 times 104 cells per well)

Virus HSV-1 strain KOS and HSV-2 strain Baylor 186were obtained from the Department ofMicrobiology Faculty

Evidence-Based Complementary and Alternative Medicine 3

Sephadex LH-20 M LPLC-RP18 WM (4 6) flow rate 1 mLmin

6(5 mg)

inactive

(CMW 10 1 0 100 10 0 100 25 25100 30 3 0 100 0)

10 mL per fraction flow rate 1 mLmin

CM (9 1) (CMW 15 5 05)

7(25 mg) active

8(34 mg) active

(HEtOAc 20 0 0 20) 10 mL per fraction

Recryst from MeOH

(100 mg)

9(60 mg) active

MCI gel CHP20P M 100 mL per fraction

F3 (64 g)

F31 (2 g)

F32 (3 g)

F33 (08 g)

C chloroform

tR 66min

R 1210ndash1750mL R 2400ndash3000mL

rarr

rarr rarr

rarr

rarr

R 10ndash90mL

LPLC-SiO2LPLC-SiO2

SiO2SiO2

EtOAc ethyl acetateH hexane

W waterM methanol


of Medicine Siriraj Hospital Mahidol University BangkokThailand The quantity of 100 plaque forming unit (PFU) permL were used for experimentsEvaluation of Cytotoxicity Serial 2-fold dilutions of the testsample in the maintenance medium were added to Veromonolayer After incubation at 37∘C for 5 days cytotoxicitywas determined by vital staining with 1 crystal violet in10 formalin for 30min The highest concentration of thetest sample which did not exhibit cytotoxicity represented themaximum nontoxic dose (MNTD) Serial 2-fold dilutions ofMNTD were used to perform the antiviral assayAntiviral Assay Antiviral activity was determined by plaquereduction assay on confluent Vero cells growing in 96-welltissue culture plates The test included three treatments thatis inactivation and pre- and posttreatments

Inactivation (To determine the neutralizing activity ofthe test sample against virus infectivity (virucidal action))virus (100 120583L) was incubated with test sample (100120583L) at37∘C for 1 h The mixture was added in duplicated wells ofmonolayer cells and incubated at 37∘C for 1 h After washingthe cells MEM and semisolid media (04 gum tragacanth)were added to the cultures whichwere then incubated at 37∘Cfor 3 days and stained with crystal violet

Pre-treatment (to determine the inhibitory activity ofviral adsorption or penetration) test sample (100 120583L) wasadded to duplicated wells of monolayer cells and incubatedat 37∘C for 24 h After washing the culture cells were infectedwith virus (100120583L) and incubated at 37∘C for 1 h Semisolidmedia were added to the duplicated wells of monolayer cells

after washing out the non adsorbed virus The cultures wereincubated at 37∘C for 3 days and then stained with crystalviolet

Post-treatment (to determine the inhibitory activity ofintracellular viral replication) the monolayer cells wereinfected with virus and incubated at 37∘C for 1 h Afterwashing the cells the test sample (100120583L) and semisolidmedia were added in duplicated wells incubated at 37∘C for3 days and then stained with crystal violetControl Cell control monolayer cell in MEM and semisolidmedia Acyclovir was used as the positive control Viruscontrol monolayer cells infected with 100 PFUmL of virusMEM and semisolid media

Calculation of plaque inhibitory capacity percent ofplaque reduction compared to the culture without treatment(virus control) was calculatedThe 50 inhibition concentra-tion (IC

50) of the active substance or sample was determined

as the lowest concentration which reduced plaque numbersby 50 in treated compared to untreated cultures

The samples were dissolved in dimethyl sulfoxide(DMSO) and diluted with the maintenance medium Thefinal concentration of DMSO in the test sample was 03Viral Culture Vero cells were grown in growth mediumMEM (Earlersquos salt JR Scientific Inc Woodland CA USA)supplemented with 10 fetal bovine serum (Gibco GrandIsland USA) and 100 unitmL penicillin G and 100 120583gmLstreptomycin (MampHManufacturing Co LtdThailand) and001M HEPES (N-2-hydroxyethyl-piperazine-N1015840-2-ethanesulfonic acid) (Gibco Grand Island USA) Maintenance


media were prepared as growth media except the concentra-tion of fetal bovine serum was reduced to 2

Vero cell monolayers in culture flask were dispersed byusing 025 (1x) trypsin (JR Scientific Inc Woodland CAUSA) It was performed after removing the growth mediaand washing twice with 5mL PBS After discarding PBS onemL of trypsin was added and cells were incubated at 37∘Cfor 2ndash5min then the culture flask was gently shaken untilthe cells were detached and 5mL of the growth media wasaddedThese cells were counted to 105 cellsmL and 15mL ofcells were added into the culture tube containing cover glassand then incubated at 37∘C in 5 CO

2overnight After the

incubation the confluent Vero cells were inoculated with 01multiplicity of infection (MOI) of HSV-1 and HSV-2 viruses(100 120583Lculture tube) (Nunclon surfaceNuncBrand productDenmark) as positive control The culture was incubatedat 37∘C in 5 CO

2for 1 h with shaking at 15min interval

time before adding the maintenance mediaThe sample fromvesicle swab collected in virus transport media (VTM) wasprocessed by refrigerated centrifugation at 10000 rpm for10min Then 100120583L of the supernatant was inoculated in105 cellsmL confluent Vero cells of culture tube containingcover glass and incubated virus at 37∘C in 5 CO

2for

1 h with shaking at 15min interval time before adding themaintenance media The confluent Vero cells of 105 cellsmLper culture tube added with maintenance media were used asnegative control Each experiment was carried out in dupli-cate tubes All culture tubes of samples positive and negativecontrol were incubated and examined for cytopathic effect(CPE) The Vero cells on the cover glass were determined forHSV-infected cells and the supernatants were kept at minus70∘Cin order to subpassage culture again The Vero cells on thecover glass were fixed with chilled acetone for 10min and air-driedThe detection ofHSV inVero cells using a specific anti-serumwas conjugatedwith fluorescein isothiocyanate (FITC)(Polyclonal antibody DAKOAS Denmark) that was dilutedto 1 40 and applied to fixed cells and incubated for 30minat 37∘C The fixed cells were washed and counterstainedwith Evans blue for 5min then washed again with distilledwater air dried and mounted with PBS-glycerol buffer Thedirect immunofluorescence of the HSV-infected Vero cellswas examined under a fluorescent microscope The negativecontrol was acetone-fixed uninfected cells which processedthe same as the test The infected cells were observed scoredand recorded for the fluorescent pattern localization andintensity of the fluorescent conjugate of antiserum

Preparation of the Plant Extract the Herbal and the PlaceboGels The plant was macerated with ethanol and evaporatedas dry extract which was standardized with 7 as a specificmarker compound or standard The standardized ethanolextract was used as drug material which was preparedas 1 and 2 herbal gels Carbopol the gelling substancewas dispersed in cool boiled water and neutralized withsodium hydroxide solution The plant extract was dissolvedin propylene glycol added to the gel and mixed thoroughlyThe placebo gel was prepared similarly but the coloring agent(caramel) was used instead of the plant extract

Patients and Method This study is a double-blind placebotrial in the treatment of recurrent herpes labialis with Gprocumbens gel compared to placebo It was approved bythe Ethical Clearance Committee on Human Rights Relatedto Researches Involving Human Subjects The clinical trialwas performed in accordance with the ICH-GCP guidelinesand the Declaration of Helsinki The patients were informedprior to commencement of the trial and provided writtenconsensus of the participation It was conducted at threehospitals that is Ramathibodi Chulalongkorn and PhraMongkutklao hospitals

The participated patients were more than 18 years ofage diagnosed as recurrent herpes labialis and the symptomappeared within 48 hours The exclusion criteria were thepatients during pregnancy or lactation and the patients withchronic diseases and HIV infection

On the first day (D0) of the trial commencement thehistory and symptoms of the patients were recorded andthe infected lesions were photographed The Tzanck smearand viral culture were performed to confirm the diagnosisThe baseline laboratory examination included CBC SGOTSGPT BUN creatinin and urine examination

The patients were allocated into three groups by blockrandomization They were group A receiving 1 herbal gelgroup B receiving 2 herbal gel and group C receiving theplacebo gel All patients were supplied with identical tubes ofgel

They were advised to apply the gel thinly on the infectedarea every two hours on the first day and four times a dayon the following days until the lesion healed The severity ofpain and itch was recorded daily by the patient on a linearvisual analogue scale of 0ndash10 from none to very severe Datesof full crusting and complete healing were also recordedThepatients were assessed by the investigators on days 2 or 4and 7 and the day after which lesions completely healed Theassessment on each follow-up visits included the severity ofpain and itch and days of full crusting and complete healingof the lesionsThe adverse reactions or any patient complaintswere also recorded On D2ndashD4 and D7 the lesions werephotographed and viral cultures were performed The bloodand the urine were collected and examined again on the lastvisit

StatisticalMethodsWe calculated that if success rate occurredin 80 of patients with drug treatment and 50 for placebopatients it would require 45 drug-treated and 45 placebopatients to detect the success rate at 5 significance level witha power of 80 We inflated our calculated sample size to50 patients to compensate for 10 of loss follow-up rate TheFisher exact testwas used to compare the success rate betweenthe drug-treated and placebo groups Descriptive statisticswere reported as mean with standard deviation Statisticalanalysis was performed on a completed study basis

3 Results

The isolation of aqueous methanol F2 and the methanol F3fractions resulted in four antiherpetic components that is 2from F2 (Figure 1 Table 1) and 7 8 and 9 from F3 (Figure 2


Table 1 Antiviral activity of the extract extract fractions and isolated compounds from G procumbens

Extractextractfractionscompounds

MNTD120583gmL

Antiviral activity (IC50 120583gmL)Inactivation

(virucidal action)Pre-treatment inhibition of viral

adsorption and penetrationPost-treatment inhibition ofintracellular viral replication

HSV-1 HSV-2 HSV-1 HSV-2 HSV-1 HSV-2Ethanol extract gt2000 625 675 minus minus 584 568F2 1000 320 366 minus minus minus minus

F3 1000 362 391 minus minus ND 266F4 1000 347 312 + + + 4461 ND ND ND ND ND ND ND2 200 + 96 minus minus + 613 200 minus minus minus minus minus minus

4 ND ND ND ND ND ND ND5 200 minus minus minus minus minus minus

6 10 minus minus minus minus minus minus

7 100 + 50 + + minus minus

8 100 minus 40 minus minus minus minus

9 500 250 minus ND ND minus minus

Positive control 50120583gmL of Acyclovir completely inhibited the plaque formation in all test methodsminus inactive at subtoxic concentration (MNTD2) (inhibition of plaque forming lt 50)+ active but IC50 is not determinedMNTD maximum nontoxic doseND not determined

Table 1)Theflavonoids (amixture of 31 and 32 and 5) whichwere isolated from F2 and 6 from F3 had no antiviral activityCompounds 2 7 8 and 9were identified using spectroscopicmethods especially NMR with field gradient technique [11ndash14]2 AMixture of Dicaffeoylquinic Acids(21 and 22)21 (Figure 4) 35-Di-O-Caffeoylquinic Acid Amorphous yel-low powder R

119891055 silica gel 60 CHCl

3MeOHH

2Oacetic

acid(21 15 3 1) UV (MeOH) 120582max 326 nm 1H-NMR(CD3OD 300MHz) 120575 762 758 (d J = 16Hz H-71015840 H-710158401015840

each) 708 706 (d J = 82 H-21015840 H-210158401015840 each) 698 696 (dd J= 8 2Hz H-61015840 H-610158401015840 each) 678 (d J = 80 H-51015840 H-510158401015840 each)641 630 (d J = 16Hz H-81015840 H-810158401015840) 555 (ddd J = 102 97 60H5ax) 538 (ddd J = 3 3 3Hz H-3eq) 390 (dd J = 7 3HzH-4ax) 228 (dd J = 151 31 Hz H-2)22 (Figure 5) 45-Di-O-Caffeoylquinic Acid Amorphous yel-low powder R


3MeOHH

2Oacetic


each) 702 698 (d J = 2Hz H-21015840 H-210158401015840 each) 689 686 (ddJ = 8 2Hz H-61015840 H-610158401015840 each) 673 671 (d J = 8Hz H-51015840 H-510158401015840 each) 626 618 (d J = 16Hz H-81015840 H-810158401015840 each) 569 (dddJ = 103 97 70Hz H-5ax) 510 (dd J = 100 30Hz H-4ax)431 (ddd J = 30 40 30Hz H-3eq) 20ndash24 (broad m H

2-2

H2-6)

7 A Mixture of 120573-Sitosteryl (71 Figure 11) and Stigmasteryl(72 Figure 12) Glucosides White powder mp 254ndash256∘C (with decomposition) R

119891048 silica gel 60

CHCl3MeOHH

2O(20 5 05) FAB-MS [M-Na]+ mz

599 and 597 (calcd for C35H60O6576 and C

35H58O6574 for

71 and 72) EI-MS (pos ion mode)mz 414 and 412 [M+H]+(corresponding aglycones) mz 396 and 394 (low intensity)[M+H-H

2O]+ 1H-NMR (C

5D5N 300 MHz) 120575 535 (dd J =

45 15Hz H-6) 523 (dd J = 155 9Hz H-22 71) 508 (dd J= 155 9Hz H-23 71) 504 (d J = 79Hz H-11015840) 455 (dd J =116 18Hz H-61015840a) 440 (dd J = 116 49Hz H-61015840b) 426 (ddJ = 156 85Hz H-31015840 H-41015840) 404 (t J = 82 79Hz H-21015840) 396(m H-3 H-51015840) 272ndash247 (m H-4) 214ndash174 (m H-2) 193(m H-1) 17 (m H-25) 14 (m H-20) 10ndash18 (m H-8 H-9H-14 H-17) 128 (m H-28) 109 (d J = 67Hz H

3-21 72) 10

(d J = 64 H3-21 72) 094 096 (m H-24) 093 (d J = 69Hz

H3-27) 092 (d J = 7Hz H

3-26) 095 (s H

3-19) 090ndash130 (m

H-23 H-23 72) 090 (d J = 61Hz H3-27) 089 (d J = 67Hz

H3-26) 088 (t J = 767 767Hz H

3-29) 08ndash22 (m H-7 H-11

H-12 H-15 H-16) 069 (s H3-18 71) 067 (s H

3-18 72)

13C-NMR (C5D5N 75MHz) 120575 140955 (C-5) 138833 (C-22)

129521 (C-23) 121921 (C-6) 102607 (C-11015840) 78605 (C-3 31015840)78440 (C-51015840) 75331 (C-21015840) 71761 (C-41015840) 62894 (C 61015840a 61015840b)56971 (C-14 72) 56889 (C-14 71) 56297 (C-17 71) 56132(C-17 72) 51460 (C-24 71) 50404 (C-9) 46097 (C-24 72)42527 (C-13 71) 42396 (C-13 72) 40783 (C-20 71) 40010(C-12) 39368 (C-4) 37526 (C-1) 36967 (C-10) 36424 (C-2072) 34269 (C-22 72) 32196 (C-7) 32114 (C-8 25 71) 30290(C-2) 29531 (C-25 72) 29317 (C-16 71) 28560 (C-16 72)25714 (C-28 71) 24579 (C-15 72) 24546 (C-15 71) 23444(C-28 71) 21502 (C-21 71) 21305 (C-11) 19446 (C-19 72)19216 (C-19 71) 19051 (C-21 72) 12191 (C-18 71) 12010(C-18 72)

8 12-bis-Dodecanoyl-3-120572-D-Glucopyranosyl-sn-Glycerol (Mo-noglucosyl Diglyceride Figure 13) Colorless waxy mass R

119891


OHOOC

HO

H

OHH

OHH

H

HO

HO

HO

1

Figure 3

OHOOC

HO

H

OHH

OH

H

HO

HO

HO

4

O

OH

OH

21

Figure 4

015 silica gel 60 CHCl3MeOHH

2O(20 5 05) 1H-NMR

(CD3OD) 300MHz) 120575 528 (dddd J = 70 64 40 41 Hz

H-2) 477 (d J = 38Hz H-1101584010158401015840) 450 (m H-1a) 414 (dd J =120 70Hz H-1b) 400 (dd J = 108 64Hz H-3a ddd J =98 90 34Hz H-5101584010158401015840) 360 (dd J = 95 90Hz H-3101584010158401015840) 356(dd J = 108 64Hz H-3b) 341 (dd J = 97 38Hz H-2101584010158401015840)328 (dd J = 143 97Hz H-6101584010158401015840a) 319 (dd J = 97 90Hz H-4101584010158401015840) 300 (dd J = 143 34Hz H-6101584010158401015840b) 230 (t J = 76 76HzH-21015840 210158401015840) 157 (m H-31015840 310158401015840) 125 (m H-41015840-131015840 410158401015840-1310158401015840) 084(t J = 64 60Hz H-141015840 1410158401015840) 13C-NMR (CD

3OD 75MHz)

120575 19231 (C-11015840 110158401015840) 9908 (C-1101584010158401015840) 7400 (C-3101584010158401015840 4101584010158401015840) 7300 (C-2101584010158401015840) 7150 (C-2) 7000 (C-5101584010158401015840) 6600 (C-3a 3b) 6400 (C-1a1b) 6100 (C-6101584010158401015840a 6101584010158401015840b) 3469 (C-21015840 210158401015840) 2952ndash3006 (C-41015840-111015840 410158401015840-1110158401015840) 2527 (C-31015840 310158401015840) 2302 (C-131015840 1310158401015840)9 A Mixture of 120573-Sitosterol (91 Figure 14) and Stigmasterol(92 Figure 15) White needles mp 145ndash148∘C R

119891075 silica

gel 60 CHCl3MeOHH

2O(20 5 05) 1H-NMR (CDCl

3

300MHz) 120575 534 (broad d J = 54Hz H-6) 515 (dd J =15 85Hz H-22 91) 501 (dd J = 15 85Hz H-23 91) 352(dddd J = 11 10 5 4Hz H-3) 226 (m H-12) 208ndash178 (mH-22 92) 13ndash08 (m H-23 92) 17 (m H-25) 128 (m H-28) 12 (d J = 64Hz H-21) 1ndash118 (m H-9 14 17 20) 10(s H3-19) 095 (m H-24) 084 (d J = 64Hz H-26lowast) 082

(d J = 64Hz H-27lowast) 080 (t J = 65 65Hz H-29) 08ndash22(m H-1 2 7 11 15 16) 068 (s H

3-18 92) 070 (s H

3-18

91) 13C-NMR (CDCl3 75MHz) 120575 14077 (C-5) 13830 (C-

22 91) 12930 (C-23 91) 12170 (C-6) 7182 (C-3) 5688 (C-1491) 5679 (C-14 92) 5609 (C-17 92) 5599 (C-17 91) 5124(C-24 91) 5019 (C-9 91) 5016 (C-9 92) 4587 (C-24 92)4233 (C-4) 4223 (C-13) 4047 (C-20) 3980 (C-12 92) 3970

OHOOC

HO

H

OH

OHH

H

HO

HO

HO O

OH

OH

22

Figure 5

O

HO

HO

HO

OOO

O

OHHOOH

OH

OH O

HO

H3C

31

Figure 6

(C-12 91) 3653 (C-10 91) 3652 (C-10 92) 3615 (C-20 92)3397 (C-22 92) 3227 (C-1) 3192 (C-7 91) 3188 (C-7 92)3169 (C-2) 2919 (C-25 92) 2891 (C-16 91) 2824 (C-16 92)2613 (C-23 92) 2540 (C-28 91) 2437 (C-15 91) 2431 (C-15 92) 2309 (C-28 92) 2121 (C-26lowast 91) 2109 (C-21 91)1981 (C-26lowast 92) 1939 (C-19) 1905 (C-27lowastlowast 91) 1899 (C-27lowastlowast 92) 1879 (C-21 92) 1223 (C-29) 1205 (C-18 91) 1186(C-18 92) lowastlowastlowastPairs interchangeable

The antiherpetic activities of 1 and 4were not determinedbecause of the minute amount The compounds 3 5 and6were inactive The chemical structures of 1 3 4 5 and 6were identified as follows1 5-O-Caffeoyl-D-Quinic Acid (Chlorogenic Acid Figure 3)Colorless amorphous powder UV (MeOH) 120582max 326 nmshoulder at 299 nm ESI MS (pos ion mode) mz 355[M+H]+ (calcd for C

16H18O9354) 1H-NMR (DMSO-d

6

300MHz) 120575 740 (d J = 160Hz H-71015840) 700 (d J = 20HzH-21015840) 694 (dd J = 80 20Hz H-61015840) 673 (d J = 80Hz H-51015840) 614 (d J = 160Hz H-81015840) 510 (ddd J = 80 70 60HzH-5ax) 390 (broad H-3eq) 348 (dd J = 70 40Hz H-4ax) 197 (m H-2a) 186 (m H-6) 171 (m H-2b) 13C-NMR(DMSO-d

6 75MHz)120575 17500 (C-7) 16590 (C-91015840) 14820 (C-

31015840) 14547 (C-41015840) 14467 (C-71015840) 12556 (C-11015840) 12118 (C-61015840)11568 (C-51015840) 11470 (C-81015840) 11445 (C-21015840) 7320 (C-1) 7150(C-3) 7119 (C-5) 6952 (C-4) 3692 (C-2)3 A Mixture of Kaempferyl-3-O-120572-L-Rhamnosyl (1rarr 6)-120573-D-Glucopyranoside (31 Figure 6) and Kaempferyl-3-O-120572-L-Rhamnosyl (1rarr 6)-120573-D-Galactopyranoside (32Figure 7) Yellow amorphous powder mp 188ndash190∘C (Lit mp198ndash200∘C) [12] UV (MeOH) 120582max 265 nm (band II) and


O

HO

HO

HO

OOO

O

HOOH

OH

OH O

HO OH

H3C

32

Figure 7

OHO

O

O

OH

OH

OH O

HOOH

HO

OH

4

Figure 8

349 nm (band I) 1H-NMR (DMSO-d6 300MHz) 120575 1248 (d

J = 37Hz 2 times 5-OH) 1010 (OH) 804 (d J = 89Hz H-21015840H-61015840 31) 796 (d J = 90Hz H-21015840 H-61015840 32) 687 (d J =89Hz H-31015840 H-51015840 31) 685 (d J = 89Hz H-31015840 H-51015840 32)618 (d J = 20Hz H-6a H-8a) 640 (d J = 18Hz H-6b)638 (d J = 20Hz H-8b) 531 (d J = 75Hz H-110158401015840) 527 (dJ = 15Hz H-110158401015840) 516 (d J = 44Hz OH) 505 (d J = 47HzOH) 503 (d J = 63Hz OH) 488 (d J = 50Hz OH) 455(d J = 53Hz OH) 452 (d J = 41Hz OH) 438 (d J =60Hz H-1101584010158401015840) 437 (d J = 60Hz H-1101584010158401015840) 368 (d J = 100HzOH) 358 (m H-210158401015840 H-410158401015840 H-610158401015840) 310ndash358 (m H-310158401015840 H-510158401015840H-2101584010158401015840 H-3101584010158401015840 H4101584010158401015840 H-5101584010158401015840) 105 (d J = 61Hz 6101584010158401015840-CH

3-Rha)

097 (d J = 63Hz 6101584010158401015840-CH3-Rha)

13C-NMR (DMSO-d6 75MHz) 120575 17700 (C-4) 16500 (C-

7) 16100 (C-5) 16000 (C-41015840) 15600 (C-2 C-9) 13300 (C-3)13100 (C-21015840 C-61015840) 12100 (C-11015840) 11501 (C-31015840 C-51015840 32) 11500(C-31015840 C-51015840 31) 10300 (C-10) 10100 (C-110158401015840) 10000 (C-1101584010158401015840)9850 (C-6) 9350 (C-8) 7565 (C-310158401015840 31) 7550 (C-510158401015840 31)7393 (C-210158401015840 31) 7331 (C-510158401015840 32) 7274 (C-310158401015840 32) 7150 (C-210158401015840 32) 7150 (C-2101584010158401015840 C-4101584010158401015840 31) 7033 (C-2101584010158401015840 C-4101584010158401015840 32)7033 (C-3101584010158401015840) 7002 (C-410158401015840 31) 6766 (C-410158401015840 32) 6766 (C-5101584010158401015840)6649 (C-610158401015840) 1741 (C-6101584010158401015840 32) 1731 (C-6101584010158401015840 31) [13]4 Quercetin-3-O-120573-D-Glucopyranoside (Figure 8) Yellowamorphous powder UV (MeOH) 120582max 256 nm (band II) and358 nm (band I) 1H-NMR (CD

3OD 300MHz) 120575 772 (d J =

21Hz H-21015840) 752 (dd J = 85 22Hz H-61015840) 689 (d J = 85HzH-51015840) 636 (d J = 20Hz H-8) 621 (d J = 20Hz H-6) 515(d J = 75Hz H-110158401015840) 371 (dd J = 119 24Hz H-610158401015840a) 356(dd J = 119 52Hz H-610158401015840b) 348 (dd J = 80 75Hz H-210158401015840)342 (dd J = 90 80Hz H-310158401015840) 334 (dd J = 100 90Hz H-410158401015840) 322 (ddd J = 105 55 25HzH-510158401015840) 13C-NMR (CD

3OD

OHO

O

O

OH

OH

OH O

HOOH

HO

5

Figure 9

O

OH

HO

OH

OH

O

6

Figure 10

75MHz) 120575 17949 (C-4) 16619 (C-7) 15904 (C-3) 15849 (C-2 C-9) 14986 (C-41015840) 14592 (C-31015840) 13560 (C-3) 12319 (C-11015840)12309 (C-61015840) 11756 (C-51015840) 11603 (C-21015840) 10539 (C-10) 10431(C-110158401015840) 9965 (C-6) 9478 (C-8) 7838 (C-310158401015840) 7811 (C-510158401015840) 7572(C-210158401015840) 7124 (C-410158401015840) 6256 (C-610158401015840)5 Kaempferyl Glucopyranoside (Figure 9) Yellow amorphouspowder mp 165ndash170∘C (with decomposition) [14] UV(MeOH) 120582max 266 nm (band II) and 349 nm (band I) IR(KBr) ]max 3350ndash3450 (OndashH) 2933 (aliph (CndashH)) 1633(C=O) 1611 (C=C) 1604 (C=O) cmminus1 ESI MSM

119877448 1H-

NMR (DMSO-d6 300MHz) 120575 1250 (broad s 5-OH) 801 (d

J = 90Hz H-21015840 H-61015840 each) 684 (d J = 90Hz H-31015840 H-51015840each) 638 (d J =2HzH-8) 617 (d J =2HzH-6) 541 (d J =71Hz H-110158401015840) 524 (210158401015840-OH) 496 (510158401015840-OH) 486 (310158401015840-OH 410158401015840-OH) 416 (610158401015840-OH) 355ndash330 (broad s H-610158401015840) 318 (H-510158401015840)316 (broad s H-210158401015840) 306 (broad s H-310158401015840 H-410158401015840) 13C-NMR(DMSO-d

6 75MHz) 17713 (C-4) 16510 (C-7) 16109 (C-5)

15643 (C-9) 15588 (C-2) 13308 (C-3) 13075 (C-21015840 C-61015840)12088 (C-11015840) 11502 (C-31015840 C-51015840) 10343 (C-10) 10097 (D C-110158401015840) 9900 (C-6) 9378 (C-8) 7741 (C-310158401015840) 7639 (C-510158401015840) 7417(C-210158401015840) 6984 (C-410158401015840) 6080 (C-610158401015840)6Kaempferol (35741015840-tetrahydroxyflavone Figure 10) Yellowamorphous powder UV (MeOH) 120582max 260 nm (shoulder)and 365 nm (band I) 1H-NMR (CD

3OD 300MHz) 120575 807

(d J = 85Hz H-21015840 H-61015840) 691 (d J = 85Hz H-31015840 H-51015840)640 (d J = 17Hz H-8) 619 (d J = 17Hz H-6) 13C-NMR(CD3OD 75MHz) 120575 17742 (C-4) 16556 (C-7) 16246 (C-5)

16056 (C-41015840) 15824 (C-9) 14810 (C-2) 13716 (C-3) 13068(C-21015840 C-61015840) 12376 (C-11015840) 11631 (C-31015840 C-51015840) 10456 (C-10)9929 (C-6) 94486 (C-8)

To evaluate the antiherpetic effectiveness of G procum-bensweperformed the double-blind randomized controlledclinical trial in patients with recurrent herpes labialis of the


OHO

HO

OH

OH

O

HH3C

CH3

CH3

CH3

CH3

CH3

71

Figure 11

OHO

HO

OH

OHO

HH3C

CH3

CH3

CH3

CH3

CH3

72

Figure 12

herbal products which contained 1 and 2 of the standard-ized plant extracts

A total of 65 patients participated in the study Fourpatients (three in placebo group one in the treated group)did not appear for the followup One patient in the treatedgroup did not record the date of full crusting and completehealing One patient in the placebo group could not continuetreatment due to allergic contact dermatitis Therefore only59 patients were evaluatedTheywere divided into the treatedgroup with 1 herbal gel (19 patients) the treated groupwith 2 herbal gel (22 patients) and the placebo group (18patients)The antiherpetic results of the treated groups (1 and2 herbal gels) were not different significantlyThe data fromboth treated groupswere thus combined (41 patients 10malesand 31 females) and compared to the placebo group whichcomprised 18 patients (3males 15 females) Both groups werecomparable for age sex pain and itching scores proportionof patient who are suffering from pain and percentage ofpatients who had positive tests for Tzanck smear and viralculture The only significant difference was the proportion ofitching patients It was greater in the treated group as shownin Table 2

The positive results of Tzanck smear of the treated andcontrol groups were 700 and 722 respectively The viralculture showed the presence of HSV-1 in the treated andcontrol groups 462 and 313 respectively The patients inthe treated and control groupswere not significantly differentapart from the itching in the treated group which wassignificantly different from the control (Table 2)

The first followup (D2 D4) indicated that the treatedgroup still suffered the pain but the number decreased from

OHO OH

OH

HOO

CHO

O

O

OH2C

H2C

CH3

H3C

( )n = 8

( )n = 8

8

Figure 13

HO

HH3C CH3

CH3

CH3

CH3 CH3

91

Figure 14

HO

HH3C

CH3

CH3

CH3

CH3

CH3

92

Figure 15

27 patients (6590) to 20 patients (3415) the itchingdecreasing from 6829 to 4878 The patients who aresuffering from pain in the placebo group decreased from 11in 18 patients (6110) to 6 in 18 patients (3333) but theproportion of patients with itching remain unchanged (7 in 18patients 3889) The average pain scores of the treated andplacebo groups were 111 and 141 respectively The averageitching scores of the treated and the placebo groups were 160and 144 respectively The results of both groups were notsignificantly different (Table 3)

Twenty-one of 41 patients in the treated group (5122)had full crusting within 4 days It was comparable to 9 of18 patients (50) in the placebo group Lesions completelyhealed within 7 days in twenty-six patients (6341) of thetreated group whereas it was 8 in 18 patients (4444) forthe placebo group In the treated group the average time forfull crusting was 49 days and for complete healing was 87days The same figures for the placebo group were 51 and 94days respectivelyThe viral culture onD7 showed the numberof patients in the treated group who had positive culture tobe reduced from 19 to 3 whereas those in the placebo group


Table 2 Patients clinical and laboratory data before treatment in the treatment and placebo groups

Patients The treated groups The placebo groups P valueSex

Male 10 3 051Female 31 15Total 41 18

Age (years)Average 3715 4172Range 16ndash71 20ndash69 020Standard deviation 1131 1525

Pain on the first day (D0)Casetotal () 2741 (6590) 1118 (6110) 072Pain score (average) 285 286 090

Itching on the first day (D0)Casetotal () 2841 (6829) 718 (3889) 003Itching score (average) 273 210 046

Tzanck smear before the treatmentPositive result () 700 722 086

Viral culture before the treatmentPositive result () 1939 (487) 516 (3125) 023

Table 3 The results of the double-blind randomized controlled clinical trial of Gynura procumbens gel product

Result The treated groups The placebo groups P valueThe pain on D2ndashD4

Number of patientstotal () 1441 (3415) 618 (3333) 095Average pain score 111 141 065

The itching from D2ndashD4Number of patientstotal () 2041 (4878) 718 (3889) 048Average itching score 160 144 080

The crust dayWithin 4 days (patientstotal) 2141 (5122) 918 (5000) 093Average time (days) 49 51 084

The healing dayWithin 7 days (patientstotal) 2641 (6341) 818 (4444) 018Average time (days) 87 94 056

The viral cultureProportion +ve culture ()On D2ndashD4 838 (2105) 315 (200) 10On D7 339 (769) 315 (200) 033

reduced from 5 to 3 patients The results differed from theplacebo insignificantly (Table 3)

Side Effects The blood chemistry (CBC UA SGOT SGPTBUN and Cr) of the participated patients was normal afterthe treatment There was one patient in the placebo groupsuffering from allergic contact dermatitis so the treatmentwas discontinued There were 10 patients (244) in thetreated group and 5 patients (278) in the placebo groupwho had mild itching and irritation but all patients couldtolerate the symptoms without disruption of the treatmentThe side effects in the treated and placebo groups were notsignificantly different

4 Discussion

The phytochemical work on G procumbens showed that theethanol extract of the aerial plant parts the drug materialhad the virucidal action againstHSV-1 andHSV-2 (IC

506250

and 6750 120583gmL resp) and prevented the viral replicationwith IC

505840 and 5680120583gmL respectively Several anti-

herpetic compounds were isolated from the extract Someof them such as dicaffeoylquinic acids (2) were knownfor the antiviral activity [15] We found that the extractcontained other antiherpetic compounds that is 7 8 and9 The antiherpetic activity against HSV-2 of 8 the gly-cosphingolipid was stronger than 2 (dicaffeoylquinic acids)


In addition several flavonoids were found in the extractThey probably exerted the activity at the concentrationabove the MNTDThe test concentrations should not exceedthe MNTD because the cultured cells would be damagedHowever the presence of the flavonoids possibly impartedanti-inflammatory effect [1 4 16] which could alleviate theinfectious symptoms

The laboratory evidence directed us to carry on the clini-cal study The double-blind randomized controlled clinicaltrial of recurrent herpes labialis with herbal and placebogels was conducted The ethanol extract was standardizedusing a mixture of sitosteryl and stigmasteryl glucosides asa marker compound (standard) because it could be isolatedin a sufficient amount without difficulty The standardizedextract was incorporated in gel base as 1 and 2 herbal gelsThe placebo gel was prepared similarly colored like herbal gelbut without the extract

We could not achieve the number of the participatedpatients as calculated (50 in each group) though the trialswere conducted at three hospitalsTherewere only 65 patientsparticipating in the trial but only 59 patients completed thestudyThus the power of this clinical trial reduced from 80to only 50 Though the antiherpetic result of herbal geldiffered insignificantly from the placebo it tended to havesome benefitsThese included the tendency of the decreasingnumber of patients who suffered from itches in the treatedgroup the greater number of patients healing within 7 daysand the lesser number of patients infected with HSV-1 Fromthe traditional laboratory and clinical pieces of evidencewe could postulate that G procumbens tended to reducethe suffering from HSV-1 infection and might have someantiherpetic action We performed the stability test of theherbal gel and found that the appearance and the contentof the marker compound remained unchanged when storingthe gel at 25∘C for 24 months and at 40∘C for 6 months Toimprove the efficacy result of this herbal gel for the treatmentof recurrent herpes labialis we recommend the change of gelbase to reduce the irritation and increase the potency of itFor further clinical investigation we recommend increasingpatients number and high concentration of the plant extractin the herbal gel

5 Conclusion

Gynura procumbens comprised antiherpetic compounds thatis caffeoylquinic acid derivatives phytosteryl glucosidesand glycoglycerolipids The flavonoids in this plant possiblyimparted anti-inflammatory effect which was advantageousto the herpetic patients The laboratory evidence and thereduction of the infection incidence in patients supported theantiherpetic effect of G procumbens The insignificant resultof the clinical study might arise from the low participatedpatient number and insufficient extract concentration in theherbal product

Conflict of Interests

All authors declare that they have no conflict of interests

Acknowledgments

The authors thank the German Academic Exchange Ser-vice (DAAD) Mahidol University the Higher EducationCommission Thailand and Thailand Research Fund for thefellowships (DBG4780002)

References

[1] Dictionary of Chinese Materia Medica Shanghai Science andTechnology Publishing House Shanghai China 1997

[2] School of Traditional Medicine Wat Phra Chetuphon Wat phraChetuphon Bangkok Thailand 1978

[3] ldquoAttributed properties and usesrdquo inMedicinal Plants of East andSoutheast Asia L M Perry Ed MIT Press London UK 1980

[4] M N Iskander Y Song I M Coupar and J W JiratchariyakulldquoAntiinflammatory screening of the medicinal plant Gynuraprocumbensrdquo Plant Foods for Human Nutrition vol 57 no 3-4pp 233ndash244 2002

[5] H Wiedenfeld ldquoTwo pyrrolizidine alkaloids from Gynurascandensrdquo Phytochemistry vol 21 no 11 pp 2767ndash2768 1982

[6] X T Liang and E Roeder ldquoSenecionine from Gynura segetumrdquoPlanta Medica vol 51 p 362 1984

[7] J R Matheson and D J Robins ldquoPyrrolizidine alkaloids fromGynura sarmentosardquo Fitoterapia vol 63 no 6 p 557 1992

[8] M Takahira Y Kondo G Kusano and S Nozoe ldquoFour new3120572-hydroxy spirost-5-ene derivatives from Gynura JaponicamakinordquoTetrahedron Letters vol 18 no 41 pp 3647ndash3650 1977

[9] F Bohlmann and C Zdero ldquoGynuron ein neues terpen-cumarin-derivat ausGynura crepioidesrdquo Phytochemistry vol 16no 4 pp 494ndash495 1977

[10] K Yoshitama M Kaneshige N Ishikura F Araki S Yaharaand K Abe ldquoA stable reddish purple anthocyanin in the leafof Gynura aurantiaca cv (Purple Passion)rdquo Journal of PlantResearch vol 107 no 3 pp 209ndash214 1994

[11] S JarikasemA phytochemical study of Anti-Herpes simplex com-ponents from Gynura procumbes Merr [PhD thesis] Facultyof Graduate Studies Mahidol University Bangkok Thailand2000

[12] K Yasukawa and M Takido ldquoA flavonol glycoside from Lysi-machia mauritianardquo Phytochemistry vol 26 no 4 pp 1224ndash1226 1987

[13] P K Agrawal Carbon-13 NMR of Flavonoids Elservier NewYork NY USA 1989

[14] J Bucking F M Macdonald and H M Bradley Eds Dictio-nary of Natural Products vol 5 ChapmanampHall London UK1994

[15] Y Li P P H But and V E C Ooi ldquoAntiviral activity and modeof action of caffeoylquinic acids from Schefflera heptaphylla (L)Frodinrdquo Antiviral Research vol 68 no 1 pp 1ndash9 2005

[16] E-J Lee G-E Ji and M-K Sung ldquoQuercetin and kaempferolsuppress immunoglobulin E-mediated allergic inflammation inRBL-2H3 and Caco-2 cellsrdquo Inflammation Research vol 59 no10 pp 847ndash854 2010

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


MPLC RP-18 (8 2 5 5) gradiently

LPLC RP-18 WM (55 45)

flow rate

5

inactive LPLC RP-8 WM 6 4 flow rate

Recryst from MeOH

F221 F222

3

inactive 4


per fraction


per fraction

MCI gel CHP20P WM (1 1)

F21 F22 F23

F2

HPLC-RP18 WM (96 4)

flow rate

HPLC-RP18 WM (8 2)

flow rate

F211

F2111

1

LPLC RP-18 WM (8 2)

flow rate

F212

21 22

2

active

W water

(123 g)

per fraction100mL

(87 g) (075g) (08 g)

20mL 5mL

1mLmin

1mLmin

R 190ndash240mL

R 150ndash165mLR 175ndash280mL

R 470ndash600mL(250mg) (80mg)

(60mg)(100mg)

(24mg)(30mg)

(90mg) (37mg)

(4mg)

(3mg) (4mg)

(8mg)

1mLmin

4mLmin

4mLmin

tR 10ndash80min

tR 20ndash40min tR 51ndash65min

NDlowast

NDlowast

not determined according to the insufficient amount of the isolated substance NDlowast

NDlowast

NDlowast

tR 80min

tR 75min

rarr

M methanol


pyrrolizidine alkaloids [5ndash7] spirostanol [8] coumarins [7ndash9] and anthocyanins [10] SinceG procumbens so far has notbeen explored towards antiherpes viral activity on the otherhand according to traditional Thai medicine the utilizationof the plant may be associated with herpes viral infectionthe investigation of the plant components for the antiherpeticactivity is performed

2 Materials and Methods

PlantMaterialGynura procumbenswas collected fromChan-thaburi province Thailand in October 1994 The plant wasidentified by Ms Leena Phupatpong an expert botanist atthe Forest Herbarium (BKF) Royal Forest Department Min-istry of Agriculture and Cooperatives Bangkok ThailandA voucher specimen (BKF no 127362) was deposited at thesame placeExtraction and Isolation The aerial parts of the plant (25 kgfresh) were washed cut into pieces dried in a hot-air oven(60∘C) and ground yielding 17 kg of the coarse-powdereddrug It was successively extracted in a Soxhlet appara-tus using petroleum ether (40ndash60∘C) dichloromethane

and ethanol The solvents were removed under reducedpressure The dry ethanol extract (114 g) was further sepa-rated using chromatographic columns with different packingmaterials MCI gel CHP20P column fractionated the extractinto the fractions F1 (water) F2 (water-methanol 1 1) F3(methanol) and F4 (ethyl acetate) F2 F3 and F4 were activeagainst herpes virus

F2 (123 g) and F3 (64 g) were progressively chro-matographed on MCI gel CHP20P using aqueous methanol(1 1) for F2 and methanol for F3 as solvent systems resultingin F21 (87 g) F22 (075 g) and F23 (08 g) and F31 (20 g)F32 (30 g) and F33 (08 g) respectively Compounds 1 2 34 and 5 were isolated from F2 (Figure 1) while compounds6 7 8 and 9 were isolated from F3 (Figure 2) F4 was notfurther studied

Cell Line The Vero cell line (African green monkey kidneycells) was grown and maintained in Eaglersquos minimum essen-tial medium (MEM) supplemented with 10 fetal calf serumand antibiotics (5 times 104 cells per well)

Virus HSV-1 strain KOS and HSV-2 strain Baylor 186were obtained from the Department ofMicrobiology Faculty



6(5 mg)

inactive

(CMW 10 1 0 100 10 0 100 25 25100 30 3 0 100 0)


CM (9 1) (CMW 15 5 05)

7(25 mg) active

8(34 mg) active


Recryst from MeOH

(100 mg)

9(60 mg) active


F3 (64 g)

F31 (2 g)

F32 (3 g)

F33 (08 g)

C chloroform

tR 66min


rarr

rarr rarr

rarr

rarr

R 10ndash90mL

LPLC-SiO2LPLC-SiO2

SiO2SiO2


W waterM methanol


















2for









3 Results





MNTD120583gmL








7 100 + 50 + + minus minus






3MeOHH

2Oacetic




3MeOHH

2Oacetic



2-2

H2-6)


119891048 silica gel 60

CHCl3MeOHH

2O(20 5 05) FAB-MS [M-Na]+ mz


35H58O6574 for


2O]+ 1H-NMR (C

5D5N 300 MHz) 120575 535 (dd J =


3-21 72) 10

(d J = 64 H3-21 72) 094 096 (m H-24) 093 (d J = 69Hz

H3-27) 092 (d J = 7Hz H

3-26) 095 (s H

3-19) 090ndash130 (m

H-23 H-23 72) 090 (d J = 61Hz H3-27) 089 (d J = 67Hz

H3-26) 088 (t J = 767 767Hz H

3-29) 08ndash22 (m H-7 H-11

H-12 H-15 H-16) 069 (s H3-18 71) 067 (s H

3-18 72)

13C-NMR (C5D5N 75MHz) 120575 140955 (C-5) 138833 (C-22)



119891


OHOOC

HO

H

OHH

OHH

H

HO

HO

HO

1

Figure 3

OHOOC

HO

H

OHH

OH

H

HO

HO

HO

4

O

OH

OH

21

Figure 4


2O(20 5 05) 1H-NMR

(CD3OD) 300MHz) 120575 528 (dddd J = 70 64 40 41 Hz


3OD 75MHz)


119891075 silica

gel 60 CHCl3MeOHH

2O(20 5 05) 1H-NMR (CDCl

3



3-18 92) 070 (s H

3-18

91) 13C-NMR (CDCl3 75MHz) 120575 14077 (C-5) 13830 (C-


OHOOC

HO

H

OH

OHH

H

HO

HO

HO O

OH

OH

22

Figure 5

O

HO

HO

HO

OOO

O

OHHOOH

OH

OH O

HO

H3C

31

Figure 6




6


6 75MHz)120575 17500 (C-7) 16590 (C-91015840) 14820 (C-



O

HO

HO

HO

OOO

O

HOOH

OH

OH O

HO OH

H3C

32

Figure 7

OHO

O

O

OH

OH

OH O

HOOH

HO

OH

4

Figure 8



3-Rha)

097 (d J = 63Hz 6101584010158401015840-CH3-Rha)

13C-NMR (DMSO-d6 75MHz) 120575 17700 (C-4) 16500 (C-


3OD 300MHz) 120575 772 (d J =


3OD

OHO

O

O

OH

OH

OH O

HOOH

HO

5

Figure 9

O

OH

HO

OH

OH

O

6

Figure 10


119877448 1H-



6 75MHz) 17713 (C-4) 16510 (C-7) 16109 (C-5)


3OD 300MHz) 120575 807


16056 (C-41015840) 15824 (C-9) 14810 (C-2) 13716 (C-3) 13068(C-21015840 C-61015840) 12376 (C-11015840) 11631 (C-31015840 C-51015840) 10456 (C-10)9929 (C-6) 94486 (C-8)



OHO

HO

OH

OH

O

HH3C

CH3

CH3

CH3

CH3

CH3

71

Figure 11

OHO

HO

OH

OHO

HH3C

CH3

CH3

CH3

CH3

CH3

72

Figure 12





OHO OH

OH

HOO

CHO

O

O

OH2C

H2C

CH3

H3C

( )n = 8

( )n = 8

8

Figure 13

HO

HH3C CH3

CH3

CH3

CH3 CH3

91

Figure 14

HO

HH3C

CH3

CH3

CH3

CH3

CH3

92

Figure 15





















4 Discussion


506250








5 Conclusion




Acknowledgments


References






















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















6(5 mg)

inactive

(CMW 10 1 0 100 10 0 100 25 25100 30 3 0 100 0)


CM (9 1) (CMW 15 5 05)

7(25 mg) active

8(34 mg) active


Recryst from MeOH

(100 mg)

9(60 mg) active


F3 (64 g)

F31 (2 g)

F32 (3 g)

F33 (08 g)

C chloroform

tR 66min


rarr

rarr rarr

rarr

rarr

R 10ndash90mL

LPLC-SiO2LPLC-SiO2

SiO2SiO2


W waterM methanol


















2for









3 Results





MNTD120583gmL








7 100 + 50 + + minus minus






3MeOHH

2Oacetic




3MeOHH

2Oacetic



2-2

H2-6)


119891048 silica gel 60

CHCl3MeOHH

2O(20 5 05) FAB-MS [M-Na]+ mz


35H58O6574 for


2O]+ 1H-NMR (C

5D5N 300 MHz) 120575 535 (dd J =


3-21 72) 10

(d J = 64 H3-21 72) 094 096 (m H-24) 093 (d J = 69Hz

H3-27) 092 (d J = 7Hz H

3-26) 095 (s H

3-19) 090ndash130 (m

H-23 H-23 72) 090 (d J = 61Hz H3-27) 089 (d J = 67Hz

H3-26) 088 (t J = 767 767Hz H

3-29) 08ndash22 (m H-7 H-11

H-12 H-15 H-16) 069 (s H3-18 71) 067 (s H

3-18 72)

13C-NMR (C5D5N 75MHz) 120575 140955 (C-5) 138833 (C-22)



119891


OHOOC

HO

H

OHH

OHH

H

HO

HO

HO

1

Figure 3

OHOOC

HO

H

OHH

OH

H

HO

HO

HO

4

O

OH

OH

21

Figure 4


2O(20 5 05) 1H-NMR

(CD3OD) 300MHz) 120575 528 (dddd J = 70 64 40 41 Hz


3OD 75MHz)


119891075 silica

gel 60 CHCl3MeOHH

2O(20 5 05) 1H-NMR (CDCl

3



3-18 92) 070 (s H

3-18

91) 13C-NMR (CDCl3 75MHz) 120575 14077 (C-5) 13830 (C-


OHOOC

HO

H

OH

OHH

H

HO

HO

HO O

OH

OH

22

Figure 5

O

HO

HO

HO

OOO

O

OHHOOH

OH

OH O

HO

H3C

31

Figure 6




6


6 75MHz)120575 17500 (C-7) 16590 (C-91015840) 14820 (C-



O

HO

HO

HO

OOO

O

HOOH

OH

OH O

HO OH

H3C

32

Figure 7

OHO

O

O

OH

OH

OH O

HOOH

HO

OH

4

Figure 8



3-Rha)

097 (d J = 63Hz 6101584010158401015840-CH3-Rha)

13C-NMR (DMSO-d6 75MHz) 120575 17700 (C-4) 16500 (C-


3OD 300MHz) 120575 772 (d J =


3OD

OHO

O

O

OH

OH

OH O

HOOH

HO

5

Figure 9

O

OH

HO

OH

OH

O

6

Figure 10


119877448 1H-



6 75MHz) 17713 (C-4) 16510 (C-7) 16109 (C-5)


3OD 300MHz) 120575 807


16056 (C-41015840) 15824 (C-9) 14810 (C-2) 13716 (C-3) 13068(C-21015840 C-61015840) 12376 (C-11015840) 11631 (C-31015840 C-51015840) 10456 (C-10)9929 (C-6) 94486 (C-8)



OHO

HO

OH

OH

O

HH3C

CH3

CH3

CH3

CH3

CH3

71

Figure 11

OHO

HO

OH

OHO

HH3C

CH3

CH3

CH3

CH3

CH3

72

Figure 12





OHO OH

OH

HOO

CHO

O

O

OH2C

H2C

CH3

H3C

( )n = 8

( )n = 8

8

Figure 13

HO

HH3C CH3

CH3

CH3

CH3 CH3

91

Figure 14

HO

HH3C

CH3

CH3

CH3

CH3

CH3

92

Figure 15





















4 Discussion


506250








5 Conclusion




Acknowledgments


References






















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























2for









3 Results





MNTD120583gmL








7 100 + 50 + + minus minus






3MeOHH

2Oacetic




3MeOHH

2Oacetic



2-2

H2-6)


119891048 silica gel 60

CHCl3MeOHH

2O(20 5 05) FAB-MS [M-Na]+ mz


35H58O6574 for


2O]+ 1H-NMR (C

5D5N 300 MHz) 120575 535 (dd J =


3-21 72) 10

(d J = 64 H3-21 72) 094 096 (m H-24) 093 (d J = 69Hz

H3-27) 092 (d J = 7Hz H

3-26) 095 (s H

3-19) 090ndash130 (m

H-23 H-23 72) 090 (d J = 61Hz H3-27) 089 (d J = 67Hz

H3-26) 088 (t J = 767 767Hz H

3-29) 08ndash22 (m H-7 H-11

H-12 H-15 H-16) 069 (s H3-18 71) 067 (s H

3-18 72)

13C-NMR (C5D5N 75MHz) 120575 140955 (C-5) 138833 (C-22)



119891


OHOOC

HO

H

OHH

OHH

H

HO

HO

HO

1

Figure 3

OHOOC

HO

H

OHH

OH

H

HO

HO

HO

4

O

OH

OH

21

Figure 4


2O(20 5 05) 1H-NMR

(CD3OD) 300MHz) 120575 528 (dddd J = 70 64 40 41 Hz


3OD 75MHz)


119891075 silica

gel 60 CHCl3MeOHH

2O(20 5 05) 1H-NMR (CDCl

3



3-18 92) 070 (s H

3-18

91) 13C-NMR (CDCl3 75MHz) 120575 14077 (C-5) 13830 (C-


OHOOC

HO

H

OH

OHH

H

HO

HO

HO O

OH

OH

22

Figure 5

O

HO

HO

HO

OOO

O

OHHOOH

OH

OH O

HO

H3C

31

Figure 6




6


6 75MHz)120575 17500 (C-7) 16590 (C-91015840) 14820 (C-



O

HO

HO

HO

OOO

O

HOOH

OH

OH O

HO OH

H3C

32

Figure 7

OHO

O

O

OH

OH

OH O

HOOH

HO

OH

4

Figure 8



3-Rha)

097 (d J = 63Hz 6101584010158401015840-CH3-Rha)

13C-NMR (DMSO-d6 75MHz) 120575 17700 (C-4) 16500 (C-


3OD 300MHz) 120575 772 (d J =


3OD

OHO

O

O

OH

OH

OH O

HOOH

HO

5

Figure 9

O

OH

HO

OH

OH

O

6

Figure 10


119877448 1H-



6 75MHz) 17713 (C-4) 16510 (C-7) 16109 (C-5)


3OD 300MHz) 120575 807


16056 (C-41015840) 15824 (C-9) 14810 (C-2) 13716 (C-3) 13068(C-21015840 C-61015840) 12376 (C-11015840) 11631 (C-31015840 C-51015840) 10456 (C-10)9929 (C-6) 94486 (C-8)



OHO

HO

OH

OH

O

HH3C

CH3

CH3

CH3

CH3

CH3

71

Figure 11

OHO

HO

OH

OHO

HH3C

CH3

CH3

CH3

CH3

CH3

72

Figure 12





OHO OH

OH

HOO

CHO

O

O

OH2C

H2C

CH3

H3C

( )n = 8

( )n = 8

8

Figure 13

HO

HH3C CH3

CH3

CH3

CH3 CH3

91

Figure 14

HO

HH3C

CH3

CH3

CH3

CH3

CH3

92

Figure 15





















4 Discussion


506250








5 Conclusion




Acknowledgments


References






















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















MNTD120583gmL








7 100 + 50 + + minus minus






3MeOHH

2Oacetic




3MeOHH

2Oacetic



2-2

H2-6)


119891048 silica gel 60

CHCl3MeOHH

2O(20 5 05) FAB-MS [M-Na]+ mz


35H58O6574 for


2O]+ 1H-NMR (C

5D5N 300 MHz) 120575 535 (dd J =


3-21 72) 10

(d J = 64 H3-21 72) 094 096 (m H-24) 093 (d J = 69Hz

H3-27) 092 (d J = 7Hz H

3-26) 095 (s H

3-19) 090ndash130 (m

H-23 H-23 72) 090 (d J = 61Hz H3-27) 089 (d J = 67Hz

H3-26) 088 (t J = 767 767Hz H

3-29) 08ndash22 (m H-7 H-11

H-12 H-15 H-16) 069 (s H3-18 71) 067 (s H

3-18 72)

13C-NMR (C5D5N 75MHz) 120575 140955 (C-5) 138833 (C-22)



119891


OHOOC

HO

H

OHH

OHH

H

HO

HO

HO

1

Figure 3

OHOOC

HO

H

OHH

OH

H

HO

HO

HO

4

O

OH

OH

21

Figure 4


2O(20 5 05) 1H-NMR

(CD3OD) 300MHz) 120575 528 (dddd J = 70 64 40 41 Hz


3OD 75MHz)


119891075 silica

gel 60 CHCl3MeOHH

2O(20 5 05) 1H-NMR (CDCl

3



3-18 92) 070 (s H

3-18

91) 13C-NMR (CDCl3 75MHz) 120575 14077 (C-5) 13830 (C-


OHOOC

HO

H

OH

OHH

H

HO

HO

HO O

OH

OH

22

Figure 5

O

HO

HO

HO

OOO

O

OHHOOH

OH

OH O

HO

H3C

31

Figure 6




6


6 75MHz)120575 17500 (C-7) 16590 (C-91015840) 14820 (C-



O

HO

HO

HO

OOO

O

HOOH

OH

OH O

HO OH

H3C

32

Figure 7

OHO

O

O

OH

OH

OH O

HOOH

HO

OH

4

Figure 8



3-Rha)

097 (d J = 63Hz 6101584010158401015840-CH3-Rha)

13C-NMR (DMSO-d6 75MHz) 120575 17700 (C-4) 16500 (C-


3OD 300MHz) 120575 772 (d J =


3OD

OHO

O

O

OH

OH

OH O

HOOH

HO

5

Figure 9

O

OH

HO

OH

OH

O

6

Figure 10


119877448 1H-



6 75MHz) 17713 (C-4) 16510 (C-7) 16109 (C-5)


3OD 300MHz) 120575 807


16056 (C-41015840) 15824 (C-9) 14810 (C-2) 13716 (C-3) 13068(C-21015840 C-61015840) 12376 (C-11015840) 11631 (C-31015840 C-51015840) 10456 (C-10)9929 (C-6) 94486 (C-8)



OHO

HO

OH

OH

O

HH3C

CH3

CH3

CH3

CH3

CH3

71

Figure 11

OHO

HO

OH

OHO

HH3C

CH3

CH3

CH3

CH3

CH3

72

Figure 12





OHO OH

OH

HOO

CHO

O

O

OH2C

H2C

CH3

H3C

( )n = 8

( )n = 8

8

Figure 13

HO

HH3C CH3

CH3

CH3

CH3 CH3

91

Figure 14

HO

HH3C

CH3

CH3

CH3

CH3

CH3

92

Figure 15





















4 Discussion


506250








5 Conclusion




Acknowledgments


References






















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















OHOOC

HO

H

OHH

OHH

H

HO

HO

HO

1

Figure 3

OHOOC

HO

H

OHH

OH

H

HO

HO

HO

4

O

OH

OH

21

Figure 4


2O(20 5 05) 1H-NMR

(CD3OD) 300MHz) 120575 528 (dddd J = 70 64 40 41 Hz


3OD 75MHz)


119891075 silica

gel 60 CHCl3MeOHH

2O(20 5 05) 1H-NMR (CDCl

3



3-18 92) 070 (s H

3-18

91) 13C-NMR (CDCl3 75MHz) 120575 14077 (C-5) 13830 (C-


OHOOC

HO

H

OH

OHH

H

HO

HO

HO O

OH

OH

22

Figure 5

O

HO

HO

HO

OOO

O

OHHOOH

OH

OH O

HO

H3C

31

Figure 6




6


6 75MHz)120575 17500 (C-7) 16590 (C-91015840) 14820 (C-



O

HO

HO

HO

OOO

O

HOOH

OH

OH O

HO OH

H3C

32

Figure 7

OHO

O

O

OH

OH

OH O

HOOH

HO

OH

4

Figure 8



3-Rha)

097 (d J = 63Hz 6101584010158401015840-CH3-Rha)

13C-NMR (DMSO-d6 75MHz) 120575 17700 (C-4) 16500 (C-


3OD 300MHz) 120575 772 (d J =


3OD

OHO

O

O

OH

OH

OH O

HOOH

HO

5

Figure 9

O

OH

HO

OH

OH

O

6

Figure 10


119877448 1H-



6 75MHz) 17713 (C-4) 16510 (C-7) 16109 (C-5)


3OD 300MHz) 120575 807


16056 (C-41015840) 15824 (C-9) 14810 (C-2) 13716 (C-3) 13068(C-21015840 C-61015840) 12376 (C-11015840) 11631 (C-31015840 C-51015840) 10456 (C-10)9929 (C-6) 94486 (C-8)



OHO

HO

OH

OH

O

HH3C

CH3

CH3

CH3

CH3

CH3

71

Figure 11

OHO

HO

OH

OHO

HH3C

CH3

CH3

CH3

CH3

CH3

72

Figure 12





OHO OH

OH

HOO

CHO

O

O

OH2C

H2C

CH3

H3C

( )n = 8

( )n = 8

8

Figure 13

HO

HH3C CH3

CH3

CH3

CH3 CH3

91

Figure 14

HO

HH3C

CH3

CH3

CH3

CH3

CH3

92

Figure 15





















4 Discussion


506250








5 Conclusion




Acknowledgments


References






















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















O

HO

HO

HO

OOO

O

HOOH

OH

OH O

HO OH

H3C

32

Figure 7

OHO

O

O

OH

OH

OH O

HOOH

HO

OH

4

Figure 8



3-Rha)

097 (d J = 63Hz 6101584010158401015840-CH3-Rha)

13C-NMR (DMSO-d6 75MHz) 120575 17700 (C-4) 16500 (C-


3OD 300MHz) 120575 772 (d J =


3OD

OHO

O

O

OH

OH

OH O

HOOH

HO

5

Figure 9

O

OH

HO

OH

OH

O

6

Figure 10


119877448 1H-



6 75MHz) 17713 (C-4) 16510 (C-7) 16109 (C-5)


3OD 300MHz) 120575 807


16056 (C-41015840) 15824 (C-9) 14810 (C-2) 13716 (C-3) 13068(C-21015840 C-61015840) 12376 (C-11015840) 11631 (C-31015840 C-51015840) 10456 (C-10)9929 (C-6) 94486 (C-8)



OHO

HO

OH

OH

O

HH3C

CH3

CH3

CH3

CH3

CH3

71

Figure 11

OHO

HO

OH

OHO

HH3C

CH3

CH3

CH3

CH3

CH3

72

Figure 12





OHO OH

OH

HOO

CHO

O

O

OH2C

H2C

CH3

H3C

( )n = 8

( )n = 8

8

Figure 13

HO

HH3C CH3

CH3

CH3

CH3 CH3

91

Figure 14

HO

HH3C

CH3

CH3

CH3

CH3

CH3

92

Figure 15





















4 Discussion


506250








5 Conclusion




Acknowledgments


References






















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















OHO

HO

OH

OH

O

HH3C

CH3

CH3

CH3

CH3

CH3

71

Figure 11

OHO

HO

OH

OHO

HH3C

CH3

CH3

CH3

CH3

CH3

72

Figure 12





OHO OH

OH

HOO

CHO

O

O

OH2C

H2C

CH3

H3C

( )n = 8

( )n = 8

8

Figure 13

HO

HH3C CH3

CH3

CH3

CH3 CH3

91

Figure 14

HO

HH3C

CH3

CH3

CH3

CH3

CH3

92

Figure 15





















4 Discussion


506250








5 Conclusion




Acknowledgments


References






















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


































4 Discussion


506250








5 Conclusion




Acknowledgments


References






















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















5 Conclusion




Acknowledgments


References






















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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