POTENTIAL THEREOF

CROSS REFERENCE TO RELATED APFLI CATiO S

[PARA 0011 The present invention is a PCT application of US appSieaipn 147051 filed on May 8., 2015 which is a coniiPoaifOn-io-paft pafefS application from USi3&446¾ fsi©d Juy 1:7, #Ι3 ife irt turn i a n n-provisionaf ling of provisional patent application ¾1672849 filed Jui.y 18, 2812 ail of which ate iPcPfpiSfafPS Mi i belo :ΐ0Ρίρί ρηρρ ip its. ©ninet .

BACKCSROU O OF THE JNVE TI0M

{PAR .002] Field of invention

PARA 0Q3J The inv ntio in general pertains to compositions for adipofenesis inriibitioa More speeffieajiy;. the present invention discloses a composition comprising sdrpusin A and soirpusin Sand anti-ae'pogenesi¾ahf S^ofeesity potential tfiereof. PARA 004] Description of; prior .¾rf

eop Yetal (260?)); FARA 006] Seirpusin 8 is a: weli established vasq-feiaxiog dimer of pteeatannoi and Has bsen obtained in iarg¾ amounts from passion fpit (Sano S e r& "ld¾ntffcaspp of ne strong yaso- relaxing substance seirpusin B, a dfmer of pieealarmoi. from passion fruit (Psss or edit ^' l ) seeds, J Agrie Food OftefO, 2Q†1 Jun §; 59{i1i:e20$--13:,SeSrpusip 8 is also noted or its mild £3SH activity {f¾aruki~ Uchitia H et ai (2013)} and abti-Hiy properties {Yang S st ai ;2005) fPARA 0Q7j i Has been eviously '^ wi iiaiihexane extact of &^®rm.fitiim$ btebis sxtr cts exbibit antf-obesit properties. (Administration of ^' ty/pe s mtur/Hus. ffsfeomes xtract prevents ¹ Weight Gain in Obese tucker rats, lemawre et ai. ¾¾f?. _: . pytether i¾es, 21;: .¾¾ ·^30 fbe h x^ has been characterized to contain a- Gypemdn , Roiundene, β-se!inene, Gala enene. Cyperene, d- egdinene.: Cypefotundotte,. Cadaiene, PatehouienoHe, tyootkatenie, Sogeonoij, §~ cai oiWie, Kofeusone, (Jyperoi, isofcobusene arid: Epi-a-seiinene (V ^' adav et a!, international- .Journal of Phar aceutica! and Cifnieai Researc 2010; 2(1 j; 20*22}.. Bui irie preserrt !nvenSors disc!oses anti* obesi y activity in ethyl acetate fraction, of Cypems roi dus. This ^Ϊ m t& i c n MQB& not contain any of the man constituents of the exane; fraction. The- p resent efcnyl aeeiafg -ffaeiiorv contains sti&eno derived compounds, : a class ef .compounds not reported to be occurring ^' €%βΜΜ$- mttmd by any inyestigatet thus: far. He ce it is the un¾up eombination of fhs uhexpeete olscover of the occurrence of stiibfe oid g ^' effyed co n uhdS: and fyrther, Iheir apti-o esity action. f is also a surprisirsg finding that following the bioactiyfty guided fractionatio of ttie rhizomes from Cyper imdus, a sub fraction of et yl acetate laye was characterize by the concentrated presence of two

[PARA Oui 0| it is yet aw^r-oi^ec8ve- .the ^; presen -Wvertlfcvf: to- -disclose- « method of managi g;: obesity in mammals using a compos on comprising scirpusin and seirpusift &

[PARA D011J it; is a feffhar Objective of the present invention to disclose method of obtaining:-, compositions cam prising A, scirpusin A and scirpusin , an d B. Piceatannoi and its dimers scirpusin A and scirpusin 8 ^' through bioactiyky guided fractionation of the rhizomes of Gypems miitMu ,

[PARA ¾i¾: f « present invention fulfils .the aforesaid objectives and resides further related: advantages. SUMMARY OF TH iHVEMT!ON

features and _: advantages of the presen invention will; become apparent . ^' from: the following, more detai!ed description, taken in con|MPCfjon th© -aece psayf rtg>- ftttsges, whic Illustrate _s b ay of example, the principle of the invention,

DETAILED DESCR TiGN OF THE DRAWI GS

{PAR&.0014] FOJ shows a flowchart outiininQ: the steps of extracting acive pfineipies from the rh!m es of Cypertjs mtutxius.

[PARA 0Q1S] FiG,f ₅ 2:8, 2b and Ft©J, 3a _? Zb and 3c show the 1G- S analysis of suia factions ill and IV respec-fivety of the ^tfeyt afieate a taaly apndhe : with ipeafaniioj dirnefs ¾i:r u$in A and SCirppSin E.

jPA A. 0018$; FK3J s ows the pftdtc¾raphs of waist circumference reduction in o ese: human (males); adfnteistered th§ ethyl acetate fraction of the: extract: of Cyper rahm&m rhizomes standardised to S% of Mai stilbenes, fpA A 0019} F!QS, Ssj Sta and Sc shows the photographs of waist efrcumferenc© reduction :in obese umans (females) administered t e eihyTa^t te fractio of th ^^a^M€ype & iU s rhizomes . standardised *o 5% of iota; stilbenes. RA 00¾0J FiQ§, §a, Sbt θο, M m$ 9» show fhe chsogSS in :s^teroid: lipids in o ese humms admin lersd the ethyl acetate: fractiori: of the extract of C pero ra^

δ¾< f total stilbertss,

£>ESCSI!»T!QM OF THE MOST PREFERRED. ESSBODt!^E T

PARA 0022) In another most preferred embodiment, the preserit iftvehtion relates, to a: method of inhibiting adipogenesis ^" m mammalian eeifs, said method comprising step ©f bringing to contac adtpogenic mammalia cells with a composition comprising scirpusin A and sci rpusih i ^: represShte3 ^: by ST 1 and St R#& festively,

PARA 0023] in another most preferred erfibo imeht, the presen t invention relates ¾ the _: melhod of ^: thera sqticaiiy if^lhiii <j sbessty c ysed by adipofjenssis in amm ls, said methad coi iprisi g: step of dietary supplementation of a c*#«pos¾&ft *ίοίη{¾½¾¾¾ -sd pu^ft- - A aad -scifjiusifi ^' 8 represented: by S f¾#1 ¾Rd 8TR#2 respectrv¾i to a maromal in eed of said therapeutic; inhibition,

{PARA 0024J in another most preferred embodiment, me present invention: ralates to fhs use of a: composition esmprising seirpisio A and scirpusin B represented by $TR#1 and SI¾#2 for inhibitin adipogenes!s in amr fen cells.

[PARA (Kl2$ In an alternate embsdfrnehi, the present invention also reiafeS, t© a process lor She bioactivity guide fractionation of the rhizomes of Cyper mtimti to obtain anti- adipcgenlc anti"- obesity eamposifcns comprising: A. sdrpysin an scirpusin δ represenferJ by STf¾#i. and STR«2 arid B. piceatanno! and. its dsmer seirpysln A and scirpwsin B represented fey ST8#1 and STRSS fespeetiveiy.,/$8ld process com rising the ste s of:

1) .¾^f¾ ^' »ie-^i2^^ f-il¾pew* rotundua and poivsrisog tbs same to form a esarse powder; 2} Extracting tine powder of ste TwHtl 3 volumes of hexane foitewed fey Seati g, reflux for

3) 3 flours and: filtering to obtain the hexafte soluble fraction and spent material;

[PARA: ivtefhpdotogy :

[PARA: DC30I Dried rhizonies Of C ems iundus were: pulverized to forrrt a coarse powcleF. The pulverized powder was them extracted with 3 \?oiumes of hexane followed fey heating, reflux for 3 hours and filtering to obtain the hexane soluble fraction and spent materia!. The spent materia! is further ext acted with 3 volumes of methanol followed by; heating, reflux for 3 hours and filtering: to obtai the methanol soluble active fraetien and spent material, the etbaRoj soluble fractio is soiuo!iised In aqueous methanol and successivel partitioned with chteroform (OHC!s):, Erhyi acetate fEtGAc) and methanol to obtain the _: chior form layer, ethyl acetate ta er and the aqueous met a ol^ layer respectively; The o oro drdi ayer,, ethyl acetat layer and »e aqu ous methanol layer are subjected: to further rjfoaetivity guided fraet natten, wherein the hldaetivity parameter was the afoiiity of the chloroform layer, etfty! acetate layer and the aqueous methahoi :iayer to inhibit adipoienesis: in 3T3-L1 mouse adipocytes ImammaMan ipoc^es}, The steps of the Gil Red Q staining technique as adapted from Salazat Olivo et al 1995}, Ws £ et a! (1998}, f M et a! {2005} to study extant of adipogenesis inhibition is explaine in EXAMPLE 1A terein below. The results are mentioned in

fPARA 8031:] EXAMPLE 1A: Terminal differentiation of adipocytes is abcompaoied by the accomuiafiOii of great amounts pi lipids in large cytoplasmic vesieies. A common ass y tp measure adipocyte differen i tion m oef culture is "with he: dye Off Red^ S { OR0), ¾RO- Is a !ipid-SQiu i bright χ&ά dye which is a reliabie indicator of adipocyte differentiation.. p ¾ 0032} PHrtCipte Oil ed: 0 Solvent R«3 27, Sudan Re SB, l 26 25. and G26H2 M 0) is a | ¾oehrome fta!-so!ubie dye} dia¾s dye used for staining of neutral triglycerides and !ipids sn frassn sections and some lipoproteins oh paraffin sections. It has the appearance of a fed powder wit maximum absorption at 319 359 nm, OS Red £3 ¾ one of the yes used for Sudan steins _* Similar dyes Include Sudan til, Sudan IV, and Sudan Black B. The staining as ttt be perfom>erj on fresh samples. a$ a!doho! fJ¾af Son removes the lipids. Oil Re O largely replaced Sudan ill and Sudan iV, a it provides much deeper ted colou and the stains: are therefore muc easier to see., ©si red Q is ah oil soluble dye. Oil soiuWe dyss exhibit greater solubility of the dye in !tooid: substances in me tissues/cells, than in the usual hydro alcoholic dye solvents. Hence, it will dee l : stain e eel!?. jPARA; 003¾ 3T34J cells 8ppr«imateiy 6δ X ί≠ ceils are ^^·¾··4$-¾· fag. 4o confluence.: After 48 hrs 200 μ of MM (Adipogenesis induction medium) eshl prepared is added. 72: hps later 200 μί of .A H (Adi ogeoesiS: progression medium) with the test ompounds in different concentrations is added to the wells. The ceils are incubated for 48 hrs in a humidified: atmosphere (37" 0} of $% GD¾ and 95% sir. The supernatant is collected and stored lo the estimation- of feptin _<: adiponesiirt, IL-S and TW-aipha. Celis are fixed by adding 106 pi of 18% formaiih and ©R0 staining. Is ds ©., 013 is read at 492 wn in rnieropiat reader,. PARA 0034] The results are expressed as !¾ values using: Qraph pad prism software:. The percentage of: Inhibition of sdipogenesls: Isoaleulated: as:f6l|&w&

bero C-absorisance of Oil red 0 in diffe entiate T-afesorbance of Gil Red Q in sampl treated ^: diffe¾niiatiFt^Und^efenS^©d-^$ . ABLE A

{PARA CS03S] The ethyl acetate layer exemplified th best bioacfivit in terms of adipogenesis infjitjitloa with an IG½ {pg/fii!} v iu© of 8.39> This fraction *^

identify the Pioacliviiy (adipogenesis inhibition): ote arker. Column fractionation involved the step of eiufing sa¾ frseiions of the ethyl acetate layer with increasing polarity of met!iaoft -c ot pier m mixture. The. sub fi¾e¾ns sf ethyi aeetate: layer ar¾TaS>eied as i, ii,. ii! and iV are subjected ts sisaeSyit {ami- adipogenssis) evafoaiion. The essential steps of af¾t-adrpsge¾iG acti ity evaluation inraive* the rocedure outlined herein atjov EXAM?¾£ 1¾. Tile results are summarized f^^^

Table 8

Sampie T !¾ ¾mli 1 j

Sub fraction 1 (Nori-po!af eo iiipertiS: : :

SuS fraction Si (Naturally endcfte in pieeatanhoi 1

8ion§ Srt imers selrpy sin A and sci usi« 8 : 1 f

Su& fraction id (Naferalfy ers iehedln ieea a fiGil 1 13.31 j

■dimfers seirpusih A ^~ 4 sdirpusin B) Sub fraction iV (Saturafly erirfchsd in piseatannef 18.75

aimers scirpusin A and sarpusiri; B}

JFARA 0036] Sub tractions Si! and V were then subjected to LC-MS wife botn fractions being enriche in ptc^ataftRdS dimsrs sdrpusi and scSqsusip { Fies _: 2 and 3), ie LC-MS S analysis was peiferrrsed on Thermo eieetroriiss . tNoftigan tCQ Advantage A spectrometer using an RP Qi coiumn (2S0 X 4,6 mm,. S μ padide -sfeej. h system consisted of a heWn^ finnigan surveyor PDA detector, aft: LC pump and n autosamp , he Mio¾e Phase : ipojucSed a ©radieft ¾n for SS rnisutes with Soimni (A) 0. 1% Acetic add in water and So!vertt i8 j Acetonitri!e,. Solvent S e ncentration increased from 5% during ©♦. ^· §. minifies, 5 - 88 % during 5÷2Q mif ^" ! tes,60 - 00 % during 20 25. minutes _f 1Q0 - S% during SS-27 minutes and remained constant at S% during 27-3S minutes. The Stationary phase teiudad Thermo: BPS hypers!, Ci& Column (Dimension- 250mm ,x 4.8niirr} Flow rate: 1 m!/rrcin; iQeteetsoR Range: 260 n ,

[PARA 003/') ionization parameters: APC positive mode,. Source voltage - 4.50 ¾ _t Capiilary tem erature- 22S degrees, Capiiiary ydita e- 43,00 V.

[PARA 0039J The first tevei of confirmation of th presence of: Rimers of Piceatannoi in the Cyperus extract v»'as based on this pre!smtnary information on mass, Scirpusin A was directly eonfir ei- by direct comparison with art authentic sampie of Scirpusin A.

{PARA G040j Su& fractions were then suh sGted through the preparative HPLG to obtain purified dimer seirpusjo 8 which was then studied usjng ths anatytfeai toois High Resolution Mass Spectroscopy {MR S}. ii uid chfomatogtlphy-mass spectrometry (LG-MS/iv S} and uclegr Magnetic Resonance: Spectroscopy Mfv5R¾ ts be confifmed as scirpusin 8, Data top the H RMS in ica ed: M*H3~4S7,138 which matched very well with the structure of the dimer and reported < a {Sane et ai., 20.111 on the same {FiQs,4i and S) add the stroeiure df scirjusih B was also confirmed using:

Scirpysih & in 0 ipems fQiun^m in the most eonvincing way.

IPA A 00411 EXAMPLE 2

[PARA 00421 EFFICACY ^" EVALUATION FOR ANTS-OBESITY EFFECT OF A:0¥PRO-AF {aeive athyl acetat* fraction) AND CVPRD-DI (ethyl acetate sub fraction naty as y enriched pieeatanno! dimefs scirp siri A and seirpusln B) EXTRACTS IN MCE.

PARA 00431 ObjeetivB of ihelest: The objective of the study ¾v¾s to: evaluate the efficacy of Cypro-AF a«d Gypro-D extracts for anti-ofoesity effect in C57 mica. PARA 004 1 test System feta is:;

{PARA 0045} Test Performance petals

481 HUSBANDRY

w Male Female

Total rsum&ef of animals : 1 60

{PARA 0048 ^" f Fom1d:lat!(Sft safa:iis:.:&¾d: dosage:

the study peried. The foltowing taiaie provided details of the test formulation.

[PARA 00511 Obesity induction: The 6 Controi group animals were fed wit normai control diet feed D124S0B 0£5:ntain:iftg 10 fceal % fat nd ths 6f ta £36 grOB animate were fed wth high fat. diet feed: D 12492 containing 80 kcai% during the utcJ oilan of obe si ty and during mai §tudy.

PARA 0052] Main Study:

PA A 0053] Main stud was stafted after t e induction of obesity. he: 3 iktSes of O prb-AF and ί dose of Cypro-D were administered to animals from Day 28 daily eonsacutive!y for a period of 27 days. The feeding of the diets continue ^' the main study was done in ( aaciton of obesity, The QT Controi and G2 High fat diet control group so ^' tmals- adminfe$^tf¾*l8*- cltsi8ted ^' - &lM while other groups animals received lest Sterns m Day 23 to Day 54 .o the study period:. The dose volume oi administration was maintaine according to the: eekly body weight of iniSividuat snimais, The total duration of the study as 61 da s t? day! ¾ inddelis of abesity* 2? days Mam study :,

PARA 0054] GSSE VAT!G S:

[PARA OQSS] Ths fpiiowirg dSssryaSo wsrs nigds for during the study erio . PA A 0056] Peed ieppsuirjipi Pd:

PARA 0057] ndividual animal feed consantptioh was: ecoi^ed^ eeidy av rage feed con um tion: was eaicuiat ed and . recorded.

pAR 00581 Body Weight

pARA 0059} tneiiwduat animal body weights were recorded on the day of receipt on Day 1 d:

weekly {:±] day) thereafter during the study period,

PARA 00603 Clinical oisseryations

pARA 0061 } All the ahiroeis. were ciinicaliy obse:n/id twice daily during the study period.

PARA 0062] OiioiGat Pafhote y

pARA 0063] At the completio of the stddy period;, blood samples were collected from the animals ih tube containing potassium ethylene di-amide tetra acetic add :( 2-Ε0ΤΑ) apieoaguiani for hematology and without anticoagulant: for clinica] shemistfy. Th blood samples collected ih tubes without anticoagulant warn eentrtfugSci at 300Q f m for 10 ^' minutes to obtain serum. Blood samples were collected fturaane^ -fom ·*9*κ «τ&»¾ί _: pfexu&-:put iur$ mehod under m9<J after aoae&tfesia w¾n. t e help of a fine capiary tube The fallowing hematology and cliptcal chemistry parameters were analyzed.

{PARA O06 | Hematology

[PARA 0065] The foiio itsg hematology parameters were estimated: eShg Sysmex, f<K-2l {Taosasa Biom dicals 1%, India):

[PARA; oG87| The following clinical chemistry jsarametes were haiyzed using the ''Efh anph ifri Cherf! Toueh analyzer ¹ ' {Transasia Sia .4edieais Ltd,, India) ttom-s im- sample

Low density lipid {LBL\ HKSidL

IP ARA G0S8j Pathology jPARA O069J After ¾e completion of humanely sacrificed by e^osih§ tfnem to g¾cs$§:ea¾ to following ext rn l and Internal jfoss peoropsy, {PARA 00701 ^Oss Necropsy

[PARA 00741: Fai p<3slte W iglts

[PARA 0075] The foJibwif¾ fat deppsits|ΤΟ _; Ε« : ail the an ais was coil-Scted ¾n : weighed,

1, Epididymai Fat

2, Brown Fat

3, Ovarian Fat

[PARA: QQ7 ] STATiSTiGAt A!¾ALySiS: A D ¾E PORT PREPERAT1Gr¾:

[PARA 0O77J The raw data obtained from the present study were safejiedstf CMiptiW-- %^iki i- processing. The computer printout of the data (in {Re form of appendix) was verified «i the originat raw data. After v tsJfcatioi¾ the data Was: s Djeetai! to One -way 8QW iAnaiysis of Variance) wit* riuaheft's post test for the data On body weig ts, hematology anet diai l chemistry parameters., orga weights using GraphPad Prism version :&0iyG¾ fi ¾

be evalu te a* the $ 5¾,ievei ef conidence (P<«Q,G¾ indicated^ the SLtpersoripts: of a where G1 is eomparsd to G3 G4. G5, and G6 and: :b where G2 is compared to S3, :G4, G5,, anct G¾ ihr tigboyi the report, as stated below: ^* : Stattstfcaly sigraf sc nt (P<6,05} wherever appSeabie..

{PARA 0078] T e data were subjected to one way - QV statisticar analyses fey comparing th following;

[FAR 0079] G1 group {Control grou {With IS kcai% f½t¾ to G3 group G¥PRG-AF SO m§fcg ^: *Hig fat diet M B0 kcsi% Pat!, G4 group: {0YPRi3--A -100 m§i¾g _: *H¾h fat diet (with 8(3 :kcai¾» Fat)}, 05 group {GYPRO- F 200 mg¾g -'High fat diet iwith ©fJ:kea!% Fat) r>d:Ge §foop {CYPRO-0 10 fli ¾a ÷Bigh fiat diel^ CYPSO--P110; mg!kg + Hig fat di t

(wits 60 kcai% Fai| FARA Qoai fiEs LTS

| A A 9083] The samoiaf of we&idy average feed feofisoro t!&n of mali and fernale animals is presentees s T BLE 1 ant! TABLE I respectively, Trse:re were no statistical sigofeafli ^fieene s ifi tie feed sonsumptfon of am ais during the study pe d.

[PAR D085] T¾&ie-2 : Bu m§ i weekly average feed eonswmptfen (<S) ! f emale animals

fi-δί Va!ues affc eart± Si«tisr evia o; P>S _: Q§ jPARA, om§] Body weight

[PARA: 0087] The summary ¾f weekly otS weight of mate afid female aftima!s is presented in Tabie 3 and Table 4 t¾sg>ecfiveiy.

[PAfM _: QQ88] ^: ¾

rpS: Values ate Pe¾ ±: §Ϊ3Π{¾'ίί t¾ev!3lf0H; F¾0, _: 0§:

(with 60 kca!% Fai)

(wi h 80 kcai% Fat) |

Va!ues are Mean ± S a dard peyiation;*-- SigniffcantdiffsreRee,: P 0.05

[PARA 008.03; in male animals, there was staiisttcai significant ne«ease in: mean ^■ weekly body weight vaiues of on Day 21 In. (33 grou fCYPRQ-AF- 50 mg% +High fat diet (with 60 kc % Fat|} _; G4 group {CYPRO-AF - 100 mg/k + High fat diet {with 60 kca!% fat}}, G5 group ,{&Y- PRG-A -200 mg&g: *Hlgh fa diet (with 80 keai Pat}) and G8 group {CYPRO-OI - 1 rog kg * High fat diet (with 80 kca!% Pat)} compare to _. £31: . groop {Se rei greiiR (wit 0 kcAi :Fat) these changes Were considered: to &e due to diiferanee in fat content of the feed .

{PARA 0091] in male ani als, t er was statistfeai signlficarii irter¾as¾ in: mean Week !y &©d eigt vaiues sf on Day 28 in m group {C¥PR0«AF <· 10 mg¾g gh fat diet (with 60 fecai% Fatj.,: G6 group fCYPRG-AF -200 ffip kg * \gb fat diet (with 80 Real % Fat}} and: 80 group {CYPRO-D1 - 0 mg/kg + High fat diet (wiiii 80 keai % Fat)} -compared to G group {Conffoi group (with 10 kea! . Fat)}. These changes were considered to he due to difference in fat content of the feed,

[PARA 0092] in male : a imals, tem was: decrease in o ean weekly body weigf¾ values of on Day 35, _: 42, 49 and 55 in QZ g owp iCVPRO-AF- 50 ng/kg -"High fat diet (Witts 80 kcai% Faf¾, , £34 οψ fCVPRG-AP" 100 rng kg ÷Fiigh: fat-diet (with 80 kcai% Fat¾ C35 group {£YPRe - Q9 n¾(kg *Nlgb fat diet: (With 60 kca!% Fat} d £56 grou {CYPRG-01 - 10 mg/kg fat diet (wit 60. kcai¾ Fat}} com ared to G1 group {Control group (with 10 keai% Fat}}, These -diaiogSS-wSre .cOn$i«iei$iH0 be due to adTOift raiion of test items,

[PARA 00MJ in femate animals, there was/Statisticai signSfieanf increase irj: rosan wesk!y po weight values of on Da 28 in G3 group {GYP R0-AF - 50 rrtg,¾g: *Btgh fat diet (with SO f ca! Pat& G4 group {GYPRG-AF - 100 mg g: * Hig fat d et (wit 60 koai% Fat}}, GS : groyp fCYPRO-AF -200: mg/¾g +H¾h fat diet (with 80 keai % Fat)} and GS group {CYPRQ-D1 - W rog kg + ¾h fat diet (with 80 kcai % Fa;}} compared to Gl group {Control group iwith 10 ^; ¾oai % Fat}) _; > These changes were eohsMered to fee due to differenoe in fat content of the feed.

PARA 0095] in female animals, there was decrease in mean weekly body weight values si on Day 35. 42:, 49 and 5§ in S3 grou {CYPRO-Af - 50 rfig kg *High fat diet (with 60 Kcai : Fat}}, B g oup {GYPF t-AF - 100 mg/kg +f¾h fat diet fwit BO; kea!% Fat}}, G5 group fCYP O^ AP -200 m&kg *High fat diet (with 60 keai% Fat}} and G8 group {GYPM-Di - 10 mg/kg: t ig fat diet wiiii 80 :feea! Fat}} compared to Gi group {Cdntroi group (with 10 feca! . Fat}}, These c ange were considered to be due to admihistiation. of test: items:.

[PARA Q0§6]: Giinicai observations

[PARA 0(597} The summary of otinseal signs of mate an fterwale- anSmaSs is presented in ahie-S a d Ta&fe-B respectively. The anirnais were found to healthy and nsrmai in rseait status during the stiniea! observations during the stady period.

{PARA 0098| Tab!e-5; Summary of Giinicai Sighs Gbsewations in Ma!e Anirnais a lSC L SiG S; GBSERVATiG S

Grou Treatment Days

1 j 2-7 . 4 I 15-21 ] 22-28 :| 2¾¾ ^" 36-42 1 so-ss

22

kcai% Fat) rs~5; ^or ai

[PARA 01081 Hema!otogy PA;RA 0101] The summary of heroaioio icai paramete s estim ions of maie -and female animate ¾ presented in Tabte? and ^" a&ie-S raspee¾vely . PAR 0 02} TaDie -7: Symftiary of hematofegy of mate anirriais

i≠S' _< Values- Mean fSiandanS: Deviation; F<G,QS iPA A 0183] TABL E -7 (Gpniinaed): BU?¾rnary-.0f fcertiaiology -of animals

^■ mio - PARAMETERS

Treat ent dotting ilfsrenii, s Leucocyte Cours time I ¾feu¾¾¾S¾ife- T ^' Lymp iQ l&¾

i (sec) §{% . j {%.} ^" (%} j f% 1 (%}

m§: Values- Mean ..±. Standard ^' Oe*|ai6fj; ;Ρ>δ ^' ;05·

|PA,RA 0 '04] Tabi^S:. Stimmat # temateilpgy of female mrnsis

n-5i ViS!ues- Msan ± Sandard Deviation; P<0.05

PARA 0105] TABLE- 8 (Continued) Summary of hematology of femaie animate

n~5. Vates- tieafi* Standard Beyia¾OiO:; P:>G>05

[PARA 01:δί¾ Hema ogy parameters statistical analy is^ S1 o GS* 64, ¾§, and

G6

{PARA: Qid7 @8f! corpuscular hemoglofeirs eoncen atioo { CHC}

[PARA.0108] t mate ansrfia!s, there was statistical significant increase In mean MCHC value of G4 group {GYPRQ-AF -100 mg¾ *Hign fat: sM (with 0kcaί%: st} ■eαr ^feί ^" to ^■ ΐ3 ^■ group CGontroS group (with 10 kcal% Fat}}, These cnanges can be considered as incidental as there %¾s «ό dose de ¾ deH.t res oiwe.

[PARA i: 09] Mean Cor usGulaf V¾i urns and Mean GorpusGuiar Hem&g!obin PARA 0 10] in. fema!e at¾ima!s. there was stafisscal significant increase if? , mean \QV and MCH vaSifes of S3 ψϋχφ {€ PPO-AF -50 Mgkg + High fat d½ gfouiS {Contrai group {with 10 kcsS .Fat)}, T ese changes can os considered as incidental as there was dose dependent fes o se.

as there was no. dose ependept response.

[PAR 0113j Clinical chemistry

[PARA 0114] The summary of clinical ¹ chemistry parameters estimations of male and femate anima!s is pesented in Tsfe!e-S and Ta ie^tO m$ *cti¾!y,

{PARA 0115] Table- 8

[PARA 01: tSITabieS ebrstiOa«d;

ri=¾ Values- Mean* Slaridaret Deviation; P 0.05

[PARAQ1;l71Tab!e-10

f»~Si Values- Ateafi* SianGartl bevistiorfi Ρ^Ο,Οδ h~¾ VaiMes* Mean± StaociaK Bviation; P<0.05

Ι ΑΗ 119] -Sl¾¾^l^iiiem^y:-is f¾rt)!ft rs- statistic^ ¾na!^ te ¾3.. G4. G5. and GS

PARA 2Q Total proteins

PARA 123] in femaSe ¾r>ir«ate., '<#ιβ& -was -statical significant decrease In ^■■■ mean Togiyeend va!ues G3 group {C¥P Q-AF -50 m§.¾g 4 High fat diet {with 60 kca1¾> Fats}, S grou {CYPRO-AF -108 mg&S +-High- m 4K* ( siith SO kc¾!% Fat)}, G5 group {C¥PRO-AF -208 mg& ♦Hig fa diet (m 60 kcaS% F-ai¾ and 66 gro p {CYPRO-D : - 16 mg¾§ f¾ fat diet {with SO keal% Fat }c0mpared to Θ1 §r©op {Control group {wiff¾ 10 kcaf¾ Fat}}, These change were :f¾o¾idafesi to pe doe to diiferer!oe i fat content of fee sed ψί Α: 24 0tal Chotesteroii

{PARA 126] in female animais, there was statistical significant increase, in: mean: Total Ghoiesteroi alues of G5 group {GYPRO-AF -200 mg/kg. + Might fat diet (with 80 kcai% Fa ¾ compared to G1 group {Contrsi group {w¾H 0 kcai Pat}}, This change can be considered due to differashce in fat content of

{PARA 127] High density lipids pARA _: 1S8J In male arjirnais, ^': ¾fwe- »s§ ^st¾i 0i: - ^ftti!iC9 i--d!ee»» fie to mean High den$i¾ iioids vaiue of GS group eYPRO-AF - 200 mg/kg + High fat diet {with 68 kca! atg^ compared to 61 group fCootroi group (with 10 Nca1 Fat)}. This change can fee cdnsiderea due to difference in fa content of the feed,

{PARA 129] Low density lipids

considered to be due to difference in fat content of the feed,: pftRA: 26] Very tow density iipids PARA 127} in female animals, there was statistical significant increase if? mean Very low density lipids value of G5 group !CYP OAF - 200 mg/kg + Nigh fat diet {with 60 kca ¾ Fat}} compared to G1 group {Control group (with 10 fceat¾> Fat}}. This change can be consletered due to tf!iier&dce m fat content of t e fee ; RARA 128] Cfinica! chemisiry parameters ststistSda! analysis: comparison befeSrt C¾ G5. rid Go jPARA 129] r^iyeer de*.

fee due the effect of the test: items, jpARA. 133] Total Cholesterol

FPAR 134 In male animals, there was decreas in mean Total Cholesteroi values of 63 group {CYPRQ-AF SO mg/kg h fat diet {with SO keaf% Fat)}, (3 group IC PRO-S - 00 mg/kg *N¾rs fat diet (with 60 kcai% Fail} a d: SB group {CYP O-AF 200 mg/kg +H¾ diet (With 60 kcal% Fat)} compared to G2 group High fat diet Gorifrol witfi 60 feeai Fat J, This decrease in tmean Toiai Cooieste oi values changes couiti fee. due the effect of the test items,

[PARS 135] in female animals, ^: ft$ers- -wa§ -.il^a^a®?. 1ft. -.mean Total C otesier i values of G3 grou¾C¥PRG-Af SO mg&g +Htgh: fat diet (with 68 kcai% Fat;}. G4 group fGYPRQ-AF 1QQ,mg/!¾g +High fat diet {with SO. te¾i% Fai¾ and G6 group (GYPRQ-DT 10 mg/kg _: +Hig fat diet (wit 60 kea!% Fat}} compared So G2 ou High fat diet oRtro (with 60 kca$¾¼ Fat); This decrease in mean Total Cnetesierol values errahges .¾u¾b# ύιϊβ the -effect -of the- test items.

{PARA. 138} High density lipid* ^'

G¾ group High fat diet Gonirol (with SO kearis Fat),

[PARA 138} The statistical significan decrease i mean High density lipid values changes _: could be due itte effect sf the test items,

TPARA. 331 in female: animals, there wa statistical significant decrease in mean Higfi density lipids values of©! group {G¥PR0*AF 50 rn,g kg _: *H¾R ^■ ■ ^■ i f6 {wth: ^{■ '} li ^■ cal%■ ^■ ¾}Ji,. ^' G group (CYPRO AF 100 mg/kg +High fat diet (With :6Q keai¾¾ Fat}}. S5 grqa {C PRG-AF 200 mg/kg *High fat die (With 60 koal Fat)} and G8 greyp C;YPRG-131 1 3 mg kg *H¾h tat list ith 60 kcai % Fat}] compares' to ■62 group High fat diet Control (with 60 keal %: Wat},. These decreases in mean High density lipid values changes could he Cue the effect of the test Items,

[PARA 140] tow density lipids jpARA: 141] in male animals there was decrease in mean Low density lipids values of £35 grou (OYPRG-AF 200 mg/kg +Htgh fat diet (with 60 kdal% Fat}} compared: to G2 grou High fat diet Control (With 6S k¾ai% Fat}. ^" This ^^a^Jft fftf&ft ^,<36n§fty-$$a values efia ges :c¾uid be due the effect of the test items.

[PARA 14.2] Very low density lipids values A A 143} in male animals, there was decrease in mean Very iow density lipids values . of GS group {CYFRO-AF 50 g!kg +High; f¾ diet (with 60 kmi% Fat)}, «34 group {CYpRO-AF -100 mg% *riglr fat diet (with ca Fat)}, Q5 group {CYRRG-AF 200. ^: m^§-*H¾b :<2iet-{^. ^' 60--€al% Fat}}- ami CSS group {CYPRO-PI 10 g/ Q +S-figh fat diet iw fh 60 kea!% Faff compared to 02 group High fat diet Control (with 6Q keai Fat), This decrease in mean ¾ery iow density, lipid -vetoes changes eouid be due the effect of the test itehYs,

{PARA: 144} in femaie anima!s, tnera _: was triarginal e^ low density SpsdS values of

£33 group {CYP O' F SO r¾¾§ + ligh: fat diet (with g0 fcca!% Fat}}, © group {OYRSQ-AP lOO rng% *Hi§h fat diet (with »0 kcai¾ Fatjj, and GB grou {GYPB -DI 10 mg/kg: +Htgn fat diet (with 80 kc&m Fat}} compared is 62 group High diet Control ( it SO keal F¾ Theses deceases in mean Very km density lipid values changes could be due the .■ effect of the test items,

(PARA 145J Conclusion; From the present study, it can tie concluded tha the test items Cyp o- Af and eypfo-DI had a affect: oh decreasing parameters: such as HDL:. Triglycerides, _. Cheiestefoi, LPL and VLOi conce trat o s in high fat diet induced: obese male and female c§7 animals at SO, 100 and 200 mg/kg body weight of Gypro-AF and 10 m:g feg body weight.ø■ Gyprd-iSi., No significa t sfafjstieai changes were observed in the organ weights and fat deposits upon necropsy dianimais _. ,

[PARA 146† EXAMPLE 3·· Toe effect of p v$ ttm us, ethyl acetate fraction comprising piceatannol and its dsmefs seirpu¾in A and seir usi 8 for weight management in fto an& ffisae , safely and ioleraialit studies; fPARA 147} STUD Y DETAILS pARA 148} Primary objectives included fi) To stud the efficacy of Cyperm mtuneim extract for weigh ma ageinent In obes patients; and ) t& study the Safety and totefebi!ity of Cyprus imtim extract for weight management in obese patients.

{PARA 149] Secondary: objective included th study of the onset of activity of Gyp ts mti d extract in the weight management of Obese patients; A A 1S0J Primary outcome measures included, ) decrease in body weight and body: mass index; (ii J decreas In waist eife wtefSftce arid waist: hip ratio {an hro ometric ^' pea®i ®i*8R¾J _> ^* : Off) decrease in CfiOfesteroi, Triglycerides, DL arid VLDL vaiues from baselina; fiv) increase id HDL values■ ^' from, jbaseiioe; and {y} photographie evideoee oiiiained of the subjects on isaselin© and on iast visit while concealing his/Mr Identity.

[PARA 1513 .Secondary oute©rae measures if¾toded assessing ..the tslerabiiii of the study materia! in terms of adverse events arid othe fjh teai signs or symptoms ¾v trie s.tudy _: su&Jecls.,

SLLUSTRATiVE FOSMULATiGSS; USED I THE: HUMA STMO^ (Tables 12 and 13}

Table 12

Table ^' 1-3

[PARA 158] The active study rftasnai and piaee¾p descri!seii; herein above were ipimufatef: as Size "00" brown/brown bard geiatiti papsuies with & fiit ve¾lil Of 560 mg Being a double Minded sty- y, boSi: ctive study brateriai ^ s ie abd !ae^ wbre ^: identical in aii :p y¾iea! ^; Sspects life size, sh pe stfid weight,. A mbdam!y selected batc of aotivs study materia! whe : a iyzed showed: 3§fS rng of Pfceaannoi and Sclrpusio 8 on an average per capsule. In pipre iiipstfative em odiment, tbe illustrative fomruistions that: coatprtse- yp&m$ mtwdtis ir&ci standardised to contain greater t an 3% of total st8ben«^- inefcdiBg .^o^tanno seirpMsin A and ctrptisln B. fa ..|f^: ^; «¾sfi¾ti*e ^' . example ertwnc ed in: Table 2, Cy ms tumius extract standardised to contain 5% of ai stiibenes ineioding pieeaiannoi, scirpusiR A. and seirpusir* B has been presented.

JPARA.157J The trial was Cfandueted in Gove rnerst A urv da Medical Goiiege,: Mysore,, india The study was initiated only after obtaining a ritten favorable opini imm tbe institutionai ethical eommittee. Mo furthe changes or amendments were made to the approved/protocol during _, the coarse of tbe study The tnas was conducted ii :apcprdane with the princlpfes enunciated in the Declaratio of Helsinki {Edinburgh; and the IJH: ^' bamtorazed tri rtite giiidsiirte bb Good llnicai Practice ( CR, VVriifen and oral information about the sisidy in a l nguage up^

tbs sbt¾8¾ was: rovide to sSI SdbjeciSv pARA 'iS8f This randomized, double blind, parallel group, placebo: controlled, study had total 5 vsits to the clinical site by the study subjects, besides screening visit, Tbe schedule of assessments is represented in Table 14,

Table 1

j Goncomitant: {dedicatio \ X I X j % X x I. x

Savers Arrhythmia in the last 6 months, &} Symptematic patient yiit ciinfes! evWehee:of "He ii :l¾i!wfe, safety outcomes were measured fey: 1) Physical Examination and VitaSs, 2) Assessment o repqrted adverse events (AEs), arty, 2} Adverse evenly if any, The efficacy outcomes we is :measui¾d by 1 ) weight, BMi, waist circumference, hi circumference and waist hip ratio.

RESULTS

(Para 162 No e of the enrolled subjects at! aOncsrn¾ai:f dEeaf history or abnprmaj h sleai findings Observed on the screening visit Or during the study visits, o statisficaiiy significant changes to yitais observed between the treatment g oups on any of the study visits. Out of 36 randomised patients,: 26 eompieted the study. The ratio of male to feniaie subjects completed, aii stud visits is 19, 3 females dr ed out of study at various time points* wherein a : anaiysis at the end of the study reveaieti - that out of 4 dropped out subjects were receiving ptecebOs The percentage: Of treatment complianc for 2© patients #¾o eoffl ietes the study was gaod> The least was S7.2|¾S and niasiimum was 1©Q% treatment compliance b 1¾ study subjects, the trial was not prematurely ^ to was stopped only after reaching the target sarnpie size of 38. "iiia-Stati^te¾ty-si R8½ai¾-^i¾es" Ss-the vta!s (Table 15} anil rio ciinieaiiy signfficant abnormal Sab yaiue¾ (Tables 18 and 1.7;} were observed from the baseline to final visits and: between the treatment groups:, There was single AE reported during the entire study period, and: as per the investigator's opinion, the event wag 'unrelated ^' to the study product. There were no serious adverse events or significant adverse events noticed in this study. Efficacy analysis of these primary parameters f!Bv¾ais that weight, BMt ahd waist clreumferenee reached statistical significance betwee the two treatment _. groups by end: -of the study,, whiie the Othe two parameters flip circumference an walst bip ratio}: did not show any signif fcance betee the two treatmen groups, fe bidohemica! assessments Ta&!e 8? iike total ShoSesteroi, thgiycarides, lo density lipoproteins, high density lipoproteins and very low density lipoproteins were found to be statisticali significant (ρ<0 _÷ 01 when compared bef eep the two iraatrtwni . group patisniS {Table: IS). Photographs of subjects before and after the study duration cisarly indicate the efficaey of fe product

Taiaie 1§ ^:

Values expressed as mean ± S.E

*p- value is estimated from p¾i rad :&test

Tabie 6

Values expressed as meaa ± '

:Sffi : 0.0 ± 0.0c

Values expressed, as mean ¾ SB

Table 8

The subjects who ^" com pSeted the fins! sit were consider d for this af a!ysis ara 63] in another most : ^ a method of reducing phesity Id nur ans, said method comprising _^

a da¾. compositi n oomptisirtg m - ethyl acetate ftaoiioh of the e¾ci of &ypew&f&im u$ rhizomes siaftdard^zed to ePPtaid 5% of total sliibenes to achi ve the effects of reduction. So pody weight, bod mass index and waist circumference, in a more specific embodiment,, fee composition comprising fee ethyi acetate fraction of ins extract of Cyperm mimdm. rhizomes standardized to contain 5% of total sti!henes consists essentfe!iy of piceatanooi add dimers thereof, in stili more specific /embodiments th composition copiprising the ethyi acetate fraction of the extract, of Qfpems mimdus. M m standardized to contain £%■ of fotai stiSpenes eonsists/ essentialy of pieeatanpoi,. seirpusio A and sclrpusio 8.

(Para 164] in yet another -most pfet®tte4 efot>9$rø<s ^i ^: ti≠ .preset itwention ieiates to a method of treating hypefpho!esteroiemia: ip■ 'humans, said method comprising step of adrfiinistedng to said 'hurns s ©ra!y twice a day. eompositipo comprising the ethyi ^' acetate . ^' fraction oi the extrast ci yp as fptii«d«s rfnzomes standardized to contain 5¾& ef total stithenes to achieve the /effects of a reducfian ih tri systemic !eveis d total chdiestdroi, Low ehsity lipoproteins >LDL). y¾ry L w Sonsft Lipoproteins {VLQL} and serum tdgiyeertdes and (b) enhancement in the systemic, levels of High Density Lipoproteins (HOI), in a more specific embodiment, the composition comprising the ethyl acetate fraetion of the extract of OiiOfys mkmdus Mzo es standardized to co tain S%. of total stiibeoss consists essentiaiiy of piceatanndl and its diroefs. in stiit pwre specific emtedinients the composition comprisiog the ethyl acetate fraction of the extract of €P9r mtun^ Ms m s standardized to contain 5% of Mat stsSbenes consists essentialiy of pieeatannoi _^ scir usin A and .scirposin 8

para 166] As further additional if lustrattve e a ple^ the pres t indention also tes†© a ^: method of

piCeatannoi, seirpusin A and seirpy«inB:. para 1.8? White the invention has been described with respect to a preferred embodiment if is to be clearly understood by those skilled in the art that the invention is not JimiterJ thereto, Rafier, scope of the in vention is to be infe f«teiS only in eoojuoci¾rs with -the ppended claims.