Phytochemical Analysis, In-vitro Anti proliferative, Anti-oxidant Part 1

Thov hu rauoscar.xiao@wecistanche.comyog xav paub ntxiv

Keeb kwm:Rumex rothschildianus yog ib tus tswv cuab ntawm ib ntu tshwj xeeb ntawm genus Rumex, hauv tsev neeg Polygonaceae. Cov tsiaj no yog ib qho me me dioecious txhua xyoo, endemic rau Palestine uas yog ib txwm siv los ua zaub mov thiab kev kho mob ntawm ntau yam kab mob. Yog li ntawd, qhov kev tshawb nrhiav tam sim no tsom rau kev tshuaj xyuas cov khoom siv tshuaj, tshuaj tua kab mob antioxidant, anti-a-amylase, anti-a-glucosidase, kev cia siab, thiab cytotoxic cuam tshuam ntawm plaub cov kuab tshuaj feem ntawm R. rothschildianus nplooj.

Txoj kev:Cov hmoov qhuav ntawm R. rothschildianus nplooj tau muab rho tawm hauv plaub cov kuab tshuaj nrog cov polarities sib txawv. Ob peb qhov zoo thiab ntau qhov kev kuaj phytochemical tau ua los txiav txim siab cov khoom ntawm cov extracts. Kev tsom xam colorimetric tau siv rau kev txiav txim ntau ntawm phenols, flavonoids, thiab tannins. Kev soj ntsuam hauv vitro tau ua los ntsuas cov tshuaj tua kab mob antioxidant, tiv thaiv-a-amylase, tiv thaiv-a-glucosidase, thiab cia siab tias yuav tiv thaiv kev ua ub no, nrog rau cytotoxicity los ntawm MTS tshuaj tiv thaiv kab mob qog noj ntshav (HeLa) thiab kab mob qog noj ntshav ntawm lub mis. (MCF7).

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Thov nias ntawm no kom paub ntxiv

Cov txiaj ntsig:Cov acetone feem ntawm R. rothschildianus nplooj pom qhov tseem ceeb tshaj plawsantioxidantkev ua haujlwm, vim muaj cov ntsiab lus siab tshaj plaws ntawm flavonoids thiab phenolics, nrog rau ICso tus nqi ntawm 6.3 ±0.4 ug/ml, piv rau 3.1±0.9 ug/ml rau Trolox, thiab hais txog lipase inhibition kev ua haujlwm ntawm acetone feem pom tau tias muaj zog tshaj plaws nrog ICso tus nqi ntawm 26.3 ±{{10}}.6 ug/ml, piv nrog orlistat zoo tswj ICso 12.3 ug/ml. Tib cov extract yog qhov muaj zog tshaj plaws inhibitor ntawm a-amylase thiab a-glucosidase, nrog ICso qhov tseem ceeb ntawm 19.1±{22}}.7 ug / ml thiab 54.9±{28}}.3 ug / ml, ntsig txog, piv txwv. rau 28.8, 37.1 ± 0.3 ug / thiab ntawm acarbose, feem. Cov feem hexane pom 99.9 feem pua inhibition ntawm HeLa hlwb thiab 97.4 feem pua inhibition rau MCF7 hlwb.

Xaus:Cov acetone feem ntawm R. rothschildianus nplooj tuaj yeem muab qhov chaw ntawm bioactive compounds rau kev kho mob ntawm oxidative kev nyuaj siab. Ib yam li ntawd, feem ntau ntawm hexane qhia txog kev cog lus antitumor peev xwm ntawm R. rothschildianus. Kom meej meej, cov lus qhia thawj zaug xav tau kev lim dej ntxiv ntawm cov tshuaj muaj peev xwm ua tau zoo, thiab thaum kawg, kev tshawb fawb hauv vivo los txiav txim siab lawv cov txiaj ntsig.

Ntsiab lus:Rumex rothschildianus, Antioxidant, Lipase, Amylase, Trolox, Phytochemistry, Anti-proliferative kev ua si

Keeb kwm

Cov nroj tsuag tau siv los ua kev kho mob txij thaum ub los. Cov hauv paus hniav, noob, tawv ntoo, nplooj, thiab paj tau siv los kho lub hom phiaj. Nyob rau niaj hnub nim no, cov tshuaj hluavtaws muaj thiab muaj txiaj ntsig zoo hauv kev kho mob ntawm ntau yam kab mob; Txawm li cas los xij, qee tus neeg tseem nyiam cov tshuaj ntsuab vim lawv pom tias tsis muaj teeb meem rau tib neeg lub cev [1,2]. Cov nroj tsuag tshuaj yog los ntawm kev txhais lub hauv paus ntawm phytochemical tebchaw uas muaj cov haujlwm kho mob. Cov khoom no tso siab rau qhov muaj cov metabolites sib txawv, xws li phenolics, terpenoids, thiab alkaloids [3]. Rumex rothschildianus Aarons. Nws yog ib tus tswv cuab ntawm ib qho tshwj xeeb ntawm genus Rumex, hauv tsev neeg Polygonaceae. Cov tsiaj no yog ib qho me me dioeciously txhua xyoo, endemic nyob rau hauv Palestine. Nws muaj qhov siab nruab nrab ntawm 45 cm thiab yog tus cwj pwm los ntawm erect stems tuav radical petiolate nplooj, uas yog luv-hastate ntawm lub hauv paus thiab luv-acuminate ntawm apex. Paj muaj ib txoj kab uas hla ntawm 3-4 hli, thaum pistillate paj yog hais txog 2 hli nyob rau hauv lub cheeb nrog ib coriaceous membranous txheej [4]. Rumex swb. yog dav nyob rau hauv ntau thaj tsam ntawm Qaib Cov Txwv, thiab yog sawv cev los ntawm 22 hom. Qee hom kab mob feem ntau yog R. patientia L, R. Crispus L, R. acetosa LR caucasicus rech, R. alpinus LR alpinus, thiab R. caucasicus yog cov nroj tsuag perennial faib nyob rau hauv nruab nrab thiab sab hnub tuaj Anatolia ntawm qhov siab ntawm {{10. } } m saum toj no hiav txwv theem. Rumex genus tau siv dav hauv cov tshuaj hauv tebchaws Turkey los kho cov kab mob, xws li cem quav, raws plab, thiab eczema [5, 6]. Cov genus tseem muaj qee cov tshuaj laxative, diuretic, antipyretic, kho qhov txhab, thiab tshuaj tiv thaiv kab mob. Ntau tus neeg nyob rau sab hnub tuaj ntawm Qaib Cov Txwv siv cov nplooj ntawm Rumex spp. raws li kev khaws cia hauv cheese, nrog rau muab cov khoom noj muaj ntxhiab [7].

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Ntau yam kev tshawb fawb tau ua tiav ntawm Rumex hom, xws li cov tshuaj tua kab mob tau tshaj tawm rau qee hom. Qee cov bioactive phytochemicals yav dhau los tau pom nyob rau hauv Rumex vicarious L, xws li carotenoids, tocopherols, polyphenols, flavonoids, thiab ascorbic acid, uas muaj lub luag haujlwm ua antioxidants thiab natural detoxifying agents. Kev noj zaub mov kom tsawg ntawm cov tshuaj antioxidant phytochemicals, zoo li carotenoids,phenolics, thiabflavonoidstuaj yeem tiv thaiv cov kab mob tsis sib kis hauv tib neeg, xws li mob qog noj ntshav, mob plawv, thiab lwm yam teeb meem kev noj qab haus huv ntsig txog oxidative stress [5, 8]. Cov teeb meem dawb radicals paub tias ua lub luag haujlwm tseem ceeb hauv ntau yam teeb meem kev noj qab haus huv, xws li mob qog noj ntshav, kab mob plawv, mob caj dab rheumatoid, cataracts,Alzheimer tus kab mob, thiab lwm yam kab mob degenerative ntsig txog kev laus. Antioxidants yog cov khoom muaj txiaj ntsig uas cuam tshuam cov dawb radicals ua ntej lawv tuaj yeem tua cov hlwb, thiab yog li lawv tiv thaiv kev puas tsuaj rau cov cell proteins, lipids, thiabcarbohydrates. Ntau yam ntawm ob qho tib si ntuj thiab hluavtaws antioxidants tau thov rau kev kho mob ntawm tib neeg cov kab mob. Xws li kev txaus siab rau lub luag haujlwm ntawm cov tshuaj antioxidants hauv tib neeg kev noj qab haus huv tau ua rau muaj kev tshawb fawb hauv kev tshawb fawb txog zaub mov thiab tshuaj ntsuab tshuaj ntsuab, tshuaj xyuas kev ua haujlwm ntawm tshuaj ntsuab raws li antioxidants. Antioxidant kev ua muaj xws li dawb radical scavenging muaj peev xwm, inhibition ntawm lipid peroxidation, hlau ion chelating muaj peev xwm thiab kuj txo lub peev xwm [9,10].

Cancer yog ib qho teeb meem kev noj qab haus huv thoob ntiaj teb. Kev txhim kho thiab kev tshawb pom ntawm cov tshuaj tiv thaiv kab mob tshiab tshiab tseem ceeb heev vim muaj ntau yam. Cov xwm txheej no suav nrog kev kho mob uas yuav ua rau muaj kev phiv loj lossis tuaj yeem kim heev. Lwm txoj hauv kev uas muaj kev nyab xeeb thiab pheej yig dua yog tseem xav tau [11-14].

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Ntau hom nroj tsuag suav hais tias muaj peev xwm ntawm bioactive molecules xws li atropine los ntawm Belladonna nplooj, cocaine los ntawm coca nplooj, vincristine los ntawm Vinca cog, thiab ntau lwm yam uas tseem ua lub luag haujlwm tseem ceeb hauv cov tshuaj niaj hnub [15,16].

Cov txiaj ntsig kho tau zoo tuaj yeem los ntawm kev sib xyaw cov khoom lag luam thib ob uas muaj nyob hauv cov nroj tsuag tshuaj. Cov tebchaw no feem ntau yog cov metabolites, xws li alkaloids, steroids, tannins, flavonoids, thiab phenolics, uas yog synthesized thiab tso rau hauv ib feem ntawm cov nroj tsuag [17,18]. Txoj kev tshawb fawb tam sim no tshawb xyuas cov in-vitro anti-a-amylase, anti- -glucosidase, anti-lipase, anti-proliferative thiab antioxidant kev ua ub no ntawm cov feem sib txawv rho tawm los ntawm R. rothschildianus nplooj.

Cov txheej txheem

Cov khoom siv cog, tshuaj, thiab cov cuab yeej siv

R. rothschildianus nplooj tau raug sau los ntawm thaj tsam sab hnub poob ntawm Palestine, thaum Lub Ob Hlis thiab Lub Peb Hlis 2018. Lawv tau txheeb xyuas los ntawm Dr. Nidal Jaradat, los ntawm Pharmacognosy Laboratory ntawm An-Najah National University, nyob rau hauv daim ntawv pov thawj code Pharm-PCT{{3} }. Tag nrho cov tshuaj tau muas los ntawm Sigma-Aldrich. Lub spectrophotometer-UV/Visible (Jenway degree 7135, Stafford-shire, UK), lim ntawv (Whitman No. 1, Washington, USA), shaker device (Memmert 531-25-1, Stockholm, Sweden), rotavap apparatus (Heidolph -VV 2000, Schwa-bach, Lub teb chaws Yelemees), grinder (Aero Plus 500 W Tov Khoom Grinder, I01, Wan Chai, Tuam Tshoj), hluav taws xob sib npaug (Radwag, AS 220/c/2, Torunska, Poland), khov tshuab-BT85 (Millrock Technology, Tuam Tshoj) thiab cryo-desiccator (Mill-pob zeb technology, BT85, Kingston, USA) tau siv.

Kev npaj ntawm extracts thiab fractionation

Cov hmoov qhuav ntawm R. rothschildianus nplooj tau muab rho tawm los ntawm kev ntxiv cov kuab tshuaj raws li lawv cov polarity, pib nrog cov tsis-polar hnyav hexane, thiab tom qab ntawd acetone (polar aprotic organic solvent), methanol (polar cawv), thiab thaum kawg distilled dej (a polar protic kuab tshuaj). Rau txhua qhov kev rho tawm, kwv yees li 25 g ntawm cov nplooj qhuav qhuav tau muab tso rau hauv 0.51 hexane rau 72h hauv lub tshuab shaker ntawm 100 kev sib hloov ib feeb ntawm 25 degree . Ua ntej, lub hexane tau hloov nrog 0.5L acetone, thiab tom qab ntawd hloov pauv koom nrog qhov sib npaug ntawm methanol thiab dej. Incubations nyob rau hauv cov kuab tshuaj yog raws li tau piav saum toj no rau hexane. Txhua feem ntawm cov organic yog lim thiab concentrated nyob rau hauv lub nqus tsev vacuum ntawm ib tug teb evaporator, thaum lub aqueous feem yog qhuav siv lub freeze dryer. Thaum kawg, tag nrho cov seem crude tau khaws cia ntawm 4 degree [19,20].

Cov txiaj ntsig ntawm txhua feem yog suav nrog cov qauv hauv qab no:

Kev ntsuam xyuas phytochemical ua ntej

Phytochemical tshuaj ntsuam xyuas ntawm R. rothschildianus nplooj nyob rau hauv plaub feem yog ua los txheeb xyuas cov nquag metabolites. Cov txiaj ntsig zoo tau qhia tias yog (ntxiv rau) rau qhov muaj thiab (-) rau qhov tsis muaj bioactive phytochemicals[10,21].

Kev txiav txim siab ntawm tag nrho cov ntsiab lus phenolic (TPC)

Cov txheej txheem los txiav txim TPC yog raws li Cheung li al. TPC tau qhia nyob rau hauv milligrams ntawm gallic acid sib npaug ib gram qhuav hnyav ntawm nplooj (mg GA / g qhuav hnyav). Freshly npaj 7.5 feem pua sodium carbonate tov yog tsim los ntawm muab 7.5g Na2CO3 nyob rau hauv lub ntim ntim thiab kho qhov ntim rau 100ml nrog dej distilled. Cov txheej txheem siv tshuaj (gallic acid tov) tau npaj los ntawm kev sib cais 100mg ntawm gallic acid hauv dej distilled mus rau qhov kawg ntim ntawm 100ml. Los ntawm qhov no, serial dilution tau ua kom tau txais kev daws teeb meem ntawm gallic acid ntawm 100,70,50,40, thiab 10ug / ml). Cov kev daws teeb meem ntawm cov feem ntawm nplooj tau npaj los ntawm kev ua kom 100mg ntawm cov nroj tsuag extract hauv dej distilled, thiab kho rau tag nrho cov ntim ntawm 100ml. Cov tshuaj tiv thaiv sib xyaw tau npaj los ntawm kev sib xyaw 0.5 ml ntawm txhua feem ntawm cov tshuaj nrog 2.5 ml ntawm 10 feem pua Folin-Ciocalteu's reagent, uas tau yaj hauv dej nrog 2.5 ml ntawm 7.5 feem pua sodium bicarbonate. Cov qauv tubes raug incubated rau 45 min ntawm 45 degree. Tom qab ntawd, qhov nqus ntawm txhua qhov tau ntsuas hauv spectrophotometer ntawm lub wavelength ntawm 765 nm. Cov qauv ua haujlwm tau npaj ua triplicate rau txhua qhov kev ntsuas ntsuas, los ntawm qhov ntsuas qhov nruab nrab thiab tus qauv sib txawv tau suav [21].

Kev txiav txim siab ntawm tag nrho cov ntsiab lus flavonoid (TFC)

Lub TFC nyob rau hauv plaub R. rothschildianus nplooj fractions raug soj ntsuam siv ib tug calibration nkhaus ntawm rutin (tus qauv siv compound). Cov txiaj ntsig tau qhia tias yog milligram ntawm rutin sib npaug ib gram qhuav hnyav ntawm nplooj extract (mg RU / g qhuav hnyav). Lub calibration nkhaus rau rutin yog tsim los siv serial dilutions generated los ntawm ib tug Tshuag tov ntawm 1{{10}}0 ug/ml. Txhawm rau ua cov tshuaj hauv Tshuag, 10 mg ntawm rutin tau yaj hauv 10 ml ntawm dej distilled thiab tom qab ntawd diluted rau 100 ml. Tom qab ntawd, cov tshuaj npaj tau diluted los muab rutin ntawm cov concentrations ntawm 10, 30, 40,50,70, thiab 100 ug / ml. Rau kev npaj ua haujlwm, 0.5ml ntawm txhua feem ntawm cov tshuaj tov nrog 3 ml methanol, 0.2ml 10 feem pua AlCl3,0.2ml 1M potassium acetate, thiab 5ml distilled dej, thiab ces incubated ntawm chav tsev kub rau 30 min. Cov kauj ruam dhau los tau rov ua dua rau txhua feem, tom qab ntawd, qhov nqus tau raug ntsuas ntawm lub wavelength ntawm 415 nm. Rau kev tswj tsis pub dhau, kev daws teeb meem ua haujlwm tau teeb tsa nrog dej distilled nyob rau hauv qhov chaw ntawm cov qauv rho tawm. Cov qauv no tau npaj rau hauv triplicate rau txhua qhov kev ntsuas ntsuas, los ntawm qhov ntsuas qhov nruab nrab thiab tus qauv sib txawv tau suav [22].

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Kev txiav txim siab ntawm tag nrho cov ntsiab lus tannin (TTC) Cov txheej txheem ntawm Sun et al. tau siv los txiav txim TTC nyob rau hauv plaub R. rothschildianus nplooj fractions, yog cov txheej txheem feem ntau siv. Catechin tau siv los ua qhov sib xyaw ua ke los tsim kom muaj qhov nkhaus calibration. Ib qho 100ug/ml Tshuag hauv methanol tau npaj, los ntawm cov tshuaj dilution tau tsim los muab catechin concentrations ntawm 10, 30, 50,70, thiab 100 ug / ml. Ib 4 feem pua ntawm cov tshuaj vanillin hauv methanol tau npaj tshiab. Cov kev daws teeb meem ntawm cov feem ntawm 100 ug / ml tau npaj siv methanol ua cov kuab tshuaj. Rau kev ua haujlwm, 0.5ml ntawm txhua feem ntawm cov tshuaj yog tov nrog 3ml vanillin tov thiab 1.5ml ntawm concentrated HCl. Cov sib tov raug tso cai sawv ntsug rau 15 feeb, thiab tom qab ntawd qhov nqus ntawm 500 nm tau ntsuas, siv cov tshuaj ua haujlwm teeb tsa nrog methanol nyob rau hauv qhov chaw ntawm cov qauv rho tawm ua ib qho khoob. Tag nrho cov qauv ua haujlwm tau txheeb xyuas hauv triplicate, los ntawm qhov nruab nrab thiab tus qauv sib txawv qhov tseem ceeb raug xam. Tag nrho cov tannin nyob rau hauv txhua feem tau qhia nyob rau hauv cov nqe lus ntawm catechin sib npaug (mg ntawm CAE / g qhuav luj ntawm nplooj) [23].

Txoj kev ua haujlwm Antioxidant

Kev kuaj dawb 2,2-diphenyl-picrylhydrazyl (DPPH) radical scavenging assay tau siv los ntsuas cov tshuaj tiv thaiv antioxidant hauv qhov sib txawv ntawm R. rothschildianus nplooj. Ib qho 1000ug/ml Tshuag cov tshuaj ntawm txhua feem ntawm cov nroj tsuag tau npaj rau hauv methanol. Tsis tas li ntawd, 1000 ug / ml ntawm Trolox kuj tau npaj (tus qauv siv). Lub dilution series tau npaj los ntawm cov kev daws teeb meem rau txhua feem, muab rau serial dilutions ntawm 2, 5, 10,20, 50, thiab 100 ug / ml. Ib ml ntawm txhua cov extract dilution yog tov nrog 1 ml 0.002 g / ml DPPH hauv methanol. Ib ml ntawm methanol tau ntxiv los muab qhov kawg ua haujlwm ntim ntawm 3 ml. Cov tshuaj DPPH tau npaj tshiab, raws li nws tau nkag siab zoo rau lub teeb. Kev tswj tsis pub muaj kev sib xyaw ua ke yog DPPH hauv methanol hauv qhov sib piv ntawm 1: 2, tsis muaj qhov sib ntxiv ntawm cov extract. Tag nrho cov kev daws teeb meem tau muab tso rau hauv qhov chaw sov (25 degree) hauv qhov tsaus rau li 30 min. Tom qab ntawd qhov ntsuas qhov muag pom tau nrog lub spectrophotometer ntawm lub wavelength ntawm 517 nm. Cov kab zauv hauv qab no tau siv los xam cov feem pua DPPH inhibition rau txhua feem ntawm cov nroj tsuag, nrog Trolox ua tus qauv compound:

nyob qhov twg, Ag yog qhov sau tseg absorbance ntawm cov tshuaj dawb paug, thiab Ats yog sau tseg absorbance ntawm cov qauv tshuaj ntsuam xyuas [21].

Porcine pancreatic lipase inhibition assay

Cov kev daws teeb meem ntawm 500 ug/ml yog tsim los ntawm txhua feem ntawm cov nroj tsuag hauv 10 feem pua DMSO. Los ntawm cov no, ib tug dilution series ntawm tsib concentrations ntawm 50,100,200, 300, thiab 400 ug / ml tau ua. Ib qho 1 mg / ml Tshuag tov ntawm porcine pancreatic lipase hauv Tris-HCl tsis yog tau npaj tshiab ua ntej siv. Lub substrate, p-nitrophenyl butyrate (PNPB) tau npaj los ntawm dissolving 20.9mg hauv 2ml acetonitrile. Rau txhua qhov kev daws teeb meem, 0.1ml porcine pancreatic lipase yog tov nrog 0.2ml cog feem los ntawm txhua tus tswv cuab ntawm dilution series. Tris-HCl tau ntxiv los ua qhov kawg ntim ntawm cov kev daws teeb meem ua haujlwm 1ml, thiab lawv tau muab tso rau ntawm 37 degree C rau 15 min. Tom qab incubation, 0.1 ml p-nitrophenyl butyrate tov tau ntxiv rau txhua lub raj kuaj. Tom qab ntawd cov sib tov yog incubated rau ib tug ntxiv 30min ntawm 37 degree. Pancreatic lipase kev ua haujlwm tau txiav txim siab los ntawm kev ntsuas qhov hydrolysis ntawm PNPB rau hauv p-nitrophenolate ntawm 410 nm, siv lub UV spectrophotometer. Tib cov txheej txheem tau rov ua dua siv orlistat raws li tus qauv siv sib xyaw. Qhov feem pua lipase inhibition los ntawm cov khoom cog qoob loo tau suav nrog cov lej hauv qab no:

nyob qhov twg, Ap yog qhov sau tseg absorbance ntawm cov tshuaj dawb paug, thiab Ats yog sau tseg absorbance ntawm cov qauv tshuaj ntsuam xyuas [24]. a-Amylase inhibitory kev soj ntsuam

A100 mg ntawm txhua feem yog yaj nyob rau hauv ob peb millimeters ntawm 10 feem pua DMSO, thiab ces yaj ntxiv mus txog 100ml hauv 0.02 M Na HPOq/ NaH-PO4,0.006M NaCl, pH 6.9 muab thaum kawg cov kev daws teeb meem nrog cov concentrations ntawm 1000 ug / ml. Los ntawm cov no, cov nram qab no dilutions tau npaj ntawm 10,50,70,100, thiab 500 ug / ml, siv 10 feem pua DMSO ua cov diluent. Ib qho 0.2ml ntim ntawm 2units / ml porcine pancreatic -amylase tau tov nrog 0.2ml

tsob nroj feem thiab tau incubated rau 10min ntawm 30 degree . Tom qab incubation, 0.2ml ntawm ib tug freshly npaj 1 feem pua ntawm starch tov nyob rau hauv dej yog ntxiv, thiab cov hlab yog ces incubated rau tsawg kawg yog peb feeb ntxiv. Thaum lub sijhawm no, cov tshuaj tiv thaiv tau nres los ntawm kev sib ntxiv ntawm 0.2ml 3, 5-dinitro salicylic acid (DNSA) colour reagent thiab diluted nrog 5ml ntawm distilled dej, ua ntej yuav rhuab ntawm 90 degree rau 10 min hauv dej. da dej. Tom qab ntawd qhov sib tov tau txias rau hauv chav sov, thiab qhov nqus tau ntsuas ntawm 540 nm. Kev tswj tsis pub dawb tau npaj siv tib yam li tau piav qhia saum toj no, tab sis hloov cov feem cog nrog 0.2ml tsis. Acarbose tau siv los ua tus qauv siv raws li cov txheej txheem tau piav qhia saum toj no. a-amylase inhibitory kev ua haujlwm tau suav nrog kev sib npaug hauv qab no: