agronomy
Article
Plant Growth Inhibitory Activity of Hibiscus sabdariffa Calyx
and the Phytotoxicity of Hydroxycitric Acid Lactone
Tugba Gonca Isin Ozkan 1, Kwame Sarpong Appiah 1,2 , Emine Akalin 3 and Yoshiharu Fujii 1,*
1 Department of Biological Production, Faculty of Agriculture, International Environmental and Agricultural
Science, Fuchu Campus, Tokyo University of Agriculture and Technology, Fuchu 183-8509, Japan;
tugbagonca_ozkan@yahoo.com (T.G.I.O.); ksappiah90@gmail.com (K.S.A.)
2 Department of Crop Science, University of Ghana, Legon, Accra P.O. Box LG 44, Ghana
3 Faculty of Pharmacy, Department of Pharmaceutical Botany, Istanbul University, Fatih, Istanbul 34116, Turkey;
akaline@istanbul.edu.tr
* Correspondence: yfujii@cc.tuat.ac.jp
Abstract: Weeds pose major constraints in crop production. The use of allelochemicals and allelopathic
species can provide an effective alternative for sustainable weed management. In a previous study
that evaluated the allelopathic activity of wild and cultivated plants in Turkey, Hibiscus sabdariffa
demonstrated the strongest inhibitory potential. This study aimed to estimate the phytotoxic influence
of the H. sabdariffa water crude extracts on Lactuca sativa L. in a bioassay experiment. High-performance
liquid chromatography (HPLC) analysis was used to identify two major compounds, hydroxycitric
acid lactone and hydroxy citric acid, and their plant growth inhibitory activities were evaluated by
bioassays. Hydroxycitric acid lactone had a stronger growth inhibitory activity on L. sativa L. and
was estimated as a major allelochemical in H. sabdariffa calyx. The high concentration (16.7% of
the dry weight of the calyx) and strong inhibitory effect (EC50, 73.7 ppm) of the hydroxycitric acid
lactone could demonstrate the growth inhibitory activity of the H. sabdariffa calyx extract. This study
showed that hydroxycitric acid lactone, a major compound in the calyx of Hibiscus sabdariffa, is a plant
growth inhibitor.
Keywords: phytotoxicity; Hibiscus sabdariffa calyx; hydroxycitric acid lactone; hydroxycitric acid;
specific activity; total activity
Citation: Isin Ozkan, T.G.; Appiah,
K.S.; Akalin, E.; Fujii, Y. Plant Growth
Inhibitory Activity of Hibiscus
sabdariffa Calyx and the Phytotoxicity
of Hydroxycitric Acid Lactone. 1. Introduction
Agronomy 2023, 13, 1746. https:// Weeds are a major impediment to the growth and development of field crops, often
doi.org/10.3390/agronomy13071746 leading to yield losses [1,2]. The annual global economic loss caused by weeds has been
Academic Editor: Kirsten Brandt estimated at more than 100 billion US dollars [3]; moreover, the worldwide expenditure on
weed management is expected to reach billions of US dollars [4]. Farmers have struggled
Received: 28 April 2023 with weeds intensively since the dawn of agriculture [5], and the control of weeds, therefore,
Revised: 20 June 2023 poses a challenging issue for sustainable agriculture [6,7]. Since the discovery of synthetic
Accepted: 24 June 2023 herbicides, they have become among the most widely used weed-control strategies in most
Published: 28 June 2023 countries [8,9]. Over two hundred functional substances had been recorded for herbicidal
applications [9,10]. However, weeds could develop resistance to synthetic herbicides [9].
Allelopathy is a biological occurrence for several plant species, in which plants release
Copyright: © 2023 by the authors. bioactive compounds into the surrounding habitat either from their aerial or underground
Licensee MDPI, Basel, Switzerland. segments through root exudates, leaching by dews and rains, and vaporizing or decompos-
This article is an open access article ing plant tissue. The released compounds could influence the growth and development
distributed under the terms and of other organisms, in particular weeds, other plants, animals, and microorganisms, in an
conditions of the Creative Commons inhibitory or stimulatory way in the environment [11,12]. These plant growth inhibitory
Attribution (CC BY) license (https:// compounds (allelochemicals) from various donor plant species could act as natural weed
creativecommons.org/licenses/by/ inhibitors. Different parts of plants, such as leaves, barks, roots, flowers, seeds, pollens,
4.0/). stems, and fruits, could contain growth-inhibitory compounds [13,14]. The concentration
Agronomy 2023, 13, 1746. https://doi.org/10.3390/agronomy13071746 https://www.mdpi.com/journal/agronomy
Agronomy 2023, 13, 1746 2 of 11
of the inhibitory compounds in the different plant parts may vary, leading to variation
in the level of plant growth inhibitory effects [13,15]. Previous studies have shown the
effective use of the species containing inhibitory substances and their growth inhibitory
compounds against weeds in good plant protection practices [16–20].
Natural products are made by syntheses in alive organisms as their answer to biolog-
ical or physical incentives. They are usually built via complicated pathways leading to
molecules not easily obtainable with ordinary synthetic methods in the laboratory [21,22].
These various motives make natural products a favorable material for the development of
replacements for long-establish synthetic agrochemicals [23].
The use of artificially produced herbicides all the time does not point out a fitting
implement to fight against resistance-developing weeds but is also asserted as harmful
to the environment [24]. Moreover, the application of the species containing inhibitory
substances and their growth inhibitory compounds against weeds offers alternatives for
the development of ecologically friendly agricultural practices to increase crop productivity
and maintain ecosystem stability [25]. Plants with growth-inhibitory properties are thought
of as potential sources of biological materials against weeds [26], and growth-inhibitory
compounds could be utilized for weed control to lessen the massive reliance on chemically
synthesized herbicides and the corresponding toxicity danger for nature [27]. Consequently,
studies for other new reliable weed-control practices have become essential, with growing
attention paid to the mechanism of plant growth inhibition [28]. The effective use of plant
growth inhibitory mechanisms has played a central role in weed control, and plant extracts
have the potential to be exploited as natural herbicides [28]. Additionally, the action of
taking out inhibitory compounds from growth-inhibitory plant materials by extraction
would be applicable to produce bioherbicides [20].
The visible changes that inhibitory compounds cause to the growth and development
of target plants, which are also called receptor or susceptible plants, are diverse. These
effects on receptor plants could consist of decreasing radicle and shoot length, becoming
darker and/or blowing up seeds, inhibited or retarded germination rate, the spiral shaping
of the root axis, color defects on seeds, the absence of root hairs, the localized death of living
tissues (necrosis), the growing total quantity of seminal roots, and the lessening in the
amount of dry weight accumulation, for example [29,30]. These morphological effects are in
many cases secondary actions of primary differentiation (the inhibition of cell division and
elongation, interference with cell membrane permeability, enzymatic activities, respiration,
photosynthesis, and so on) at the cellular or molecular level in susceptible plants [29].
Hibiscus sabdariffa L. is a member of the Malvaceae family of plants, and it is often
recognized as Roselle. The plant is an all-around living annual plant in the tropics and
subtropics of both hemispheres and many areas of Central and West Africa, Southeast Asia,
America, and elsewhere as it is relatively easy to grow. The red calyx of the plant is used in
several ways, including in herbal teas, herbal medicines, syrups, and food coloring [31].
H. sabdariffa calyx had shown strong growth inhibitory activity on lettuce seedling elon-
gation; it reduced the lettuce radicle and hypocotyl elongation to 3.2% and 6.6% of the
control, respectively, in previous research [32]. The chemical composition of H. sabdar-
iffa has been described as consisting of hydroxycitric acid and its lactone in the aqueous
extracts [31,33–37]. Although the whole plant (leaves, stem, and roots) and isolated chemi-
cals from the whole plant indicated strong inhibitory activity on the growth of test plant
species [38–41], the inhibition effect of the calyx alone and its substances has not been
researched. (2S,3R)-hydroxycitric acid has been shown to prevent the action of pancreatic-
amylase and small intestine glucosidase [34]. H. sabdariffa is implemented in traditional
medicine in the form of herbal teas or cold beverages for its hypotensive and diuretic effects
and to drop body temperature and blood viscosity [36]. H. sabdariffa dried calyx ethanolic
extract in addition to the diet of rats showed a reduction in serum lipids on their serum
lipid profile [42].
Hydroxycitric acid (Figure 1a) is a citric acid molecule with a hydroxyl group at the
second carbon [35]. This acid has four stereoisomers: (2S,3S), (2R,3R), (2S,3R), and (2R,3S),
Agronomy 2023, 13, x FOR PEER REVIEW 3 of 12 
 
diuretic effects and to drop body temperature and blood viscosity [36]. H. sabdariffa dried 
calyx ethanolic extract in addition to the diet of rats showed a reduction in serum lipids 
Agronomy 2023, 13, 1746 on their serum lipid profile [42]. 3 of 11
Hydroxycitric acid (Figure 1a) is a citric acid molecule with a hydroxyl group at the 
second carbon [35]. This acid has four stereoisomers: (2S,3S), (2R,3R), (2S,3R), and (2R,3S), 
and theiar nladctthoneier floarcmtosn e[3f7o]r. mIns g[3e7n]e.rIanl sgoelnuetiroanl ,s ohlyudtiroonx,yhciytrdirco axcyidci tirsi ca amciidxtiusrae mofi xntounr-e of non-
lactone alancdt olnacetoannde floacrmtosn.e (2foSr,3mSs).-h(2ySd,r3oSx)y-hcyitdrirco axcyicdi t(raic macixidtu(rae mofi x(t2uSr,e3So)f-h(2ySd,r3oSx)-yhcyitdrirco xycitric
acid steraecoiidsosmteerero ainsodm itesr laacntdonites floarcmto)n ies ffoourmnd)  iisnf Gouarncdiniina cGaamrcbiongiaiac. a(2mSb,o3gRia)-.h(y2Sd,r3oRx)y-hciytdrirco xycitric
acid (a macixidtu(rae mofi x(t2uSr,e3Ro)f-h(2ySd,3roRx)y-hcyitdrirco axcyicdi tsrtiecraecoiidsosmteerer oainsodm itesr laacntdonites floarcmto)n ies ffoorumn)di s found
enrichede ninr itchhee dcailnyxthees coafl yHx. essabodfaHri.ffsaa b[3da5r]i. fHfay[3d5r]o.xHyycidtrriocx aycciidtr ilcacatcoidnel a(cFtiognuere(F 1ibg)u, rwe h1bic)h, w hich is
is the latchtoenlaec ftoornme  foofr m(+)o-fal(l+o)--haylldo-rhoyxdyrcoitxryicc iatrciicda (cFidig(uFrieg u1rae),1 aan),da nitds idtserdievraitviavteisv easrea rtehteh e major
major orogragnainc iccocmompopuonudnsd isni nthteh elelaevaevse asnadn dcaclaylcyecse esxetxrtarcatcst os fo Hf H. s.asbadbadraiffriaff a[3[13]1. ]. 
  
(a) (b) 
Figure 1.F Tighuer eta1r.geTt hceomtaproguetndcos minp othuins dsstuidnyt.h (ias) s(t2uSd,y3.R()a-h) y(d2Sro,3xRy)c-ihtryidc raocxiydc i[t4r3ic]; a(cbi)d (2[4S3,]3;R()b-) (2S,3R)-
hydroxychityrdicr oaxciydc iltarcictoanceid [3la4c,4to3,n4e4][.3 4,43,44].
Total actTivoittayl acntdiv siptyecainfidc aspcteicviifityc arceti vtwityo ianrde itcwatooirns dfoicra tthoer safsosrestshme eansste osfs mthen gtrofwtthhe growth
inhibitoriyn hciabpitaocriytyc oafp a cpiltaynotf aandpl an tinadnidviadnuianld ciovmidpuoaul ncodm [1p5o,u26n,d45[–151,2]6. T,4h5e– 5to1t]a. lT ahcetitvoitayl activity
of a comopfoauncodm inp oa upnladnitn isa tphlea pnrtoisptohretiopnro opfo trhteio cnonofcethnetrcaotinocne notfr anti oinnhoibf iatnoriyn hcoibmitpooryuncodm pound
in the priondtuhceinpgro pdluancitn tgo pitlsa nspt etocifiitcs sapcteicviifityc a(EctCiv50i)t,y w(EhCic5h0 )i,sw thhei cehffiescthiveee cfofenccteivnetrcaotinocne notfr ation of
the inhibthiteoriyn hciobmitoproyucnodm repqouinreddr etoq uexireerdt ato heaxlfe-rmt axhimalfu-ma exffimecut mone aff reeccteoivnear rpelcaenitv [e4r4p].l ant [44].
The activTihtye oafc itnivhiitbyitofryin choimbiptoruyncdosm anpdo uthned sspaencidest hcoenstpaeinciinesg cinohnitbaiitnoirnyg suinbhsitbaintocersy csaunb stances
be evalucaatnedb ebye vthaleuira tsepdecbiyfict haecitrivsiptyec. iTfihcea ccotimviptyo.uTnhdes cwomithp ohuignhd bs iwoliothgihcaigl hacbtiivoiltoyg ipcearl activity
unit wepigehrtu onfi ttwhee igcohmt opfotuhnedc osmhopuoludn bdes hcounltdembpelcaotendte mtop dlaetveedlotop dheevrebliocpidhese.r bTihciedsees . These
compoucnodms pshoouunldds hsahvoeu sldmhaallv EeCs5m0 avlalluEeCs5,0 wvhailcuhe si,nwdihciacthe ihnidghic astpeehciifigch ascpteivciifityc [a4c8t,i5v2i]t.y [48,52].
On the Ootnhtehre hoathnedr, htahned ,tothtael toatcatlivaicttyiv oitfy coofncsoindseidreedre dinihnihbiibtoitroyr yccoommppoouunnddss ccoouulldd bbeea ssessed
assessedt otod deetteerrmiinnee tthhee ccoonnttrriibbuuttiioonn ooff ssuucchh ccoomppoouunnddss toto ththee ggrorowwthth inihnihbiibtoitroyr yeffeeffcetcs ts of the
of the spsepceiecsie csocnotnatianiinnign ginihnihbiibtoitroyr ysusubbstsatnanceces s[5[500].] .TThhee tototatal laacctitvivitityy aasssseessssmmeenntt ccoouulldd bbee applied
applied ttoo aapppproroxximimaatetet htheei mimpapcatcot foaf cao mcopmoupnoudnodn othne tihneh iibnihtoibryitoerfyfe cetff[e5c0t] .[5T0h]e. cTohme pounds
compouwnditsh whiigthh tohtiaglha cttoivtaitly ,awcthivicithyi, swa hfuicnhc tiiosn aal ifnudnicctaiotonratlo idnedcilcaarteotrh etoi ndhiebciltaorrey pthoet ential of
inhibitorayc poomtepnotuianld o,fs ah ocoumldpboeudnidre, cstheoduflodr bfue rdthireerctpeldan fot rg rfuowrththeri nphlaibnitt ogrryowretshe ainrchhibointotrhye path of
researchh eornb icthidee pdaetvhe loopf mheenrbt i[c5i2d,e5 3d].eIvneplorpevmioeunst s[t5u2d,5ie3s],. sIenv eprarlevcoiomups osutnuddsiehsa, vseebveeeranl isolated,
compouinddesn htiafiveed b, eaennd iesovlaaltueadt,e iddebnytitfiheedt,o atanlda ecvtiavliutyataedp pbryo athceh taostainl ahcibtiivtoitryy acpopmropaocuhn adss . Based
inhibitoroyn ctohmeptootuanldasc.t Bivaisteyda opnp rthoea cthot,aml aacntiyviptyla anptpgrrooawchth, minahniyb iptolarnytc gormowpothu inndhsibsiutocrhya s L-3,4-
compoudnidhsy dsurocxhy apsh eLn-y3l,4al-adnihinyed(rLo-xDyOphPeAn)yilnalManuicnuen a(Lp-rDuOriePnAs)[ 4i6n] ,Mcyuacnuanma ipdreuirnienVsic i[a46v]i,l losa [47],
cyanamicdise- icnin Vniacima ovyilllogsalu [c4o7s],i dciess-ciinnnSapmiroaeyal gthluucnobseirdgeiis [i4n8 S],priruateian thinunFbaegrogpiiy [r4u8m], reusctuinle innt um [13],
Fagopyruamng eeslciucilnenitnuHme r[a1c3l]e,u amnsgoeslnicoiwns kinyi Hfreuriatc[l4e9u]m, c saorsnnooswicskaycii dfriuniRt o[4sm9]a,r cinaurns oofsfiicci naacliids  liena ves [15],
Rosmarinaunsd offiincdinigalois ilneafvreusi t[1p5u],l panodf inCdouigroou inpi ftraugitu piaunlepn osifs C[o2u6r]owupeirtae giudieannteinfiseisd [2a6s] twheerpe rincipal
identifieidn hasib tihtoer pyrcinocmippaolu inndhsibfirtoomry rceopmorptoeudnindhs ifbriotmor yrepploanrttesd.  inhibitory plants. 
The intrTohdeucintotrroyd succrteoernyinscgr esetnuidnyg sfotur dtyhefo irnthhiebiitnohriyb ietoffreyctesf foecf tsploafnpt lasnptecsipeesc i[e3s2][ 32] indi-
indicatedca tHed. Hsa.bsdaabrdiaffraif facaclaylxy xaass aa ppootetnentitailapl lapnlatngtr ogwrtohwitnhh ibinithoirbyitcoarnyd icdaantde itdhaatted ethmaot nstrated
demonsttrhaetesdtr otnggrowthhineh 
essit
trionnhgibesitto irnyheifbfietcotrbye tewffeeectn boethtwereseonn otthheetest plant, L. sativa. However, the plantbitory effect of calyx and the contributiorns oofni ttshtew toescth parlaanctte, rLis. tiscatciovam. pounds
Howevetro, tthhee ipnlhainbti togrryoweftfhe citnohfibciatloyrxy reeffmeacitn oufn ckanloywx na.nIdn tthheis csotundtryib, tuhteioinn hoifb iittos rytwaoc tivity of
H. sabdariffa calyx was demonstrated by specific activity, where the contribution of two com-
pounds (hydroxycitric acid lactone and hydroxycitric acid) of calyx to its inhibition effect
 was evaluated by total activity. Consequently, current research focused on (i) the evaluation
of the plant growth inhibitory effects of H. sabdariffa calyx water crude extract on L. sativa
Agronomy 2023, 13, 1746 4 of 11
and (ii) the estimation of the contribution of hydroxycitric acid and hydroxycitric acid
lactone to the phytotoxicity of H. sabdariffa calyx.
2. Materials and Methods
2.1. Plant Material and Extraction Procedure
The crude extract was obtained from the air-dried Hibiscus sabdariffa calyx plant sample.
The air-dried samples were finely ground, and 160 mg of the sample was extracted with
20 mL distilled water (MilliporeSigmaTM, Burlington, MA, USA) at room temperature. The
solution was sonicated (10 min), filtered (No. 1 filter paper, Advantec Toyo, Tokyo, Japan),
and operated as a working solution.
2.2. Chemicals and Test Plant for Bioassay
Potassium hydroxycitrate tribasic monohydrate (hydroxycitric acid tripotassium salt) and
(+)-garcinia acid ((−)-hydroxycitric acid lactone, (2S,3S)-3-hydroxy-5-oxotetrahydrofuran-2,3-
dicarboxylic acid, Garcinia lactone) were bought from Sigma-Aldrich Chemie GmbH (Steinheim,
Germany). Both compounds were analytical standards and were used without further purifica-
tion. Lettuce seeds (Lactuca sativa, cultivar Legacy) were bought from a domestic seed company
in Japan, Takii Seed Co., Ltd. (Kyoto, Japan) and used as test plant in the bioassay to assess the
phytotoxic effect of pure compounds and Hibiscus sabdariffa calyx extract.
2.3. Inhibitory Effects of H. sabdariffa Crude Extract and Test Compounds
The specific activity of the crude extract and the total activity of the examined standard
compounds were estimated using lettuce as a test plant as described by Golisz et al. [13]
and Appiah et al. [15]. Concentrations of the crude extracts (10, 50, 100, 250, 500, 1000,
and 4000 ppm), hydroxycitric acid (5, 10, 25, 50, 100, 200, 1000, 2000, and 4000 ppm), and
hydroxycitric acid lactone (5, 10, 25, 50, 100, and 200 ppm) were prepared with distilled
water for the inhibitory activity bioassays. Filter paper (27 mm ø, Toyo Roshi, Ltd., Tokyo,
Japan) was laid down in a glass Petri dish (30 mm ø). A total of 0.7 mL of test solution was
put into the filter. Five pre-germinated seedlings were left on the filter paper and incubated
(CN-25C, Mitsubishi Elec., Tokyo, Japan) for 48 h at 22 ◦C in dark conditions.
Distilled water was used as the control treatment without crude extract or pure
compound. Each treatment was replicated three times. After the incubation period, the
lengths of hypocotyls and radicles were determined by millimeter paper with 1 mm
accuracy, and the means were put into numbers as a percentage of the control. The
elongation percentage was calculated using the Equation (1) below:
X
Elongation% = × 100 (1)
Y
where X = treated (crude extract or pure compound) means radicle or hypocotyl length
and Y = control means radicle or hypocotyl length.
The contribution of hydroxycitric acid lactone and hydroxycitric acid to the allelopathy
of H. sabdariffa calyx crude extract was estimated through the total activity concept, which
is derived from the inhibition and concentration of the compounds in the crude extract.
Total activity was estimated by the division of hydroxycitric acid lactone or hydroxycitric
acid concentration in H. sabdariffa calyx by the specific activity (EC50) of the compound.
The concentrations of these compounds in H. sabdariffa calyx and their specific activities
have the same unit. Therefore, the total activity is without any unit. Total activity was
calculated using Equation (2) below [51]:
Total Activity = Concentration/Speci f ic Activity (2)
2.4. HPLC Analysis of H. sabdariffa Calyx
The ground 160 mg of the H. sabdariffa calyx samples were accurately weighed into the
Erlenmeyer and extracted with 20 mL distilled water as shown in the extraction procedure
Agronomy 2023, 13, x FOR PEER REVIEW 5 of 12 
 
activities have the same unit. Therefore, the total activity is without any unit. Total activity 
was calculated using𝑇 E𝑜q𝑡u𝑎a𝑙 t𝐴io𝑐n𝑡𝑖 𝑣(2𝑖𝑡)𝑦 be=lo𝐶w𝑜 𝑛[5𝑐𝑒1𝑛]:𝑡 𝑟 𝑎𝑡𝑖𝑜𝑛 ⁄𝑆𝑝𝑒𝑐𝑖𝑓𝑖𝑐 𝐴𝑐𝑡𝑖𝑣𝑖𝑡𝑦 (2)
2.4. HPLC Analysis of H. sabdariffa Calyx 
Agronomy 2023, 13, 1746 The ground 160 mg of the H. sabdariffa calyx samples were accurately weighed 5inofto11 
the Erlenmeyer and extracted with 20 mL distilled water as shown in the extraction 
procedure to analyze hydroxycitric acid lactone and hydroxycitric acid concentration by 
HPLC. An aliquot of the extract was filtered through a 0.2 µm syringe filter before injection 
to(5a nµaLly).z eHhPyLdCro xayncailtyriscisa cwidasla cptoernfeoramndedh yudsrionxgy caintr icLaCc-i2d0AcoDn celinqturaidti ocnhbroymHaPtoLgCra. pAhn 
a(liSqhuimotaodfztuh, eKeyxottroa,c Jtawpaans)fi. Altenr eIndetrhtsriol uOgDhSa 30 .c2olµummnsy (r2i5n0g e× fi4.l6te mr bme,f o5r eµmin jpecatritoicnle(s5, µGLL) .
HSPcLieCncaensa Ilnysci, sTwokasyop,e Jrafopramn)e dwauss iunsgeadn inL Cis-o2c0rAatDicl icqounidditcihornosm. Tahtoeg mraopbhil(eS hpihmasaed zwua,sK 0y.1o%to ,
JaHpaPnO). An Inertsil3 4 (phosphoricO aDciSd3) icno wluamtenr.( T25h0e ×co4lu.6mmn mte,m5pµemratpuarret iwclaess ,mGaLinStaciinenedce astI 3n0c°,CT,o aknydo ,
Jathpea nfl)owwa rsautes eodf tihnei smocorbaitleic pchoansdei twioans ss.eTt haet 0m.5o bmilLe/mphina.s eThwea asn0a.l1y%sisH w3PasO m4 (opnhitoosrpehdo bryic 
aucisdin) gin anw SaPteDr.-MT2h0eA cdoelutemctnort aetm 2p1e0r natmu.r eThwea csommpaionutnained at 30
◦
ds were quanCt,ifiaendd btyh ecoflmopwarrinatge 
otfhteh epmeaokb ialreeapsh aosfe twhea stasergt eatt c0o.5mmmLe/rcmiailn .coTmhpeoaunnadlsy swisiwtha sthme oanbiutonrdeadnbcey oufs inthgesaen 
ScPoDm-Mpo2u0nAdsd eitnec ttohrea tc2o1r0rensmpo.nTdhinegc omstapnoduanrddss wuesreedq uinan ttihfiee dcbalyibcroamtiopna ricnugrvthe.e pTehaek 
arqeuaasnotiffitchaetitoanrg wetacso ombmtaienrecida lbcyo cmopmopuanrdinsgw tihteh ptheeaka baurenadsa onfc ethoef ttahregseet ccoommppoouunnddss iwn itthhe 
cothrree asbpuonnddainngces toafn tdhaesrde scoumsepdoiunntdhse icna tlhiber caotirornescpuornvdei.nTgh setaqnudaanrtdifis cuasteiodn inw tahseo cbatlaibinraetdiobny 
cocumrpvaer. iAnlgl tchheempeiacakla arneaalsyosfetsh weetarerg deotncoe minp tohurneed srewpiltichatthees.a bundance of these compounds
in the corresponding standards used in the calibration curve. All chemical analyses were
d3o.n Reeisnutlhtsr eaenrde pDliicsacutesss.ion 
3.3.R1.e Esuffletcstsa onfd thDe iCsrcuudses Eioxntracts of Hibiscus sabdariffa Calyx on Lettuce Growth 
3.1. EfTfehcets opflathnet Cgrruodwe Ethx trinachtisboitfoHryib isecffuescstsa bdoaf riHffa. CsablydxaroinffaL ectatulycxe Gcrouwdteh extracts were 
inveTshtiegaptleadn tognr othwet hraidnihclieb iatonrdy heyffpeoctcsotoyfl He.losnabgdaatiroifnfa ocfa llyettxuccreu d(Feiegxutrrea c2t)s. wTheere sipnevceifistci -
gaactetidviotyn (tEhCe5r0)a odfi ctlheea cnruddhey epxotrcaoctty flreolmon tghaet cioanlyox folfe Httu. scaebd(Farigiffuar we 2as). eTsthime sapteedc iffiocr aracdtiivciltey 
(EaCnd h) yopfothcoetycrl utod beee 8x7tr7a pctpmfro amndt h1e88c0a lpypxmo,f rHes.pseacbtdivaerilfyfa. Twhaes aepsptliimcaattieodn ofof 4000 ppm 50 r radicle anodf 
htyhpeo ccroutdyel  tcoalybxe e8x7t7rapcpt mresaunltded1 8in8 0thpep hmig,hreesstp inechtiibvietiloyn.  T(9h6e.5a%p)p olfi claetttiuonceo rfad40ic0l0e gprpomwtohf 
th(ie.ec.,r uledtteuccael yraxdeixclter aecltornegsautlitoend oinf 3t.h5e%h).i gPhreesvtioinuhs isbtiutidoines( 9s6h.o5w%e)do fthleattt uthcee lreaadf,i cslteemgr,o awntdh 
(ir.eo.o, tl eottf uHce. sraabddiacrlieffeal ohnagda itniohniboitfo3r.y5 %ac)t.iPvirteyv ioonu sthset ugdrioews tshh oowf etedstt hpaltatnht eslpeeacfi,esst e[m38,,3a9n]d. 
roHootwofeHve.rs,a bthdearrief faish ando isnthuidbyit oorny athctei vpitlyanotn gthroewgtrho winthhiobfittoersyt  pelffanect ts poefc itehse [3c8a,l3y9x] .oHf oHw. -
evsaebrd, athrieffrae. iTshnios ssttuuddyy oansstehsesepdl atnhteg irnoflwutehncineh oibf iHto. rsyabedffaercifft ao fcathlyexc aolny xploafnHt .gsraobwdatrhi fffao.r Tthheis 
stfiurdsty taimssee.s sed the influence of H. sabdariffa calyx on plant growth for the first time.
 
FiFgiguurere2 2. .E Effffeecctstso offw waatteerr ccrruuddee eexxttrraacctt ffrroomm HH.. ssaabbddaarriiffffaa ccaallyyxx ((HHssCCrruuddee)),, hhyyddrrooxxyycciittrriicc aaccidid (H(HCCAA),) ,
and hydroxycitric acid lactone (HCAL) on the radicle (a) and hypocotyl (b) growth of lettuce 
ansedehdylidnrgosx. yTchiter idcaatcai darlea ctthoen me (eHanC ±A sLt)aonndathrde rdaedviicaletio(an), ann =d 3h.y pocotyl (b) growth of lettuce seedlings.
The data are the mean ± standard deviation, n = 3.
The specific activity (EC50) of H. sabdariffa calyx obtained in this study (877 ppm) was of
a lower value than many species in the existing studies. In other studies, the ethanolic crude
extracts of Rosmarinus officinalis [15] and of Phragmites communis Trin., and the methanolic
 crude extracts of Gliricidia sepium (Jacq.) Kunth, Pachysandra terminalis Siebold and Zucc.,
Samanea saman (Jacq.) Merr, Brachiaria brizantha (A. Rich.) Stapf, and Tamarindus indica L.,
demonstrated lower inhibitory effects (EC50 values of 1280, 2130, 1780, 1920, 2200, 2500,
and 2510 ppm, respectively) [15,54–56]. The species containing inhibitory substances could
be evaluated by their specific activity, and such species ought to have small EC50 values
for their utilization in weed control [13,15,48]. H. sabdariffa calyx could be assessed as a
prospective candidate for this purpose.
Agronomy 2023, 13, x FOR PEER REVIEW 6 of 12 
 
The specific activity (EC50) of H. sabdariffa calyx obtained in this study (877 ppm) was 
of a lower value than many species in the existing studies. In other studies, the ethanolic 
crude extracts of Rosmarinus officinalis [15] and of Phragmites communis Trin., and the 
methanolic crude extracts of Gliricidia sepium (Jacq.) Kunth, Pachysandra terminalis Siebold 
and Zucc., Samanea saman (Jacq.) Merr, Brachiaria brizantha (A. Rich.) Stapf, and Tamarindus 
indica L., demonstrated lower inhibitory effects (EC50 values of 1280, 2130, 1780, 1920, 2200, 
2500, and 2510 ppm, respectively) [15,54–56]. The species containing inhibitory substances 
could be evaluated by their specific activity, and such species ought to have small EC50 
values for their utilization in weed control [13,15,48]. H. sabdariffa calyx could be assessed 
Agronomy 2023, 13, 1746 as a prospective candidate for this purpose. 6 of 11
3.2. The Content of Pure Compounds in Hibiscus sabdariffa Calyx 
3.2H. TyhderCoxonytceintrtico faPcuidr eaCnodm hpyodunrodxsyinciHtriicb iascuids  slabcdtoarnieff awCearley xfound in the crude extracts 
of the eHxaymdrionxedyc citarliycxa ocifd Ha. nsdabhdayrdiffroa.x HycPitLrCic cahcridomlaacttoognreamwes roef fsotuannddairndt hpeurcer ucdheemexictaralsc ts
anodf tHhe. esxaabdmairniffead ccaallyyxx owf Hat.esra bcdraurdifefa .eHxtPraLcCt cwhirtohm aa toregtreanmtisonof stitmaned aorfd 1p0u.5r emchinem fiocra ls
hyadnrdoxHy.cistarbicd aarciifdfa (caa) laynxdw 1a4t.e0r mcriun dfoere hxytrdarcotxwycitihtriac raectiedn ltaiocntotniem (eb)o afr1e0 s.5homwinn fionr Fhigyudrreo x-
3. yTchiter icreascuildts( ao)fa tnhde 1H4.P0LmCi nanfoarlyhsyisd roofx Hyc. itsraibcdaacriiffdal accatloynxe w(ba)tearr eesxhtroawctn cionnFfiirgmureed3 t.hTeh e
prreesseunlctes ooff thhyedHroPxLyCcitarnica laycsiids aonf dH .hsyadbrdoaxriyffcaitcraicly axciwda ltaecrtoenxter ainct tchoen cfiarlmyxe, dFitghuerper 3ecs.e nTchee of
cohnycednrotrxaytcioitnri ocfa hciyddarnodxyhcyitdrirco xaycicdit rlaicctaocnide lwacatso nesetiimn tahteedc aalty 1x6, 7F imgugr eg−31 cd. rTyh we ecoignhcte noftr tahteio n
caolyfxh y(d16ro.7x%yc),i trwichaicleid tlhaactt onofe whyads reosxtiymcaittreidc aatc1id6 7wmags g9−11.5d rmy wg egig−1h tDoWf t h(e9c.1a5ly%x).( 1T6.h7e% ),
hywdhroilxeytchitartico fahciydd rloaxctyocniter icwaacsi dmwoarse 9a1b.5unmdgangt− 1inD Wthe( 9c.1a5ly%x) . oTf hHe .h ysdabrdoaxryiffcait ritchaanc id
hyldacrotoxnyeciwtraics amciodr.e Saimbuilnadrlayn, pt rinevtihoeucs asltyuxdioefsH h.avsaeb adlasroi frfeapthoratnedh yhdydrorxoyxcyictirtircica accidid.  Slaicmtoilnaer ly,
[35p,r5e7v,5io8u] sansdtu hdyiedsrohxayvceitarlisco arceipdo [r3t5e,d57h,5y9d]r aosx ythceit rmicaajocri dorlagcatnoinc eac[3id5s,5 i7n,5 t8h]ea cnadlyhxy edxrtoraxcytc oitfr ic
H.a scaibdda[3ri5ff,5a.7 ,59] as the major organic acids in the calyx extract of H. sabdariffa.
 
(a) 
 
(b) 
 
(c) 
FigFuigreu r3e. 3H. PHLPCL Cchcrhormomataotgorgarmams s(a()a )hhyyddroroxxyyccitirtricic aacciidd;; ((bb)) hhyyddrrooxxyycciittrriicc aacciidd lalaccttoonnee;;a anndd( c()c)H H. s.a b-
sabddaarriiffffaac caallyyxxe exxttrraacctt.. 
 3 .3. Plant Growth Inhibitory Effects of Pure Compounds Present in H. sabdariffa Calyx
Hydroxycitric acid and hydroxycitric acid lactone were investigated for their plant
growth inhibitory effect on lettuce. The examined compounds revealed different degrees of
 inhibitions on the lettuce radicle and hypocotyl elongation (Figure 2). The lettuce radicle
elongation was inhibited more than hypocotyl elongation for both compounds examined in
this study. Similar to the results of this study, some other reported allelochemicals had very
minimal or no influence on the hypocotyl growth of lettuce seedlings [15,26,46]. Previous
studies showed that the isolated chemicals (trimethyl allo-hydroxycitrate and β-sitosterol)
from the leaf, stem, and root of H. sabdariffa had inhibitory activity on the growth of test
plant species [41,42]. However, the allelopathy of the calyx’s substances had not been
studied before. Current research assessed the worth of the influence of H. sabdariffa calyx’s
characteristic compounds (hydroxycitric acid lactone and hydroxycitric acid) on the plant
growth for the first time.
Agronomy 2023, 13, 1746 7 of 11
The specific activity (EC50) on lettuce radicle elongation was estimated at 1730 ppm
for hydroxycitric acid and 73.7 ppm for hydroxycitric acid lactone, whereas EC50 on lettuce
hypocotyl elongation of hydroxy citric acid lactone was about ≈36-fold lower than that
of hydroxycitric acid. Moreover, the hormesis effect can explain a low-dose stimulatory
effect, and a high-dose inhibitory effect of hydroxycitric acid on the hypocotyl growth
(Figure 2b). Calabrese et al. [60] described this kind of dose-response relationship formerly.
Hydroxycitric acid lactone demonstrated a stronger inhibitory effect on the L. sativa seedling
growth than hydroxycitric acid had. Current research is reporting for the first time that
hydroxycitric acid lactone could be an important plant growth inhibitor from the calyx
of H. sabdariffa.
This study showed that hydroxycitric acid lactone, a saturated lactone form, is a
stronger plant growth inhibitor than its non-lactone form, hydroxy citric acid. Previous
studies have also shown that lactone-form compounds of some species have plant-growth
inhibitory properties. Some bioactive natural chemicals, such as coumarin [61,62], um-
belliferone [63,64], esculetin [14], scopoletin [65,66], patulin [67,68], psoralen [69,70], and
angelicin [49], are α-β unsaturated lactones and have allelopathic potentials. Many unsat-
urated lactones are potent antimicrobial compounds. It was presumed that unsaturated
lactone antibiotics may prevent enzyme activity by uniting with SH and possibly amino
groups of enzyme proteins. This could be a basic mechanism of all unsaturated lactone
inhibitors as well as coumarins and protoanemonin [71].
Moreover, sesquiterpene lactones and hydroxycitric acid lactone have the same lactone
ring structure, which is in saturated form. Sesquiterpene lactones are broadly distributed
in plants and the potential phytotoxicity [72] of dehydrocostus lactone [73,74], inulox-
ins A–D [75], artemisinin [76], and cnicin [77] has been reported. Momilactone [78] and
acremolactone A, B, C [79,80] are other natural-origin compounds that demonstrated plant
growth inhibitory effects on target plants in previous studies. Considering all the above
presented, it could be thought that the lactone units of the compounds could be the core
functional group required for biological activity such as the plant growth inhibitory effects
of hydroxycitric acid lactone and the above-mentioned natural compounds.
Other organic acids such as citric acid, malic acid, and ascorbic acid were reported in
the calyx of H. sabdariffa, aside from hydroxycitric acid lactone and hydroxycitric acid [31].
However, these organic acids have not shown the plant growth inhibitory effect; rather,
they have revealed the plant growth improving effect on the target plants in several
studies [52,81–84]. The application of citric acid has enhanced physiological parameters
in numerous plant species such as Polianthes tuberosa, Lilium spp., and Phaseolus vulgaris
(ordinary bean) [81] and had a growth stimulatory effect on L. sativa, for example [52].
Treatment with malic acid has significantly enhanced the growth of Miscanthus sacchariflorus
under cadmium stress [82]. The foliar application of ascorbic acid has improved the shoot
fresh and dry weights of cucumber [83] and early spring maize seedling establishment [84]
in previous studies. Hence, the low EC50 of hydroxycitric acid lactone not only indicates
the plant growth inhibitory effect of this compound but also further shows the influence
and power of the compound for the plant growth inhibitory effect of calyx.
3.4. Estimation of Contributions of the Compounds to the Growth Inhibitory Activity of
H. sabdariffa Calyx
The contribution of the inhibitory effects of hydroxycitric acid and hydroxycitric
acid lactone in the crude extracts of H. sabdariffa calyx on the radicle elongation of lettuce
seedlings was weighed up by the total activity approach. This approach was used to
estimate which of the two compounds of H. sabdariffa calyx had a stronger contribution to
the growth inhibitory activity of H. sabdariffa calyx on the radicle elongation of L. sativa.
The total activity approach of evaluating the contribution of a plant growth inhibitor
compound is based on the concentration and inhibitory effect (specific activity, EC50)
of the compound [45]. The inhibitory effect of H. sabdariffa calyx extract on the radicle
growth of lettuce could be described by the existence of hydroxycitric acid lactone in
Agronomy 2023, 13, 1746 8 of 11
the extract (Figure 2). The total activity of hydroxycitric acid lactone and hydroxycitric
acid was approximated at 2270 and 53.0, respectively. Thus, the plant growth inhibitory
effect of hydroxycitric acid lactone was about ≈43-fold stronger than the inhibitory effect
of hydroxycitric acid. One of the fundamental findings of this study is that the high
concentration of hydroxycitric acid lactone in H. sabdariffa calyx coupled with low EC50
further shows the influence and dominance of the compound in the plant growth inhibition
recognized. The inhibitory effect of H. sabdariffa calyx on both the radicle and hypocotyl
growth of lettuce could be described by hydroxycitric acid lactone in the extract (Figure 2).
The total activity (TA) of hydroxycitric acid lactone in this study is 2270, and this value
is relatively high when compared to many other plant growth inhibitory compounds in the
existing literature. Based on the total activity evaluation, hydroxycitric acid lactone showed
higher inhibitory effects than juglone (TA: 2000) [85], coumarin (TA: 2000) [86], L-DOPA
(TA: 250) [46], indigo (TA: 58) [26], and cyanamide (TA: 40) [47]. However, goniothalamin
(TA: 3600) [87] had a higher total activity value than hydroxycitric acid lactone.
4. Conclusions
H. sabdariffa calyx demonstrated a strong plant growth inhibitory effect on lettuce
growth elongation in the initial screening. The inhibitory effect of the calyx and its sub-
stances had not been studied before. The result showed that the calyx of H. sabdariffa
had plant growth inhibitory potential, and this is the first report. The crude extract from
the calyx of H. sabdariffa and the pure hydroxycitric acid lactone inhibited lettuce radicle
elongation by 50% at 877 ppm and 73.7 ppm, respectively. The inhibitory contribution of
hydroxycitric acid lactone to the effect of H. sabdariffa calyx extract was estimated (using
the total activity approach) to be higher than hydroxycitric acid showed. This is the first
report of hydroxycitric acid lactone as a plant growth inhibitor from H. sabdariffa calyxes.
Future studies should focus on evaluating the herbicidal potential of H. sabdariffa calyx and
hydroxycitric acid lactone under both greenhouse and field conditions.
Author Contributions: Conceptualization, T.G.I.O. and Y.F.; methodology, T.G.I.O., K.S.A. and
Y.F.; software, Microsoft Office 2016; validation, T.G.I.O., E.A. and Y.F.; formal analysis, T.G.I.O.;
investigation, T.G.I.O.; resources, T.G.I.O. and Y.F.; data curation, T.G.I.O. and Y.F.; writing—original
draft preparation, T.G.I.O.; writing—review and editing, T.G.I.O., K.S.A. and Y.F.; visualization,
T.G.I.O. and K.S.A.; supervision, Y.F.; project administration, T.G.I.O. and Y.F.; and funding acquisition,
Y.F. All authors have read and agreed to the published version of the manuscript.
Funding: This research was partly supported by JST CREST Grant Number JPMJCR1702 and JSPS
KAKENHI Grant Number 26304024.
Data Availability Statement: Not applicable. Data related to this publication is shared in the manuscript.
Acknowledgments: We would like to thank Kohinoor Begum for her technical support during
the experiments. We also thank our other colleagues and laboratory members of the International
Environmental and Agricultural Sciences, Tokyo University of Agriculture and Technology.
Conflicts of Interest: The authors declare no conflict of interest.
References
1. Oerke, E.-C.; Dehne, H.-W. Safeguarding production—Losses in major crops and the role of crop protection. Crop Prot. 2004, 23,
275–285. [CrossRef]
2. Oerke, E.-C. Crop losses to pests. J. Agric. Sci. 2006, 144, 31–43. [CrossRef]
3. Appleby, A.P.; Muller, F.; Carpy, S. Weed control. In Agrochemicals; Muller, F., Ed.; Wiley: New York, NY, USA, 2000; pp. 687–707.
4. Kraehmer, H.; Baur, P. Weed Anatomy; Wiley-Blackwell: Chichester, UK, 2013.
5. Scavo, A.; Mauromicale, G. Integrated Weed Management in Herbaceous Field Crops. Agronomy 2020, 10, 466. [CrossRef]
6. Gawronska, H.; Ciarka, D.; Bernat, W.; Gawronski, S.W. Sunflower-desired Allelopathic Crop for Sustainable and Organic
Agriculture. In Allelopathy New Concept and Methodology; Fujii, Y., Hiradate, S., Eds.; Science Publishers: Enfield, CT, USA, 2007;
pp. 185–210.
7. Scavo, A.; Mauromicale, G. Crop Allelopathy for Sustainable Weed Management in Agroecosystems: Knowing the Present with a
View to the Future. Agronomy 2021, 11, 2104. [CrossRef]
Agronomy 2023, 13, 1746 9 of 11
8. Duke, S.O. Why have no new herbicide modes of action appeared in recent years? Pest Manag. Sci. 2012, 68, 505–512. [CrossRef]
9. Cárdenas, D.M.; Bajsa-Hirschel, J.; Cantrell, C.L.; Rial, C.; Varela, R.M.; Molinillo, J.M.G.; Macías, F.A. Evaluation of the phytotoxic
and antifungal activity of C17-sesquiterpenoids as potential biopesticides. Pest Manag. Sci. 2022, 10, 4240–4251. [CrossRef]
10. Vencill, W.; Nichols, R.; Webster, T.; Soteres, J.; Mallory-Smith, C.; Burgos, N.; Johnson, W.G.; McClelland, M. Herbicide Resistance:
Toward an Understanding of Resistance Development and the Impact of Herbicide-Resistant Crops. Weed Sci. 2012, 60, 2–30.
[CrossRef]
11. Fujii, Y.; Parvez, S.S.; Parvez, M.M.; Ohmae, Y.; Iida, O. Screening of 239 medicinal plant species for allelopathic activity using the
sandwich method. Weed Biol. Manag. 2003, 3, 233–241. [CrossRef]
12. Iqbal, Z.; Nasir, H.; Fujii, Y. Allelopathic Activity of Buckwheat: A ground Cover Crop for Weed Control. In Allelopathy New
Concept and Methodology; Fujii, Y., Hiradate, S., Eds.; Science Publishers: Enfield, CT, USA, 2007; pp. 173–183.
13. Golisz, A.; Lata, B.; Gawronski, S.W.; Fujii, Y. Specific and total activities of the allelochemicals identified in buckwheat. Weed Biol.
Manag. 2007, 7, 164–171. [CrossRef]
14. Haig, T. Allelochemicals in Plants. In Allelopathy in Sustainable Agriculture and Forestry; Zeng, R.S., Mallik, A.U., Luo, S.M., Eds.;
Springer: New York, NY, USA, 2008; pp. 63–104.
15. Appiah, K.S.; Mardani, H.K.; Omari, R.A.; Eziah, V.Y.; Ofosu-Anim, J.; Onwona-Agyeman, S.; Amoatey, C.A.; Kawada, K.;
Katsura, K.; Oikawa, Y.; et al. Involvement of Carnosic Acid in the Phytotoxicity of Rosmarinus officinalis Leaves. Toxins 2018,
10, 498. [CrossRef]
16. Vyvyan, J.R. Allelochemicals as Leads for New Herbicides and Agrochemicals. Tetrahedron 2002, 58, 1631–1646. [CrossRef]
17. Barney, J.N.; Hay, A.G.; Weston, L.A. Isolation and Characterization of Allelopathic Volatiles from Mugwort (Artemisia vulgaris).
J. Chem. Ecol. 2005, 31, 247–265. [CrossRef] [PubMed]
18. Weston, L.A.; Duke, S.O. Weed and Crop Allelopathy. CRC Crit. Rev. Plant Sci. 2003, 22, 367–389. [CrossRef]
19. Soltys, D.; Krasuska, U.; Bogatek, R.; Gniazdowsk, A. Allelochemicals as Bioherbicides—Present and Perspectives. In Herbicides:
Current Research and Case Studies in Use; Price, A.J., Kelton, J.A., Eds.; IntechOpen: London, UK, 2013; pp. 444–466. [CrossRef]
20. Scavo, A.; Pandino, G.; Restuccia, A.; Mauromicale, G. Leaf extracts of cultivated cardoon as potential bioherbicide. Sci. Hortic.
2020, 261, 109024. [CrossRef]
21. Duke, S.O.; Dayan, F.E. Modes of action of microbially-produced phytotoxins. Toxins 2011, 3, 1038–1064. [CrossRef] [PubMed]
22. Duke, S.O.; Dayan, F.E.; Romagni, J.G.; Rimando, A.M. Natural products as sources for new mechanisms of herbicidal action.
Crop Prot. 2000, 40, 99–111. [CrossRef]
23. Cimmino, A.; Masi, M.; Evidente, M.; Superchi, S.; Evidente, A. Fungal phytotoxins with potential herbicidal activity: Chemical
and biological characterization. Nat. Prod. Rep. 2015, 60, 1629–1653. [CrossRef] [PubMed]
24. Macías, F.A.; Molinillo, J.M.G.; Galindo, J.C.G.; Varela, R.M.; Simonet, A.M.; Castellano, D. The Use of Allelopathic Studies in the
Search for Natural Herbicides. J. Crop Prod. 2001, 4, 237–255. [CrossRef]
25. Scavo, A.; Abbate, C.; Mauromicale, G. Plant allelochemicals: Agronomic, nutritional and ecological relevance in the soil system.
Plant Soil 2019, 442, 23–48. [CrossRef]
26. Begum, K.; Motobayashi, T.; Hasan, N.; Appiah, K.S.; Shammi, M.; Fujii, Y. Indigo as a Plant Growth Inhibitory Chemical from
the Fruit Pulp of Couroupita guianensis Aubl. Agronomy 2020, 10, 1388. [CrossRef]
27. Wu, H.; Pratley, J.; Lemerle, D.; Haig, T.; An, M. Screening method for the evaluation of crop allelopathic potential. Bot. Rev. 2001,
67, 403–415. [CrossRef]
28. Scavo, A.; Pandino, G.; Restuccia, A.; Lombardo, S.; Pesce, G.R.; Mauromicale, G. Allelopathic potential of leaf aqueous extracts
from Cynara cardunculus L. on the seedling growth of two cosmopolitan weed species. Ital. J. Agron. 2019, 14, 78–83. [CrossRef]
29. Bhadoria, P. Allelopathy: A Natural Way towards Weed Management. Am. J. Exp. Agric. 2011, 1, 7–20. [CrossRef]
30. Appiah, K.S.; Li, Z.; Zeng, R.S.; Luo, S.; Oikawa, Y.; Fujii, Y. Determination of allelopathic potentials in plant species in
Sino-Japanese floristic region by sandwich method and dish pack method. Int. J. Basic Appl. Sci. 2015, 4, 381–394. [CrossRef]
31. Da-Costa-Rocha, I.; Bonnlaender, B.; Sievers, H.; Pischel, I.; Heinrich, M. Hibiscus sabdariffa L.—A phytochemical and pharmaco-
logical review. Food Chem. 2014, 165, 423–443. [CrossRef]
32. Isin Ozkan, T.G.; Akalin Urusak, E.; Appiah, K.S.; Fujii, Y. First Broad Screening of Allelopathic Potential of Wild and Cultivated
Plants in Turkey. Plants 2019, 8, 532. [CrossRef]
33. Zarrabal, O.; Barradas-Dermitz, D.M.; Orta-Flores, Z.; Hayward-Jones, P.M.; Nolasco-Hipólito, C.N.; Aguilar-Uscanga, M.G.;
Miranda-Medina, A.; Bujang, K.B. Hibiscus sabdariffa L., roselle calyx, from ethnobotany to pharmacology. J. Exp. Pharmacol. 2012,
4, 25–39. [CrossRef]
34. Hida, H.; Yamada, T.; Yamada, Y. Absolute Configuration of Hydroxycitric Acid Produced by Microorganisms. Biosci. Biotechnol.
Biochem. 2006, 70, 1972–1974. [CrossRef]
35. Yamada, T.; Hida, H.; Yamada, Y. Chemistry, physiological properties, and microbial production of hydroxycitric acid. Appl.
Microbiol. Biotechnol. 2007, 75, 977–982. [CrossRef]
36. Zheoat, A.M.; Gray, A.I.; Igoli, J.O.; Kennedy, A.R.; Ferro, V.A. Crystal structures of hibiscus acid and hibiscus acid dimethyl ester
isolated from Hibiscus sabdariffa (Malvaceae). Acta Cryst. 2017, 73, 1368–1371. [CrossRef]
37. Hida, H.; Yamada, T.; Yamada, Y. Genome shuffling of Streptomyces sp. U121 for improved production of hydroxycitric acid. Appl.
Microbiol. Biotechnol. 2007, 73, 1387–1393. [CrossRef] [PubMed]
Agronomy 2023, 13, 1746 10 of 11
38. Piyatida, P.; Kato-Noguchi, H. Screening of Allelopathic Activity of Eleven Thai Medicinal Plants on Seedling Growth of Five Test
Plant Species. Asian J. Plant Sci. 2010, 9, 486–491. [CrossRef]
39. Pukclai, P.; Kato-Noguchi, H. Evaluation of allelopathic activity of Hibiscus sabdariffa L. Adv. Biol. Res. 2011, 5, 366–372.
40. Suwitchayanon, P.; Pukclai, P.; Ohno, O.; Suenaga, K.; Kato-Noguchi, H. Isolation and identification of an allelopathic substance
from Hibiscus sabdariffa. Nat. Prod. Commun. 2015, 10, 765–766. [CrossRef] [PubMed]
41. Piyatida, P.; Kimura, P.; Sato, M.; Kato-Noguchi, H. Isolation of β-sitosterol from Hibiscus sabdariffa L. Allelopath. J. 2013, 32,
289–300.
42. Carvajal-Zarrabal, O.; Waliszewski, S.M.; Barradas-Dermitz, D.M.; Orta-Flores, Z.; Hayward-Jones, P.M.; Nolasco-Hipólito, C.;
Angulo-Guerrero, O.; Sánchez-Ricaño, R.; Infanzón, R.M.; Trujillo, P.R. The consumption of Hibiscus sabdariffa dried calyx ethanolic
extract reduced lipid profile in rats. Plant Foods Hum. Nutr. 2005, 60, 153–159. [CrossRef]
43. PubChem. Available online: https://pubchem.ncbi.nlm.nih.gov/compound/ALLO-2-hydroxycitric-acid (accessed on 23
June 2023).
44. PubChem. Available online: https://pubchem.ncbi.nlm.nih.gov/compound/6481826 (accessed on 23 June 2023).
45. Hiradate, S.; Ohse, K.; Furunayashi, A.; Fujii, Y. Quantitative evaluation of allelopathic potentials in soils: Total activity approach.
Weed Sci. 2010, 58, 258–264. [CrossRef]
46. Fujii, Y.; Shibuya, T.; Yasuda, T. Allelopathy of mucuna: Its discrimination and identification of L-DOPA as a candidate of
allelopathic substance. Jpn. Agric. Res. Q. 1992, 25, 238–247.
47. Kamo, T.; Hiradate, S.; Fujii, Y. First Isolation of Natural Cyanamide as a Possible Allelochemical from Hairy Vetch Vicia villosa.
J. Chem. Ecol. 2003, 29, 275–283. [CrossRef]
48. Hiradate, S.; Morita, S.; Sugie, H.; Fujii, Y.; Harada, J. Phytotoxic Cis-Cinnamoyl Glucosides from Spiraea thunbergii. Phytochemistry
2004, 65, 731–739. [CrossRef]
49. Mishyna, M.; Laman, N.; Prokhorov, V.; Fujii, Y. Angelicin as the Principal Allelochemical in Heracleum sosnowskyi Fruit. Nat. Prod.
Commun. 2015, 10, 767–770. [CrossRef] [PubMed]
50. Hiradate, S.; Morita, S.; Furubayashi, A.; Fujii, Y.; Harada, J. Plant Growth Inhibition by Cis-Cinnamoyl Glucosides and
Cis-Cinnamic Acid. J. Chem. Ecol. 2005, 31, 591–601. [CrossRef] [PubMed]
51. Fujii, Y.; Hiradate, S. A Critical Survey of Allelochemicals in Action—The Importance of Total Activity and the Weed Suppression
Equation. In Establishing the Scientific Base; Harper, J.D.I., An, M., Wu, H., Kent, J.H., Eds.; Centre for Rural Social Research,
Charles Sturt University: Wagga Wagga, Australia, 2005; pp. 73–76.
52. Syed, S.; Ahmed, Z.I.; Al-haq, M.I.; Mohammad, A.; Fujii, Y. The possible role of organic acids as allelochemicals in Tamarindus
indica L. leaves. Acta Agric. Scand. 2014, 64, 511–517. [CrossRef]
53. Hiradate, S. Isolation strategies for finding bioactive compounds: Specific activity vs. total activity. In Natural Products for Pest
Management; Rimando, A.M., Duke, S.O., Eds.; American Chemical Society: Washington, DC, USA, 2006; pp. 113–126.
54. Takemura, T.; Sakuno, E.; Kamo, T.; Hiradate, S.; Fujii, Y. Screening of the Growth-Inhibitory Effects of 168 Plant Species against
Lettuce Seedlings. Am. J. Plant Sci. 2013, 4, 1095–1104. [CrossRef]
55. Li, F.; Hu, H. Isolation and Characterization of a Novel Antialgal Allelochemical from Phragmites communis. J. Appl. Environ.
Microbiol. 2005, 71, 6545–6553. [CrossRef]
56. Kobayashi, A.; Kato-Noguchi, H. The Seasonal Variations of Allelopathic Activity and Allelopathic Substances in Brachiaria
brizantha. Bot. Stud. 2015, 56, 25. [CrossRef]
57. Rodríguez-Medina, I.C.; Beltrán-Debón, R.; Molina, V.M.; Alonso-Villaverde, C.; Joven, J.; Menéndez, J.A.; Fernández-Gutiérrez, A.
Direct characterization of aqueous extract of Hibiscus sabdariffa using HPLC with diode array detection coupled to ESI and ion
trap MS. J. Sep. Sci. 2009, 32, 3441–3448. [CrossRef]
58. Ramirez-Rodrigues, M.M.; Plaza, M.L.; Azeredo, A.; Balaban, M.O.; Marshall, M.R. Physicochemical and phytochemical properties
of cold and hot water extraction from Hibiscus sabdariffa. J. Food Sci. 2011, 76, C428–C435. [CrossRef]
59. Beltrán-Debón, R.; Alonso-Villaverde, C.; Aragones, G.; Rodriguez-Medina, I.; Rull, A.; Micol, V.; Segura-Carretero, A.; Fernández-
Gutiérrez, A.; Camps, J.; Joven, J. The aqueous extract of Hibiscus sabdariffa calices modulates the production of monocyte
chemoattractant protein-1 in humans. Phytomedicine 2010, 17, 186–191. [CrossRef]
60. Calabrese, E.J.; Bachmann, K.A.; Bailer, A.J.; Bolger, P.M.; Borak, J.; Cai, L.; Cedergreen, N.; Cherian, M.G.; Chiueh, C.C.;
Clarkson, T.W.; et al. Biological stress response terminology: Integrating the concepts of adaptive response and preconditioning
stress within a hormetic dose–response framework. Toxicol. Appl. Pharmacol. 2007, 222, 122–128. [CrossRef]
61. Haig, T.J.; Seal, A.N.; Pratley, J.E.; An, M.; Wu, H. Lavender as a source of novel plant compounds for the development of a
natural herbicide. J. Chem. Ecol. 2009, 35, 1129–1136. [CrossRef] [PubMed]
62. Wu, C.; Zhao, G.; Liu, D.; Liu, S.; Gun, X.; Tang, Q. Discovery and Weed Inhibition Effects of Coumarin as the Predominant
Allelochemical of Yellow Sweet clover (Melilotus officinalis). Int. J. Agric. Biol. 2016, 18, 168–175. [CrossRef]
63. Real, M.; Gamiz, B.; Lopez-Cabeza, R.; Celis, R. Sorption, persistence, and leaching of the allelochemical umbelliferone in soils
treated with nanoengineered sorbents. Sci. Rep. 2019, 9, 9764. [CrossRef]
64. Morikawa, C.I.O.; Miyaura, R.; Kamo, T.; Hiradate, S.; Chávez Pérez, J.A.; Fujii, Y. Isolation of Umbelliferone as a Principal
Allelochemical from the Peruvian Medicinal Plant Diplostephium foliosissimum (Asteraceae). Rev. Soc. Quim. Peru 2011, 77, 285–291.
65. Narwal, S.S. Allelochemicals. In Allelopathy in Crop Production; Kumar, P., Ed.; Scientific Publishers: Jodhpur, India, 2004; pp. 6–17,
ISBN 978-8172-337-77-3.
Agronomy 2023, 13, 1746 11 of 11
66. Fay, P.; Duke, W. An Assessment of Allelopathic Potential in Avena Germ Plasm. Weed Sci. 1977, 25, 224–228. [CrossRef]
67. Einhellig, F. Allelopathy-A Natural Protection, Allelochemicals. In CRC Handbook of Natural Pesticides: Methods Volume I: Theory,
Practice, and Detection; Mandava, N.B., Ed.; CRC Press: Boca Raton, FL, USA, 2017; pp. 161–200, ISBN 9781351072717.
68. Chen, J.; Wei, J.; Gao, J.M.; Ye, X.; Mcerlan, C.S.P.; Ma, Y. Allelopathic inhibitory effects of Penicillium griseofulvum produced patulin
on the seed germination of Orobanche cumana Wallr. and Phelipanche aegyptiaca Pers. Allelopath. J. 2017, 41, 65–80. [CrossRef]
69. Putnam, A.R. Weed Allelopathy. In Weed Physiology: Volume I: Reproduction and Ecophysiology; Duke, S.O., Ed.; Reissued 2018; CRC
Press: Boca Raton, FL, USA, 1985; Volume 1, pp. 131–156, ISBN 0-8493-6313-6.
70. Shilling, D.G.; Dusky, J.A.; Mossier, M.A.; Bewick, T.A. Allelopathic Potential of Celery Residues on Lettuce. J. Am. Soc. Hortic.
Sci. 1992, 117, 308–312. Available online: https://journals.ashs.org/jashs/downloadpdf/journals/jashs/117/2/article-p308.pdf
(accessed on 25 December 2022). [CrossRef]
71. Rice, E.L. Allelopathy, 2nd ed.; Academic Press, Inc.: Orlando, FL, USA, 1984; pp. 233, 268, 344, ISBN 978-0-12-587055-9.
72. Macias, F.A.; Fernandez, A.; Varela, R.M.; Molinillo, J.M.G.; Torres, A.; Alves, P.L.C.A. Sesquiterpene Lactones as Allelochemicals.
J. Nat. Prod. 2006, 69, 795–800. [CrossRef]
73. Toda, Y.; Shigemori, H.; Ueda, J.; Miyamoto, K. Isolation and identification of polar auxin transport inhibitors from Saussurea
costus and Atractylodes japonica. Acta Agrobot. 2017, 70, 1700. [CrossRef]
74. Toda, Y.; Okada, K.; Ueda, J.; Miyamoto, K. Dehydrocostus lactone, a naturally occurring polar auxin transport inhibitor, inhibits
epicotyl growth by interacting with auxin in etiolated Pisum sativum seedlings. Acta Agrobot. 2019, 72, 1779. [CrossRef]
75. Andolfi, A.; Zermane, N.; Cimmino, A.; Avolio, F.; Boari, A.; Vurro, M.; Evidente, A. Inuloxins A-D, phytotoxic bi-and tri-cyclic
sesquiterpene lactones produced by Inula viscosa: Potential for broomrapes and field dodder management. Phytochemistry 2013,
86, 112–120. [CrossRef] [PubMed]
76. Bai, H.; Wang, C.; Chen, J.; Peng, J.; Cao, Q. A novel sensitive electrochemical sensor based on in-situ polymerized molecularly
imprinted membranes at graphene modified electrode for artemisinin determination. Biosens. Bioelectron. 2015, 64, 352–358.
[CrossRef] [PubMed]
77. Schabes, F.I.; Sigstad, E.E. A calorimetric study of the allelopathic effect of cnicin isolated from Centaurea diffusa Lam. on the
germination of soybean (Glicine max) and radish (Raphanus sativus). Thermochim. Acta 2007, 458, 84–87. [CrossRef]
78. Xu, M.; Galhano, R.; Wiemann, P.; Bueno, E.; Tiernan, M.; Wu, W.; Chung, I.M.; Gershenzon, J.; Tudzynski, B.; Sesma, A.; et al.
Genetic evidence for natural product-mediated plant–plant allelopathy in rice (Oryza sativa). New Phytol. 2012, 193, 570–575.
[CrossRef] [PubMed]
79. Sassa, T.; Kinoshita, H.; Nukina, M.; Sugiyama, T. Acremolactone A, a novel herbicidal epoxydihydropyranyl γ-lactone from
Acremonium roseum I4267. J. Antibiot. 1998, 5, 967–969. [CrossRef] [PubMed]
80. Sassa, T.; Ooi, T.; Kinoshita, H. Isolation and Structures of Acremolactones B and C, Novel Plant-Growth Inhibitory γ-Lactones
from Acremonium roseum I4267. Biosci. Biotechnol. Biochem. 2004, 68, 2633–2636. [CrossRef] [PubMed]
81. Tahjib-Ul-Arif, M.; Zahan, M.I.; Karim, M.M.; Imran, S.; Hunter, C.T.; Islam, M.S.; Mia, M.A.; Hannan, M.A.; Rhaman, M.S.;
Hossain, M.A.; et al. Citric Acid-Mediated Abiotic Stress Tolerance in Plants. Int. J. Mol. Sci. 2021, 22, 7235. [CrossRef]
82. Guo, H.; Chen, H.; Hong, C.; Jiang, D.; Zheng, B. Exogenous malic acid alleviates cadmium toxicity in Miscanthus sacchariflorus
through enhancing photosynthetic capacity and restraining ROS accumulation. Ecotoxicol. Environ. Saf. 2017, 141, 119–128.
[CrossRef]
83. Naz, H.; Akram, N.A.; Ashraf, M. Impact of Ascorbic Acid on Growth and Some Physiological Attributes of Cucumber (Cucumber
sativus) Plants under Water-Deficit Conditions. Pak. J. Bot. 2016, 48, 877–883.
84. Ahmad, I.; Basra, S.M.A.; Afzal, I.; Farooq, M.; Wahid, A. Growth improvement in spring maize through exogenous application
of ascorbic acid, salicylic acid and hydrogen peroxide. Int. J. Agric. Biol. 2013, 15, 95–100.
85. Jung, K.; Fujii, Y.; Yoshizaki, S.; Kobori, H. Evaluation of total allelopathic of heartseed walnut (Juglans ailanthifolia Carr.) and its
potential to control black locust (Robinia pseudoacacia L.). Allelopath. J. 2010, 26, 243–253.
86. Yamamoto, Y.; Fujii, Y. Exudation of allelopathic compound from plant roots of sweet vernal grass (Anthoxanthum odoratum).
J. Weed Sci. Technol. 1997, 42, 31–35. [CrossRef]
87. Raihan, I.; Miyaura, R.; Baki, B.B.; Fujii, Y. Assessment of allelopathic potential of goniothalamin allelochemical from Malaysian
plant Goniothalamus andersonii J. Sinclair by sandwich method. Allelopath. J. 2019, 46, 25–40. [CrossRef]
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