Characterisation and properties of the inclusion complex of24-epibrassinolide with �-cyclodextrin

M.B.M. De Azevedo1,4,*, M.A.T. Zullo1, J.B. Alderete2, M.M.M. De Azevedo3, T.J.G.Salva1 and N. Durán3

1Phytochemistry Laboratory, Instituto Agronômico – IAC, P.O. Box 28, Campinas, CEP: 13001-970, SP,Brazil; 2Organic Chemistry Department, Universidad de Concepción, Concepción, Chile, ; 3BiologicalChemistry Laboratory, Instituto de Química, Universidade Estadual de Campinas – UNICAMP, P.O. Box6154, Campinas, CEP 13083-970, SP, Brazil; 4Current address: STQ – Scientia Tecnologia Química, CIETEC– Centro Incubador de Empresas Tecnológicas, Av. Prof. Lineu Prestes 2242, Cicade Universitária USP, CEP05508-000 São Paulo, SP, Brazil; *Author for correspondence (e-mail: mbmazevedo@uol.com.br; phone: +5511 30398372; fax: +55 11 38127093)

Received 12 October 2001; accepted in revised form 19 December 2001

Key words: �-cyclodextrin, Brassinosteroids, Inclusion complex formation, Rice lamina inclination assay, Ther-mal analysis, X-ray diffraction analysis

Abstract

This paper reports the first study of an inclusion complex of a brassinosteroid with �-cyclodextrin. The forma-tion of inclusion complexes between 24-epibrassinolide and �-cyclodextrin was confirmed by their physicochem-ical properties and the compounds were analysed by differential scanning calorimetry, powder X-ray diffraction,nuclear magnetic resonance spectrometry and scanning electron microscopy. Theoretical calculations using theMM+ HyperChem force field showed a preference for inclusion of the side chain of the epibrassinolide moleculeinto the �-cyclodextrin cavity to form a 1:1 inclusion complex, although complexes involving inclusion of thesteroidal nucleus also possess a favourable interaction energy. Rice lamina inclination assay, employing IAC-103and IAC-104 cultivars, showed an improved activity for the epibrassinolide-cyclodextrin complex compared tothe epibrassinolide itself. The results suggest that brassinosteroid complexation with cyclodextrins may enhancethe biological activity of these plant growth regulators.

Introduction

Brassinosteroids (BS) are natural products with plantgrowth promoting activity (Mandava 1988; Sakuraiand Fujioka 1993). These compounds occur at verylow concentrations in plants (Clouse and Sasse 1998;Cutler et al. 1991; Sasse 1997) and have several reg-ulatory activities on plant growth and development(Khripach et al. 1999). At the molecular level, BS en-hance photosynthesis and nucleic acid and proteinsynthesis (Braun and Wild 1984; Brutti et al. 2000;Kalinch et al. 1985; Mandava 1988; Sairam 1994;Vardhini and Rao 1999). They also regulate the genesencoding xyloglucan endotransglycosylase, an en-

zyme related to membrane permeability (Fujioka andSakurai 1997; Okii 1993).

Several reports on the agricultural use of BS andtheir analogues showed that they may increase agri-cultural productivity (Fujioka et al. 1998; Sairam1994; Sasse 1997) and increase plant resistance to bi-otic and abiotic stresses (Cutler et al. 1991; Ikekawaand Zhao 1991; Marquart and Adam 1991).

Natural BS are lipophylic compounds, character-ised by a 5�-cholestane moiety oxygenated at least atcarbons 3, 22 and 23. The most active BS are alsooxygenated at carbon 6, as a ketone or a lactone.

Cyclodextrins (CD) are toroidally cyclic oligomersconstituted of 6, 7 or 8 carbohydrate units and arenamed �-, �- and �-CD, respectively. These oligosac-

233Plant Growth Regulation 37: 233–240, 2002.© 2002 Kluwer Academic Publishers. Printed in the Netherlands.

charides are able to act as hosts in inclusion com-plexes for allowing the solubilisation of many organiccompounds in aqueous solutions (Connors 1997; Ra-jagopalan et al. 1986; Stella et al. 1999).

Recently, a comprehensive review on CD com-plexes described their structural properties, stabilities,and their potential use in drug formulation and deliv-ery (Connors 1997; Koehler et al. 1996; Stella andRajewski 1997). The interaction of CDs and steroidshave also been investigated (Ahmed 1998), such asthe complexation of homologous derivatives of ste-roid hormones (Alberts and Muller 1992) and theiraplication in the bioconversion of 17�-estradiol to4-hydroxyestradiol (Uden and Woerdenbag 1994).

The inclusion constants of a series of 18 steroidhormones in cyclodextrins were analyzed as a func-tion of structure by the Partial Least Squares (PLS)method (Marzona et al. 1992). Physicochemical prop-erties of compounds and physicochemical parametersof substituents were used to analyse the structuralfeatures.

Cyclodextrins were also used in the formulation ofinclusion complexes of plant growth regulators, likeindole- (Szejtli et al. 1989) and naphthaleneacetic(Brutti et al. 2000) acids, 1-triacontanol (Sawamoto2000), cinnamic acid (Hayashi et al. 1998) and ben-zamide (Gosset and Gauvrit 1992) herbicides, to ex-amine the potential of their effects in appplicationssuch as plant tissue cultures (Durzan and Ventimiglia2000) and germination of carrots (Huet and Jullien1992).

BS are only soluble in organic solvents and to in-crease their solubility in aqueous solutions and to pre-vent early drying in BS formulations, adjuvants, suchas long chain polyamines are used (Sawamoto 2000).

An entirely new approach for the formulation ofBS using cyclodextrin complexes will be described inthis paper.

Here we describe the inclusion of 24-epibrassino-lide in �-CD and tested its activity against 24-epi-brassinolide itself, using the rice lamina inclinationassay.

Materials and methods

Materials

24-Epibrassinolide (EBL) was a gift of Prof. Dr.Nobuo Ikekawa (Niigata College of Pharmacy).�-Cyclodextrin (�-CD) was purchased from Sigma

Chem. Co. (St. Louis, MO) and it was used withoutfurther purification. Seeds of the rice cultivars IAC-103 and IAC-104 were obtained at the InstitutoAgronômico (IAC), Campinas, Brazil.

Methods

Preparation of the inclusion complexThe inclusion complex was prepared with a 1:1 mo-lar ratio of EBL to �-CD. �-CD (113.5 mg, 0.1 mmol)was dissolved in 100 mL of acetone containing 48.0mg (0.1 mmol) of EBL. To obtain the solid inclusioncomplex, the solution was evaporated immediately af-ter its preparation under vacuum in a rotatory evapo-rator at 45 °C (De Azevedo et al. 2000).

Theoretical studiesA molecular mechanics study of the complex struc-tures between �-CD and EBL was performed usingthe MM+ force field and the HyperChem software.The EBL/�-CD interaction was evaluated by takinginto account the energy involved in this process(Zhou et al. 2000).

No cut-off was used and geometry optimisationwas carried out until the energy gradient was less than0.01 kcal mol−1 Å−1, for both isolated and complexstructures. Several orientations of EBL were consid-ered for the 1:1 complex. The optimised structures ofthe inclusion complex are given in the Figure 1 to-gether with the notation used to designate the differ-ent conformations of 1:1 inclusion complexes.

Thermal analysis studiesDifferential scanning calorimetry (DSC) curves wererecorded on a differential scanning calorimeter (DTA-1600, TA Instruments). Samples (of approximately 5mg) were heated in crimped aluminium pans at ascanning rate of 15 °C min−1 using dry nitrogen aspurge gas (40–50 mL min−1) (De Azevedo et al. 2000;Durán et al. 2000).

X-Ray diffractometryX-Ray powder diffraction patterns were recorded us-ing a 6000-XRD (Shimadzu X-ray diffractometer) un-der the following conditions: Cu anode material tar-get, Ni filter, voltage 40 kV, current 30 mA for thegenerator, scanning speed 2 °C min−1, and countrange 1000 CPS. The detector was a proportionalcounter with 1.7 kV detector voltage (De Azevedo etal. 2000; Durán et al. 2000).

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Morphological AnalysisThe morphological analyses of the EBL crystals,�-CD, the physical mixture and the solid inclusioncomplexes were determined by scanning electron mi-croscope (SEM XL 40; Philips, Eindhoven). The sam-ples were mounted on aluminium stubs (TAAB Lab-oratories Equipment, Berks) using double-side stickytab (TAAB Laboratories Equipment, Berks) and vac-uum coated with gold for 60 sec (Polaron E-500;Balzers Union, Liechtenstein).

Nuclear magnetic resonance studies (1H-NMR)1H-NMR spectra were recorded on a Varian INOVA-500 spectrometer operating at 500 MHz. Sampleswere dissolved in deuterated dimethyl sulphoxide

(DMSO-d6, 30 mM), and tetramethyl silane was usedas internal standard.

Rice Lamina Inclination BioassayRice lamina inclination bioassays (Wada et al. 1984)were carried out using seedlings of IAC-103 andIAC-104 cultivars. Test solutions were prepared atconcentrations of 0.0, 1.0, 10−1, 10−2, 10−3 and 10−4

mg EBL L−1 from stock solutions of EBL and EBL/�-CD at 0.5 mg EBL mL−1 of ethanol:water 1:1.

Results and discussion

Theoretical studies

The formation of EBL/�-CD inclusion complexes canbe represented by the equation:

EBL � � � CD → EBL/� � CD

and the energy change associated with the complexformation is given by the relation:

�EC � E�complex� � �E�EBL� � E�� � CD��

where E(complex), E(EBL) and E(�-CD) are the to-tal energies of the complex, of the isolated guest(EBL) and of the isolated host (�-CD), respectively.

A negative value for DEC indicate that a stable in-clusion complex can be formed between EBL and�-CD. Table 1 shows the molecular mechanic valuesobtained for the inclusion of EBL into the �-CD cav-ity. Four different 1:1 complexes were studied (Fig-ure 1B–E). All these complexes have a negative �EC

value, which indicates that their formation is possible.Both partial (complex EBL-C3, Figure 1D and com-plex EBL-C4, Figure 1E) and total ring inclusion(complex EBL-C1, Figure 1B) present negative val-ues for the interaction energy. However, the forma-tion of the complex EBL-C2 (Figure 1C), which in-volves the inclusion of the EBL side chain into the�-CD cavity, is favoured by approximately 4kcal mol−1 over the most stable complex involvinginclusion of the steroidal nucleus into the �-CD cav-ity. Although a total inclusion of the rings A and Bleads to a less stable conformation (Figure 1B), thisconformation also posses a favourable interaction en-ergy. The results show that an inclusion complex of1:2 stoichiometry (complex EBL-CC1) is alsoposible, i.e., the EBL can complex with two �-CD

Figure 1. Theoretical calculation by Hyperchem force field forEBL alone (A), EBL-C1 (B); EBL-C2 (C), the lowest energy ar-rangement, EBL-C3 (D), EBL-C4 (E) and EBL-CC1 (F) inclusioncomplexes between EBL and �-CD. Bold structures in the com-plexes refer to EBL, whose structural formula is presented in G.

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molecules, in which both rings and acyclic parts areincluded into �-CD cavities.

Solid state studies

The DSC curves revealed some information on thesolid state interations of EBL with �-CD (Figure 2).The EBL crystals showed the characteristic endother-mic peak at 250.6 °C, corresponding to the 24-epi-brassinolide melting point (Figure 2A). The DSC pro-file of �-CD exhibited a broad endothermic peak(between about 100 °C and 120 °C) which was attrib-uted to the release of water molecules entrapped in-side the cavity (Figure 2C). The DSC thermogram ofEBL/�-CD complex shows that the steroid meltingendotherm appeared with a substantial reduction inpeak area in case of co-evaporated products (Figure2B). This implies that the molecular arrangement ofthe brassinosteroid in the solid complex is differentfrom the pure compound in their own crystal. Theendothermic peak attributed to the host molecule wasslightly distorted. This may be due to exchange ofwater molecules entrapped inside the CD cavity withthe steroid molecules as a result of complex forma-tion (De Azevedo et al. 2000; Durán et al. 2000).

Figures 3A and 3B show the powder X-ray diffrac-tion pattern of the EBL and co-evaporates of EBL/�-CD in 1:1 molar ratio, respectively. The diffractionpattern of the 1:1 physical mixture (not shown) wassimply a superposition of those of both components,corresponding to EBL (Figure 3A) and �-CD (Figure3C). The diffraction pattern of the co-evaporate indi-cated crystalline properties with two new crystallinepeaks at 2� = 11.5 ° and 24 °, as it can be observedin Figure 3B. The diffraction pattern of the EBL

shown in Figure 3A was a neat crystalline peak at 2�= 8 °, which was not present in the 1:1 molar ratiomixture of the co-evaporate (De Azevedo et al. 2000;Durán et al. 2000).

The interferences in X-ray diffraction pattern ofthe inclusion complex were attributed to the inclusionof EBL within the cyclodextrin cavity, by similarityto the amphotericin/�-CD inclusion complex (Raja-gopalan et al. 1986). The thermal behaviour (absenceof the melting peak of EBL) and same flat X-ray dif-fraction pattern in the co-evaporate confirm the for-mation of the inclusion complex by the evaporationprocess, with evidence of the inclusion of EBL intothe cyclodextrin cavity. In fact, the evaporation ofEBL solution without �-CD does not produce theamorphisation.

The morphological analyses of the EBL crystals,EBL/�-CD inclusion complexes and the solid �-CDare shown in Figure 4.

Table 1. Total energy for the isolated and inclusion complexes be-tween EBL and �-CD and energy change associated with inclusionphenomena.

Species Etotal (kcal mol−1) �EC (kcal mol−1)

EBL (Figure 1A) 99.13

�-CD 83.94

1:1 Complex

EBL-C1 (Figure 1B) 160.95 −22.12

EBL-C2 (Figure 1C) 154.92 −28.15

EBL-C3 (Figure 1D) 159.84 −23.23

EBL-C4 (Figure 1E) 158.89 −24.18

1:2 Complexa

EBL-CC1 (Figure 1F) 218.65 −48.36

aFor the 1:2 complex �EC = E(complex) − [E(EBL) + 2 E(�-CD)]

Figure 2. Differential scanning calorimetry analysis (DSC) of EBL(A); EBL/�-CD (B) and �-CD (C).

236

The plant hormone crystals showed an elongatedfeature with a dimension ranging from a few mi-crometers up to 150–200 �m. The inclusion complex(evaporated inclusion complex) is characterised bycrystals of a different product, in which no EBL crys-tal appears, as is shown in Figure 4.

1H-NMR studies

The 1H-NMR data of EBL, EBL/�-CD inclusioncomplex and �-CD in deuterated dimethylsulfoxide(DMSO-d6) solutions are presented in Table 2. Thespectrum of �-CD has a broad peak at approximately� 4.39 ppm due to the presence of small amounts ofwater. In the spectrum of the complex, the broad peakof water into the �-CD cavity is absent. Also, the sig-nal of H-23, that appears at d 3.34 ppm in EBL alone,was shifted to higher field. It appears that most of theprotons are shifted, suggesting that a portion of theside chain in the inclusion complex is in proximity ofthe �-CD protons. This fact is in agreement with the

results obtained from molecular mechanics calcula-tions.

Rice Lamina Inclination Bioassay

The results of rice lamina inclination assay (Figure 5)using IAC-103 cultivar revealed that the complex-ation of EBL with �-CD produced a more pronouncedlamina inclination than EBL alone at concentrationsof 1.0 - 10−4 mg L−1. The IAC-104 cultivar was lessresponsive, since inclinations obtained with the EBL/�-CD complex were higher than those obtained byEBL alone only at higher concentrations (1.0 - 0.1mg L−1).

Analysis of variance of these experiments showedthat the responses in rice lamina inclination assaywere dependent on the cultivar, the EBL concentra-tion, the complexation with �-CD and the interactionbetween concentration and complexation.

Figure 3. X-Ray powder diffraction of EBL (A), EBL/�-CD in-clusion complex (B) and �-CD (C).

Figure 4. Scanning electron micrographies of EBL (A–C) andEBL/�-CD (D–F) magnified 350× (A, D), 750× (B, E) and 1000×(C, F).

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Conclusion

In the present study, EBL was found to form inclu-sion complexes with �-cyclodextrin, leading to an in-crease of BS activity. The rice lamina inclination bio-

assay results indicate that EBL/�-CD complexes maybe used to improve the bioavailability of the guestmolecule and for the control of the dosage, whichmay have great potential for future application in cropproduction.

Table 2. 1H-NMR peaks (� ppm) of EBL, �-CD and EBL/�-CD inclusion complex in DMSO-d6 solutions.

Protons Compound EBL/�-CD

EBL1-H, 2Ha 1.91/1.65 1.75/1.69

2-H, 1H 3.47, ddd 3.34-3.28

3-H, 1H 3.76, br, s 3.89, d (J = 12.5 Hz)

4-H, 2Ha 1.96/1.91 2.11/1.90

5-H, 1H 3.16, dd (J = 12 Hz; 4.4 Hz) 3.07, dd (J = 12.5 Hz, 4.5 Hz)

7-H, 2Ha 4.12 4.17/4.14

8-H, 1H 1.64 1.65

9-H, 1H 1.17 1.34-1.14

11-H, 2Ha 1.67/1.37 1.85/1.44

12-H, 2Ha 1.93/1.26 1.92/1.23

14-H, 1H 0.92, s 1.13

15-H, 2Ha 1.49/1.35 1.68/1.23

16-H, 2Ha 1.26/1.19 1.34

17-H, 1H 1.47 1.44

18-H, 3H 0.67, s 0.74, s

19-H, 3H 0.86, s 0.85

20-H, 1H 1.37 1.35

21-H, 3H 0.89, d (J = 6.6 Hz) 0.84, d, J = 4.2

22-H, 1H 3.48, br, dd 3.71-3.56

23-H, 1H 3.34, dd (J = 4.5 Hz; 4.5 Hz) not apparent

24-H, 1H 1.38 1.36

25-H, 1H 1.92 1.90

26-H, 3H 0.85, d (J = 6.9 Hz) 0.80, s

27-H, 3H 0.81, d (J = 6.9 Hz) 0.79, d (J = 5.2 Hz)

28-H, 3H 0.76, d (J = 6.9 Hz) 0.76, d (J = 5.2 Hz)

�-CDH-1, 7H 4.82, d (J = 3.3 Hz) 4.82, d (J = 3.4 Hz)

H-2, 7H 3.28, d (J = 3.9 Hz) 3.28, d (J = 3.2 Hz)

H-3, 7H 3.65, d (J = 8.8 Hz) 3.65, d (J = 6.8 Hz)

H-4, 7H 3.33, dd (J=17.6 Hz, 9.2 Hz) 3.30, d (J = 3.2 Hz)

H-5, 7H 3.56, d (J = 10.6 Hz) 3.56, d (J = 9.8 Hz)

H-6a,b, 14H 3.62, d (J = 9.2 Hz) 3.61, d (J = 9.1 Hz)

a chemical shifts of � and � hydrogens

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Acknowledgements

This research was supported by Brazilian AgencyFAPESP (99/05119-7, 99/11678-9 and 99/07907-2).We thank Prof. Dr. Nobuo Ikekawa (Niigata Collegeof Pharmacy) for EBL samples.

References

Ahmed S.M. 1998. Improvement of solubility and dissolution of19-norprogesterone via inclusion complexation. J. InclusionPhenom. Mol. Rec. Chem. 30: 111–125.

Alberts E. and Muller B.W. 1992. Complexation of steroid-hor-mones with cyclodextrin derivatives. Substituent effects of theguest molecule on solubility and stability in aqueous-solution.J. Pharm. Sci. 81: 756–761.

Braun P. and Wild A. 1984. The influence of brassinosteroids ongrowth and parameters of photosynthesis of wheat and mustardplants. J. Plant Physiol. 116: 189–196.

Brutti C., Apostolo N.M., Ferrerotti S.A., Llorente B.E. andKrymkiewicz N. 2000. Micropropagation of Cynara scolymusL. employing cyclodextrins to promote rhizogenesis. Sci. Hor-tic. 83: 1–10.

Clouse S.D. and Sasse J.M. 1998. Brassinosteroids: essential regu-lators of plant growth and development. Annual Rev. PlantPhysiol. Plant Molec. Biol. 49: 427–451.

Connors A. 1997. The stability of cyclodextrin complexes in solu-tion. Chem. Rev. 97: 1325–1357.

Cutler H.G., Yokota T. and Adam G. (eds) 1991. Brassinosteroids:Chemistry, Bioactivity and Applications. ACS Symposium Se-ries 474. American Chemical Society, Washington, DC.

De Azevedo M.B.A., Alderete J.B., Lino A.C.S., Loh W., Faljoni-Alario A. and Durán N. 2000. Violacein/�-cyclodextrin inclu-sion complex formation studied by measurements of diffusioncoefficient and circular dichroism. J. Incl. Phenom. Macrocy-clic Chem. 37: 67–74.

De Azevedo M.B.M., Alderete J., Zullo M.A.T., Salva T.J.G. andDuran N. 2000. Brassinosteroids: a new class of plant hor-mones. The biological activity of 24-epibrassinolide and an in-clusion complex of 24-epibrassinolide and �-cyclodextrin. Pro-ceed. Int’l. Control. Rel. Bioact. Mater. Controlled ReleaseSociety, Inc.: 5006–5007.

Durán N., De Azevedo M.B.M., Zullo M.A.T., Salva T.J.G. andAlderete J.B. 2000. Process of cyclodextrin/brassinosteroidsformulation, for agricultural application, used as plant hor-mones, Brazilian Patent BR9906202-A.

Durzan D.J. and Ventimiglia F.F. 2000. Cyclodextrin nutrients inplant tissue cultures, US Patent US 6087176 [Chem. Abstr.133: 88976 (2000)].

Fujioka S., Noguchi T., Takatsuto S. and Yoshida S. 1998. Activityof brassinosteroids in the dwarf rice lamina inclination bioas-say. Phytochemistry 49: 1841–1848.

Fujioka S. and Sakurai A. 1997. Biosynthesis and metabolism ofbrassinosteroids. Physiol. Plant. 100: 710–715.

Gosset S. and Gauvrit C. 1992. Activity enhancement of benzamideherbicides by cyclodextrins. French 9222204 A1 [Chem. Abstr.118: 75387 (1997)].

Hayashi T., Iijima Y., Hoshino A. and Nakamura M. 1998. Agentsand method for flowering acceleration using cinnamic acid-cy-clodextrin inclusion compounds Japanese Patent. Kokai Tok-kyo Koho JP 10273404 A2 [Chem. Abstr. 129: 327304 (1999)].

Huet H. and Jullien M. 1992. The �-cyclodextrins delay the ger-mination of the somatic embryos of carrot (Daucus carota L.).Acad. Sci., Ser. III 314: 171–177.

Ikekawa N. and Zhao Y. 1991. Application of 24-epibrassinolide inagriculture. In: Cutler H.G., Yokota T. and Adam G. (eds),Brassinosteroids: Chemistry, Bioactivity and Applications. ACSSymposium Series 474. American Chemical Society, Washing-ton, DC, pp. 280–305.

Kalinch F.N., Mandava N.B. and Todhunter J.A. 1985. Relation-ship of nucleic acid metabolism to brassinolide-induced re-sponses in bean. J. Plant Physiol. 120: 207–214.

Khripach V.A., Zhabinskii V.N. and De Groot A.E. 1999. Brassi-nosteroids, a new class of plant hormones. Academic Press,Harcourt Brace & Company, USA.

Koehler G., Grabner G., Klein C.T.H., Marconi G., Mayer B.,Monti S. et al. 1996. Structure spectroscopic properties of cy-clodextrin inclusion complexes. J. Inc. Phenom. Mol. Rec.Chem. 25: 103–108.

Lipkowitz K.B. 1998. Applications of computational chemistry tothe study of cyclodextrins. Chem. Rev. 98: 1829–1873.

Mandava N.B. 1988. Plant growth promoting brassinosteroids.Ann. Rev. Plant Physiol. Plant Mol. Biol. 39: 23–52.

Marquart V. and Adam G. 1991. Recent advances in brassinoster-oids research. In: Ebing W. (ed.), Chemistry of Plant Protec-tion, Herbicide Resistance - Brassinosteroids, Gibberellins,Plant Growth Regulators. Vol. 7. Springer-Verlag, Berlin, pp.104–139.

Marzona M., Carpignano R. and Quagliotto P. 1992. Quantitativestructure-stability relationships in the inclusion complexes ofsteroids with cyclodextrins. Annal. Di Chim. 82: 517–537.

Okii M. 1993. Cyclodextrins for enhanced callus tissue culture ofrice Japanese Patent. Kokai Tokkyo Koho JP 05292955 A2[Chem. Abstr. 120: 1324504 (1997)].

Rajagopalan N., Chen S.C. and Chow W.S. 1986. A study of theinclusion complex of amphotericin-B with cyclodextrin. Int. J.Pharm. 29: 161–168.

Figure 5. Rice lamina inclination assay of cultivars IAC-103 andIAC-104 with 24-epibrassinolide (EBL) and its complex with �-cy-clodextrin (EBL/�-CD) (mean standard deviations of 6.91 ° for cv.IAC-103 and 9.18 ° for cv. IAC-104).

239

Sairam P.K. 1994. Effect of homobrassinolide application on plantmetabolism and grain yield under irrigated and moisture stressconditions of two wheat varieties. Plant Growth Regul. 14:173–181.

Sakurai A. and Fujioka S. 1993. The current status of physiologyand biochemistry of brassinosteroids. Plant Growth Regul. 13:147–159.

Sasse J.M. 1997. Recent progress in brassinosteroid research. Phys-iol. Plant. 100: 696–701.

Sawamoto T. 2000. Plant growth-stimulating and disease-prevent-ing agents containing 1-triacontanol and their manufacture Jap-anese Patent 2000128707 A2 [Chem. Abstr. 132: 304657].

Stella V.J. and Rajewski R.A. 1997. Cyclodextrins: their future indrug formulation and delivery. Pharm. Res. 14: 556–567.

Stella V.J., Rao V.M., Zannou E.A. and Zia V. 1999. Mechanismsof drug release from cyclodextrin complexes. Adv. Drugs Del.Rev. 36: 3–16.

Szejtli J., Szente L., Harshegyi J., Daroczi I., Vorashazy L. andTorok S. 1989. Inclusion complexes and mixtures of plantgrowth regulators with cyclodextrins., Hung. Patent Teljes HU47961 A2 [Chemical Abstracts 112: 50398 (1997)].

Takeno K. and Pharis R.P. 1982. Brassinosteroid-induced bendingof the leaf lamina of dwarf rice seedlings: an auxin-mediatedphenomenon. Plant Cell Physiol. 23: 1275–1281.

Uden W.V. and Woerdenbag H.J. 1994. Cyclodextrins as a usefultool for bioconversions in plant cell biotechnology. Plant CellTissue Organ Cult. 38: 103–113.

Vardhini B.V. and Rao S.S.R. 1999. Effect of brassinosteroids onnodulation and nitrogenase activity in groundnut Arachis hy-pogaea L. Phytochemistry 48: 927–930.

Wada K., Marumo S., Abe H., Morishita T., Nakamura K.,Uchiyama M. et al. 1984. A rice lamina inclination test - a mi-cro-quantitative bioassay for brassinosteroids. Agric. Biol.Chem. 48: 719–726.

Wada K., Marumo S., Ikekawa N., Morisaki M. and Mori K. 1981.Brassinolide and homobrassinolide promotion of lamina incli-nation of rice seedlings. Plant Cell Physiol. 22: 323–326.

Zhou D., Wu Y., Xu Q., Yang L., Bai C. and Tan Z. 2000. Molecu-lar mechanics study of the inclusion of trimethylbenzene iso-mers in �-cyclodextrin. J. Inc. Phenom. Macrocyclic Chem. 37:273–279.

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