Synthesis, characterization and biological activity of Rhein-cyclodextrin conjugate

Article abstract of DOI:10.1016/j.molstruc.2016.08.047

Cyclodextrin conjugate complexation is a useful method to enhance the solubility and absorption of poorly soluble drugs. A series of new Rhein-β-cyclodextrin conjugates (Rh-CD conjugates) have been synthesized and examined. Rhein is covalently linked with the β-CD by amido linkage in a 1:1 molar ratio. The conjugates were characterized by ¹H NMR, ¹³C NMR, HRMS, powder X-ray diffraction (powder XRD) as well as thermogravimetric analysis (TGA). The results reveal that incorporation of β-CD could improve the aqueous solubility of Rhein and the cytotoxicity against hepatocellular carcinoma (HepG2) cell line as well as antibacterial activity against three organisms. The improved biological activity and the satisfactory water solubility of the conjugates will be potentially useful for developing novel drug-cyclodextrin conjugates, such as herbal medicine.

Full text of DOI:10.1016/j.molstruc.2016.08.047

Accepted Manuscript
Synthesis, characterization and biological activity of Rhein-cyclodextrin conjugate
Manshuo Liu, Pin Lv, Rongqiang Liao, Yulin Zhao, Bo Yang
PII:
S0022-2860(16)30877-8
10.1016/j.molstruc.2016.08.047
MOLSTR 22873
DOI:
Reference:
To appear in: Journal of Molecular Structure
Received Date: 19 April 2016
Revised Date: 14 August 2016
Accepted Date: 16 August 2016
Please cite this article as: M. Liu, P. Lv, R. Liao, Y. Zhao, B. Yang, Synthesis, characterization and
biological activity of Rhein-cyclodextrin conjugate, Journal of Molecular Structure (2016), doi: 10.1016/
j.molstruc.2016.08.047.
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ACCEPTED MANUSCRIPT
Graphical abstract
Synthesis, characterization and anticancer activity of
Rhein-cyclodextrin conjugate
a1
a1
a
b*
a∗
Manshuo Liu , Pin Lv , Rongqiang Liao ,Yulin Zhao , Bo Yang
a
Faculty of Life Science and Technology, Kunming University of Science and
Technology, Kunming 650500, P.R. China
b
Faculty of Chemical Engineering, Kunming University of Science and Technology,
Kunming 650500, P.R. China
∗
Corresponding author. Tel.:+86 13064281879
E-mail: zhaoyulin919@sina.com; yangbo6910@sina.com
1

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Synthesis, characterization and biological activity of
Rhein-cyclodextrin conjugate
a1
a1
a
b*
a∗
Manshuo Liu , Pin Lv , Rongqiang Liao ,Yulin Zhao , Bo Yang
a
Faculty of Life Science and Technology, Kunming University of Science and
Technology, Kunming 650500, P.R. China
b
Faculty of Chemical Engineering, Kunming University of Science and Technology,
Kunming 650500, P.R. China
Correspondence to: Bo Yang (yangbo6910@sina.com Tel.:+86 13064281879)
Abstract Cyclodextrin conjugate complexation is a useful method to enhance the
solubility and absorption of poorly soluble drugs. A series of new
Rhein-β-cyclodextrin conjugates (Rh-CD conjugates) have been synthesized and
examined. Rhein is covalently linked with the β-CD by amido linkage in a 1:1 molar
1
13
ratio. The conjugates were characterized by H NMR, C NMR, HRMS, powder
X-ray diffraction (powder XRD) as well as thermogravimetric analysis (TGA). The
results reveal that incorporation of β-CD could improve the aqueous solubility of
Rhein and the cytotoxicity against hepatocellular carcinoma (HepG2) cell line as well
as antibacterial activity against three organisms. The improved biological activity and
the satisfactory water solubility of the conjugates will be potentially useful for
developing novel drug-cyclodextrin conjugates, such as herbal medicine.
Keywords: Rhein; β-cyclodextrin; Conjugate; Biological activity.
∗
Corresponding author. Tel.:+86 13064281879
E-mail: zhaoyulin919@sina.com; yangbo6910@sina.com
1
These authors contributed equally to this work.
1

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1
. Introduction
Rhein (4,5-dihydroxy-9,10-dioxoanthracene-2-carboxylate)
is
a
natural
anthraquinone derivative (Scheme 1), that exists as the free form glucoside in different
species of plant such as Polygonaceae and Leguminosae [1-2]. The biological study of
Rhein has been reported, including anti-angiogenic, anti-microbial, anti-inflammatory,
anti-cancer activities and it also acts as an effective material in experimental treatment
of diabetic nephropathy [3-7]. It is reported that Rhein has the better antibacterial
activity towards P.gingivalis. Furthermore, it was also found to inhibit the proteolytic
activity of the bacterium [8]. However, the clinical application of Rhein in
chemotherapy is limited by the low water solubility, poor oral bioavailability and
photo-sensitivity [9]. Therefore, it is urging to develop an efficient and nontoxic
method to tackle those limitations for clinical applications.
β-cyclodextrin (β-CD) is a cyclic oligosaccharide which consists of glucose units
interconnected by α-1,4 linkages. Due to its unique chemical structure, β-CD can form
inclusion complexes with small molecule that its size allows it fit into the cavities of
β-CD. It has been applied to modify the properties of guest molecules in many fields,
such as food, chemical, textile industrial, agricultural and pharmaceutical area. β-CD
is also widely used in advanced drug delivery systems because of the versatility of
biodegradability, low toxicity, biocompatibility, and facile chemical modification [10].
Therefore, β-CD-tagged drugs are extensively studied as the drug delivering strategy
[
11].
Based on the promising properties of Rhein, a simple procedure for the structure of
the compound modification was designed. In the present study, we developed a series
of new active Rhein-β-CD conjugates (Rhein-1N-β-CD conjugate, Rhein-2N-β-CD
2

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conjugate and Rhein-3N-β-CD conjugate, Scheme 2) as prodrug to improve water
solubility and enhance anticancer activity comparing with free Rhein. The structures
of Rhein and their conjugates were characterized by NMR and mass spectrometry.
The compounds were also studied by powder XRD and TGA. Then the solubility,
stabilization effects and their anti-cancer activities on hepatocellular carcinoma
(HepG2) cell lines were investigated. The results demonstrate the potential
application of β-CD-tagged drugs as the drug delivery system. Furthermore, the
antibacterial activity of Rhein-β-CD conjugates was also studied. The results show
Rhein-β-CD conjugates have improved antibacterial activity.
2
. Results and Discussion
2
.1. Chemistry
The mono (6-deoxy-6-amino)-β-CD (1N-β-CD) was synthesized through the
method reported in our previous work [12]. Mono (6-deoxy-6-ethyleneamino)-β-CD
2N-β-CD) and mono (6-deoxy-6-diethyleneamino)-β-CD (3N-β-CD) were obtained
(
according to the reported procedure [13]. The three types of CDs (1N-β-CD, 2N-β-CD
and 3N-β-CD) was respectively coupled to the carboxyl group of Rhein which was
achieved by EDCI and HOBt in ice bath for 3 hours. And the Rhein-CD conjugates
were obtained by purification with acetone. Their structures were characterized by
NMR spectra (Fig. 1, Fig. 2). Seen from the high resolution mass spectrum, there are
+
+
+
1
422.7087 [M+Na] (Fig. S1), 1465.7570 [M+Na] (Fig. S2) and 1486.8189 [M+H]
Fig. S3), respectively. The results show that the Rhein is incorporated to one of the
primary hydroxyl groups of β-CD through a 1:1 molar ratio.
(
3

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2
.2. Powder X-ray diffraction (powder XRD)
The Powder XRD analysis allows examination of the medium and long range
ordering of materials [14]. It could provide insightful characterization of the
conjugates and drug molecules, such as powder XRD pattern of Rhein, 1N-βCD,
Rhein–1N-βCD physical mixture and Rhein-1N-βCD. As shown in Fig. 3, Rhein
showed a high degree of crystallinity and exhibited characteristic peaks between 5°
and 60° (2θ=9.8°, 9.9°, 17.5°, 23.0°, 27.4°, 31.5°, 41.7°)(A). In contrast, 1N-βCD (B)
and Rhein-1N-βCD (D) were of amorphous form. Their physical mixture (C) showed
a superposition of the above and displayed the characteristic peaks of Rhein
(2θ=27.4°), indicating the formation of the conjugate. We also performed different
powder XRD experiments (scan with mixed solvent) in order to evaluate the
crystallinity of their samples under different environments (Fig S5). The results
showed that the conjugates in solvent mixtures were all amorphous.
2
.3. Thermogravimetric analysis (TGA)
TG analysis was used to study the thermal properties of the Rhein-βCD conjugates
(Fig. 4). TG curve of Rhein (A), Rhein-1N-βCD (B), Rhein-2N-βCD (C) and
Rhein-3N-βCD (D) was determined. The analysis showed that the three conjugates
o
decomposed at ca. 248 C. In contrast, decomposition temperature of Rhein was ca.
o
3
20 C.
2
.4. Water solubility
The water solubility of three conjugates was assessed by the preparation of its
saturated solution. The Rhein-β-CD conjugates were obviously more soluble than
4

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Rhein (ca. 0.0052 mg/mL) [15], and remarkably rose up to 0.91, 1.02, 1.12 mg/mL
because of solubilizing effects of 1N-βCD, 2N-βCD and 3N-βCD respectively as
shown in Table 1. The satisfactory water solubility of Rhein-β-CD conjugates was
regarded as the phase change of Rhein from a crystalline state to an amorphous state
of the conjugates [16]. In Fig. 3, the powder XRD patterns could further support this
hypothesis.
2
.5. In vitro cytotoxicity studies
In vitro cytotoxicity on HepG2 results were showed in Table 2. As we expected, the
conjugates exhibited better anti-cancer activity than the free Rhein. The IC values of
5
0
Rhein-1N-βCD, Rhein-2N-βCD, and Rhein-3N-βCD were 18.45 ꢀmol/L, 15.26
mol/L, 16.51 ꢀmol/L respectively, which were better than Rhein (37.23 ꢀmol/L). On
the control tests, cisplatin was used as the positive drug which IC value was 9.75
ꢀ
5
0
ꢀ
mol/L, while β-CD derivative carriers showed no activity against HepG2. Overall,
2
N-βCD is the most suitable carrier among the three β-cyclodextrin derivatives for
Rhein in HepG2 system.
However, the exact working mechanisms remain unconfirmed. In previous report,
the β-CD conjugate was cleaved in endosomes, and then the released drug moved to
the nucleus [17]. There are also some reports that drug–βCD conjugate could activate
autophagy and autophagic cell-death in cancer cells [18]. In our previous work, the
anticancer activities of scutellarin–βCD conjugates were better than scutellarin
because β-CD cavity on these conjugates showed the capability of including lecithin
or cholesterol of the damaged cell membranes [19]. It is still necessary to study the
5

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working mechanism of drug–βCD conjugate for enhancing the cytotoxic effect.
2
.6. Antibacterial activity
The antibacterial activity of Rhein-β-CD conjugates was measured by the agar disk
diffusion method with three organisms (S. aqureus，B. subtilis and E. coli aureus) in
aqueous. The concentration of Rhein and Rhein-β-CD conjugates water solutions was
0
.044 mol/L. The results were summarized in Table 3. The diameter of inhibitory zone
represented antibacterial activity. According to this method, Rhein-β-CD conjugates
displayed better inhibitory effect than Rhein, and β-CDs controls presented no
antibacterial activity. In other words, the conjugates showed better inhibitory effect.
The results are probably attributed to that Rhein did not completely dissolve into the
water. So it also cannot diffuse through the agar media [20]. Meanwhile the
Rhein-β-CD conjugates have good water solubility, which can result in better
inhibitory effect.
3
. Experimental
3
.1.Materials
β-CD was purchased from Mengzhou Huaxing Biochemistry Co., which was
recrystallized three times by double distilled water. N, N-dimethylformamide was
purchased from Chengdu Kelong Chemical Co., which was dried over calcium
hydride for 2 days. 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide hydrochloride
(EDCI), 1-hydroxybenzotriazole (HOBt) and Rhein were purchased from Aladdin
Industrial Co.. All experiments were carried out using ultrapure water and analytical
reagent. Staphylococcus aureus, Bacillus subtilis and Escherichia coli were obtained
6

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from First People’s Hospital in Yunnan Province.
.2. Measurements
3
1
13
H NMR and C NMR spectra were recorded on a Bruker Avance DRX 600 MHz
spectrometer at the temperature of 298 K. Chemical shifts were referenced internally
1
1
to solvent shift (D O: H δ4.80 ppm; DMSO-d : H δ2.50 ppm). ESI MS were
2
6
performed on Agilent LC/MSD TOF. Powder XRD were measured in a D/max-3B
diffractometer using Cu-κα (κ = 15460 Å, 30 mA) with 40 kV. The powder diffraction
data was examined by PANalytical Highscore Plus v. 2.2c software package, the
mixed solvents (H O:EtOH, H O:ACN) were used in a 1:1 ratio. TGA was performed
2
2
under the NETZSCH STA449F3 instrument. The heating rate was 10 ºC /min in the
temperature range from 30 to 450 ºC with 6.0 - 10.0 mg samples.
3
.3. Synthesis Rhein-β-CD conjugates
Mono (6-deoxy-6-amino)-β-CD (1N-β-CD) was prepared using the parent β-CD
as starting material, and purified by Sephadex chromatography. A solution of
amino-β-CD (1.1 g, 1 mmol), Rhein (0.34 g, 1.2 mmol), EDCI (0.23 g, 1.2 mmol),
and HOBt (0.16 g, 1.2 mmol) in 30 mL DMF solutions was stirred in an ice bath for
2
h, and then continued reaction for 24 h at room temperature in dark condition
under N protection. Then insoluble materials were filtered to remove, and the
2
filtrate was poured into 200 mL acetone solution. This step was repeated three times.
T
he eluent was removed under reduced pressure and Rhein-1N-β-CD conjugate was
collected. The synthetic steps for Rhein-2N-β-CD and Rhein-3N-β-CD were similar
to the Rhein-1N-β-CD described above.
7

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Mono(6-Rhein-amino-6-deoxy)-β-cyclodextrin (Rhein-1N-β-CD)
Rhein-1N-β-CD was further purified by Sepha-dex LH-20 column
1
chromatography, the yield was 62.3% (1.042 g, dark red solid). H NMR (600 MHz,
D O) δ 7.76 (s, H-2 of Rhein), 7.67 – 7.61 (m, H-5,6 of Rhein), 7.42 (s, H-4 of Rhein),
2
7
1
1
1
8
.35-7.33 (m, H-7 of Rhein), 4.96 (s, H-1 of 1N-β-CD), 3.87 – 3.61 (m, H-3,5,6 of
13
N-β-CD), 3.63 – 3.29 (m, H-2,4 of 1N-β-CD); C NMR (151 MHz, D O) δ 188.86,
2
84.00, 172.55, 166.08, 164.91, 163.67, 143.27, 135.88, 133.10, 132.99, 128.93,
27.76, 126.23, 125.36, 118.47, 117.84, 117.73, 116.56, 101.91, 101.72, 82.87, 81.12,
1.02, 73.15, 73.08, 72.16, 71.89, 71.47, 60.22, 41.15, 36.94, 31.44. HRMS (ESI,
+
m/z): [M+H] calcd for C H NO 1399.4037; found, 1399.7087.
57
77
39,
Mono(6-Rhein-ethylenedi-amino-6-deoxy)-β-cyclodextrin (Rhein-2N-β-CD)
Rhein-2N-β-CD was further purified by Sepha-dex LH-20 column chromatography,
1
the yield was 56.8% (0.983 g, dark red solid). H NMR (600 MHz, D O) δ 7.76 (s,
2
H-2 of Rhein), 7.67 – 7.61 (m, H-5,6 of Rhein), 7.42 (s, H-4 of Rhein), 7.35 – 3.33(m,
H-7 of Rhein), 4.94 (s, H-1 of 2N-β-CD), 3.86 – 3.60 (m, H-3,5, 6 of 2N-β-CD), 3.64
1
3
–
1
1
8
3.27 (m, H-2, 4 of 2N-β-CD); C NMR (151 MHz, D O) δ 188.89, 184.02, 172.57,
2
66.09, 164.93, 163.68, 143.28, 135.89, 133.11, 133.00, 128.94, 127.77, 126.23,
25.38, 118.48, 117.84, 117.74, 116.57, 101.92, 101.88, 101.73, 82.87, 81.15, 81.12,
1.02, 73.15, 73.08, 72.16, 72.09, 71.90, 71.48, 60.22, 41.14, 36.94, 31.44. HRMS
+
(
ESI, m/z): [M+H] calcd for C H N O 1442.4495; found, 1442.7570.
5
9
82
2
39,
Mono(6-Rhein-diethylenetriamino-amino-6-deoxy)-β-cyclodextrin (Rh-3N-β-CD)
Rhein-3N-β-CD was further purified by Sepha-dex LH-20 column chromatography,
8

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1
the yield was 52.1% (0.926 g, dark red solid). H NMR (600 MHz, D O) δ 7.97 (s,
2
H-2 of Rhein), 7.72 – 7.64 (m, H-5,6 of Rhein), 7.59 (s, H-4 of Rhein), 7.33 – 7.21 (m,
H-7 of Rhein), 4.98 (s, H-1 of 3N-β-CD), 3.88–3.63 (m, H-3,5,6 of 3N-β-CD), 3.61 –
1
3
3
1
1
5
.27 (m, H-2,4 of 3N-β-CD); C NMR (151 MHz, D O) δ 187.79, 185.83, 173.81,
2
42.06, 134.51, 133.99, 133.66, 130.28, 119.84, 118.32, 116.80, 115.42, 102.01,
01.94, 101.73, 101.48, 83.42, 81.07, 80.93, 73.10, 72.21, 72.08, 71.84, 70.06, 60.16,
+
0.63, 50.51, 48.92, 47.76, 47.42, 39.80. HRMS (ESI, m/z): [M+H] calcd for
C H N O 1485.4917; found, 1485.8007.
59
82
2
39,
3
.4. Water solubility
The water solubility of the conjugates was studied by preparation of its saturated
solution [21]. Excessive amounts of the conjugates were placed in 5 mL water, and
the mixture was stirred for 2 h at room temperature (25 ± 0.5 ºC). Then insoluble
materials were removed and the filtrate was evaporated under reduced pressure to
produce product.
3
.5. Cells and medium
Hepatocellular carcinoma (HepG2) cell line was suspended in T-25 flasks,
supplemented with 10% fetal bovine serum (FBS) and seeded into the wells of
6-well culture plates. Antibiotics (100 U/mL penicillin and 100 mg/L-streptomycin)
were used as control in a humidified atmosphere under 5% CD in air at 37 ºC [22].
9
2
3
.6 Cytotoxicity assay
Cytotoxicity was evaluated by MTT assay as previously described [23]. Cells
4
-1
were seeded at 4×10 mL and using the indicated amounts of the complex in a flat
9

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bottom 96-well plate. Cells were added with different concentrations of Rhein and the
conjugates (5 - 115 µM) for 48 h. Then the medium was replaced with fresh medium
and 20 ꢀL of MTT stock solution (5 mg/mL in PBS) was added. Then the medium
was incubated for 4 h at 37 ºC. Formazan was dissolved in 150 µL DMSO. Results
were read at 570 nm in a spectrometer, finally the means of triplicates were
calculated.
3
.7. Preparation of standard bacterial suspension
S. aqureus B. subtilis and E. coli were inoculated three times for activation. Then
0 mL of sterile butter solutions (pH = 7) was added to the bacterial solutions
，
1
6
7
respectively. Finally the bacterial suspensions were adjusted to 10 - 10 cfu/mL by
blood counting chamber.
3
.8. Antibacterial activity
S. aqureus，B. subtilis and E. coli bacterial suspension (200 ꢀL) were seeded with
a 9 cm layer of nutrient agar (7 mL). Meanwhile, 0.044 mol/L stock solutions of
Rhein and Rhein-β-CD conjugates were prepared in water, respectively. And 100 ꢀL
volumes of Rhein-β-CD solutions, Rhein controls and CDs controls were placed into
the holes. Ampicillin (5 mg/mL) acted as positive controls and the plates were
o
incubated at 37 C for 18h. The procedure was conducted in triplicate for each
bacterium in three independent assays.
4
. Conclusions
In this study, we have successfully prepared three types of cyclodextrin (CD)-based
novel
carrier-drug
conjugates
(Rhein-1N-β-CD,
Rhein-2N-β-CD,
and
1
0

ACCEPTED MANUSCRIPT
Rhein-3N-β-CD), which showed much improved aqueous solubility than Rhein.
Moreover, the results of the testing-agar disk diffusion method showed that the
Rhein-β-CD conjugates had a satisfactory antibacterial activity for three organisms. In
addition, our results indicate that the conjugates exhibit cytotoxic effects in HepG2
cells. The incorporation of β-CD allows Rhein to improve the solubility and to
increase its biological activity. Above all, the biological activity of conjugates was
relatively better than Rhein. Further study will be performed to study the mechanism
to improve the cytotoxic effect in future. These conjugates provide a useful approach
for the development of a novel form of Rhein as drug. Therefore these conjugates
enable Rhein have more application in pharmaceutical fields.
Acknowledgments
This work was supported by the National Natural Science Foundation of China
(NNSFC) (No. 21362016, 21361014, 21302074), which are gratefully acknowledged.
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22] B.L. May, S.D. Kean, C.J. Easton, S.F. Lincoln, Preparation and characterization
of 6A-polyamine-mono-substituted β-cyclodextrins. J
1997) 3157.
. Chem. Soc. Perk. T. 1
(
23] M.V.G. De Araújo, E.K.B. Vieira, G. Silva Lázaro, L.S. Conegero, L. E. Almeida,
L.S. Barreto, Sulfadiazine/hydroxypropyl-beta-cyclodextrin host-guest system:
Characterization, phase-solubility and molecular modeling. Bioorg. Med. Chem.
16 (2008) 5788.
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4

ACCEPTED MANUSCRIPT
Table 1. Aqueous solubility of Rhein and three conjugates
a
Compound
Rhein
MW
284
Aqueous solubility (mg/mL)
0.0052
0.91
Rhein-1N-β-CD
Rhein-2N-β-CD
Rhein-3N-β-CD
In water at 25 ºC, pH7.0
1399
1442
1485
1.02
1.12
a
Table 2. IC (ꢀM) of Rh and Rhein-cyclodextrin conjugates in Human hepatoma cell
5
0
line (HepG2)
a
Samples
Cisplatin
Rhein
IC (ꢀM)
5
0
9.75
37.23
>100
18.45
15.26
16.51
1
N-β-CD/2N-β-CD/3N-β-CD
Rhein-1N-β-CD
Rhein-2N-β-CD
Rhein-3N-β-CD
a
The concentrations of free Rhein, Rhein-1N-β-CD, Rhein-2N-β-CD and
Rhein-3N-β-CD

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Table 3. Inhibitory effect of Rhein-1N-β-CD, Rhein-2N-β-CD and Rhein-3N-β-CD,
Rhein and CDs controls (X±s, n =3)
Drug (0.044
Diameters of inhibitory zone (mm)
a
mol/L)
S. aqureus
0
B. subtilis
0
E. coli
0
1
2
3
N-β-CD
N-β-CD
N-β-CD
Rhein
0
0
0
0
0
0
10.0±0.23
15.7±0.66
17.5±0.27
16.8±0.56
11.3±0.35
16.8±0.85
17.0±0.73
15.3±0.45
9.2±0.33
14.3±0.35
15.2±0.38
13.6±0.53
Rhein-1N-β-CD
Rhein-2N-β-CD
Rhein-3N-β-CD
a
The concentrations of free Rhein, Rhein-1N-β-CD, Rhein-2N-β-CD and
Rhein-3N-β-CD

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O H
O
O H
CO O H
O
Scheme. 1. The structures of Rhein
HO
C6
C5
O
HO
O
C4 OH
C1
O
O
OH
S
C3 C2
O
O
C6
NaN3, PPh3, H2O, DMF (1N- CD)
ethylenediamino (2N- CD)
O
OH
6
OH
diethylenetriamino (3N- CD)
TsCl
OTS CD
HO
C6
Pyridine
-
CD
OH O OH
H
N
O
O
O
H
H N
C6
2
n
COOH
N
O
N
C6
n
H
derivatives
OH
OH
EDCI, HOBt
DMF
Conjugates
n = 0,1,2,3
n = 0,1,2,3
Scheme. 2. The synthesis of Rhein-β-cyclodextrin conjugates.

ACCEPTED MANUSCRIPT
A
1
0
8
6
4
2
H-1of CD
B
1
0
8
6
4
2
Rh
C
1
0
8
6
4
2
δ
/ppm
1
Fig. 1. H NMR spectra of Rhein (A, DMSO-d ), 2N-βCD (B, D O) and
6
2
Rhein-2N-βCD (C, D O).
2
1
3
Fig. 2. C NMR spectra of Rhein-1N-βCD (A), Rhein-2N-βCD (B) and
Rhein-3N-βCD (C) in D O.
2

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2
000
0
D
0
0
20
20
7.440
60
60
80
80
100
2
2
000
0
C
40
100
2
000
B
0
0
20
7.5
40
60
80
100
23.0
1
27.4
9.9
31.5
2000
9.8
41.7
A
0
0
20
40
60
80
100
degree
Fig. 3. X-ray diffractograms (Cu-κα) for Rhein (A), 1N-βCD (B), Rhein/1N-βCD (1:1
ratio) physical mixture (C) and Rhein-1N-βCD (D).
1
10
00
1
90
80
70
60
50
40
30
A
C
B
D
100
200
300
400
T(℃）
Fig. 4. TG curves for: Rhein (A); Rhein-1N-β-CD (B); Rhein -2N-β-CD (C) and
Rhein -3N-β-CD (D).

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Highlight
1
2
3
. A series of new Rhein-β-cyclodextrin conjugates have been successfully
synthesized through amido linkage in a 1:1 molar ratio.
. The Rhein-β-cyclodextrin conjugates shows improved water solubility which is
important for development of drug.
. The Rhein-β-cyclodextrin conjugates shows enhanced biological activity compared
with Rhein.
1