A Luminescence Study of the Interaction of Sulfobutylether-β-Cyclodextrin with Rutin

Min Wu, Zhenghua Song* and Jingjing Zhang

Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of Ministry of Education, College of Chemistry & Material Science, Northwest University, Xi’an, 710069, China

Abstract: The luminol/sulfobutylether-β-cyclodextrin (SBE7M-β-CD) chemiluminescence (CL) system and the interaction of SBE7M-β-CD/rutin were first described by flow injection (FI) CL method. It was found that SBE7M-β-CD with luminol could form 1:1 complex online, which could accelerate the electrons transferring rate of excited 3-aminophthalate, giving the enhanced CL intensity of luminol. The enhancement of CL intensity was proportional to the concentrations of SBE7M- β-CD with a linear range from 25 to 1750 μmol L-1. It was also found that rutin could inhibit the CL intensity from luminol/SBE7M-β-CD system, and the decrement of CL intensity was logarithm over the concentrations of rutin ranging from 0.1 to 100.0 nmol L-1, giving the regression equation AI = 32.90lgCrutin + 16.26 (R2 = 0.9952) with a detection limit of 0.03 nmol L-1 (3σ). According to the proposed CL model, the binding constant (KCD-R) and the stoichiometric ratio of SBE7M-β-CD/rutin complex were obtained as 1.6 × 106 L2 mol-2 and 2:1. The possible mechanism of luminol/SBE7M-β- CD/rutin interaction was also discussed. The method was successfully applied to monitor rutin in human urine samples after ingesting SBE7M-β-CD/rutin complex, with a total excretion of 68.8% within 8.0 h.
Keywords: Chemiluminescence, flow injection, luminal, rutin, pharmacokinetics, sulfobutylether-β-cyclodextrin.

Cyclodextrins (CDs) are cyclic oligosaccharides


consisting of 6, 7 or 8 (a-, β- or y-CD, respectively) D- glucopyranose units linked by R-(1→4) bonds. As the internal cavity is relatively non-polar, CDs can accommodate hydrophobic molecules of suitable sizes as guests [1, 2]. It is well known that studies of the interaction of CD and drug with different methods have become a hot spot in the field of analytical chemistry, agriculture, pharmaceutical field and
food chemistry in recent years [3-6]. Sulfobutylether-β-




cyclodextrin (SBE -β-CD) is a negatively charged


derivative of β-CD (shown in Fig. 1), the SBE groups variably substitute at the 2-, 3- and 6-positions of the total 21 hydroxyl groups of β-CD [7], and the subscript of 7M indicates an average total degree of substitution (TDS) of seven SBE groups per CD molecule. The SBE7M-β-CD provides an extended hydrophobic cavity and an extremely




hydrophilic exterior surface. The solubility in water for SBE7M-β-CD (excess 70 g/100 mL at 25°C) is significantly higher than the parent β-CD (1.85 g/100 mL at 25°C) [8, 9]. Additionally, SBE7M-β-CD exhibits better bioavailability and lower nephrotoxicity than β-CD which makes it appear to be more effective and safe adjuvant for targeted drug delivery [10-13]. SBE7M-β-CD is becoming one of the most popular β-CD derivatives used as an excipient material to improve the physiochemical properties of drugs.

Fig. (1). The structure of SBE7M-β-CD sodium salt form.

Rutin, a flavanoid glucoside known as vitamin P (shown in Fig. 2), has been proven to exhibit strong antioxidation and free-radical-scavenging activities [14-19]. However, the poor solubility substantially influenced its bioavailability. As one of model drugs, the interaction of native and modified β- CD with rutin has often been reported [20-25]. However, to the best of our knowledge, no FI-CL method has been reported yet for studying the interaction between rutin and


*Address correspondence to this author at the Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of Ministry of Education, College of Chemistry & Material Science, Northwest University, Xi’an, 710069, China; Tel: (+86) 029 88303798; Fax: (+86) 029 88302604;
E-mail: [email protected], [email protected]

In this work, the photochemical reaction mechanism of SBE7M-β-CD with rutin was investigated with luminol used as a luminescence probe by Flow Injection (FI) analysis. It was found that SBE7M-β-CD and luminol could form 1:1

1874-0758/11 $58.00+.00 ©2011 Bentham Science Publishers

complex online, which could accelerate the rate of the luminescent intermediate of the reaction, excited 3- aminophthalate’s electrons transferring, producing the steady and enhanced CL signal of luminol. The enhancement of CL intensity was proportional to the concentrations of SBE7M-β- CD with a linear range from 25 to 1750 μmol L-1 with the RSD less than 4.0%. It was also found that the CL intensity from luminol/SBE7M-β-CD system could be quenched in the presence of rutin, and the decrement of CL intensity was logarithm over the concentrations of rutin ranging from 0.1

China. Rutin was purchased from National Institute for the Control of Pharmaceutical and Biological Products, China.
Luminol stock solution of 2.5 × 10-2 mol L-1 was prepared by dissolving 0.44 g luminol with 0.01 mol L-1 NaOH solution in a 100 mL brown calibrated flask and was then kept in dark place. 2.5 mol L-1 stock solution of NaOH was prepared by dissolving 25 g NaOH powder and diluting it to 250 mL calibrated flask. Working solutions of luminol/NaOH were prepared daily from the above stock
solution. 2.5× 10-4 mol L-1 of SBE -β-CD was prepared by

to 100.0 nmol L-1 with a detection limit of 0.03 nmol L-1 (3σ). The interaction of SBE7M-β-CD/rutin was discussed, the binding constant KCD-R of 1.6 × 106 L2 mol-2 and the stoichiometric ratio of 2:1 were obtained. The method was successfully applied to monitor rutin in human urine samples after ingesting SBE7M-β-CD/rutin complex, with a total excretion of 68.8% within 8.0 h.



Fig. (2). The structure of rutin

All reagents used in this work were of analytical reagent grade. Doubly deionized water was purified in a Milli-Q system (Millipore, Bedford, MA, USA) and it was used for the preparation of solutions in the whole procedure. Luminol (Fluka, Biochemika) was obtained from Xi’an Medicine Purchasing and Supply Station, China. SBE7M-β-CD (TDS = 5) was purchased from Zibo Qianhui Fine Chemical Co., LTD,

dissolving 0.241g SBE7M-β-CD in 500 mL water. Finally, a series of testing standards solutions of rutin was prepared daily from the 10 mg L-1 stock solution by appropriate dilution as required.

The FI-CL system used in this work is shown schematically in Fig. (3). A peristaltic pump of an IFFL-DD Luminescence Analyzer (Xi’an Remax Electronic Science- Tech. Co. Ltd., Xi’an, China) was applied to deliver all streams. PTFE tubing (1.0 mm i.d.) was used throughout the manifold for carrying the CL reagents. A six-way valve with a loop of 100.0 μL was used for sampling. The CL signal produced in flow cell was detected without wavelength discrimination, and the photomultiplier tube (PMT) output was recorded by PC with the IFFL-DD client system. A Hitachi F-4500 fluorescence spectrofluorimeter (Tokyo, Japan) was applied for the fluorescence measurements.

General Procedures
As shown in Fig. (3), flow lines were inserted with luminol/NaOH, carrier (water), SBE7M-β-CD and sample solutions, respectively. The pump was started with the sample stream of water at a constant speed of 2.0 mL min-1 to wash the whole system until a stable baseline was recorded. Then, rutin solutions were injected and CL intensities were recorded. The concentration of rutin could be quantified on the basis of the decrement of CL intensity, AI = Io – Is, where Is and Io were CL signals in the presence and in the absence of rutin, respectively.




Flow Cell




Mixing Tubing




Fig. (3). Schematic diagram of the present FI-CL Luminol: 2.5×10-5 mol L-1 (NaOH 0.025 mol L-1); SBE7M-β-CD: 1×10-4 mol L-1; Flow rate:
2.0 mL min-1; Mixing tube: 10.0 cm; High voltage: –750 V.

Relative CL Intensity-Time Profile
The relative CL intensity-time profile of luminol/SBE7M- β-CD/rutin was presented in Fig. (4). It was clear that the time of reaching the maximum CL intensity (Tmax) for luminol (curve a) was 8.4 s; the Tmax of luminol/SBE7M-β-CD system (curve c) changed to 7.8 s, giving a more lasting and a 3-fold maximum CL intensity (Imax) as that in the absence of SBE7M-β-CD. The Tmax of luminol/SBE7M-β-CD system did not change in the presence of rutin (curve b) and the Imax decreased from 630 to 508 by 19%.

indicated that the CL signal could reach a maximum value in the NaOH concentration of 0.025 to 0.050 mol L-1. Hence, 0.025 mol L-1 NaOH was chosen as the optimum concentration of this system and used in the subsequent experiments.










0.0 0.5 1.0 1.5
CSBE-β-CD /mmol L-1





9 18 27 36

Fig. (5). Effect of SBE7M-β-CD concentration on CL intensity Luminol: 2.5 × 10-5 mol L-1; NaOH: 0.025 mol L-1.

Analytical Performances for Determinations of SBE7M-β- CD/Rutin
Under the optimum condition given above, the solutions of rutin were tested by FI-CL using luminol/SBE7M-β-CD system. It was found that the decrement of ICL was

Fig. (4). Relative CL intensity-time profile in different CL systems
The concentrations of luminol, SBE7M-β-CD and rutin were 2.5 × 10-5, 2.5 × 10-4 and 1.0 × 10-7 mol L-1, respectively. a: CL system of luminol/dissolved oxygen reaction; b: CL system of luminol/ SBE7M-β-CD/rutin reaction; c: CL system of luminol/SBE7M-β-CD reaction.

Optimum Conditions of Luminol/SBE7M-β-CD CL System
The effects of concentrations of luminol and SBE7M-β- CD on the CL intensity were tested (shown in Fig. 5). The increment of luminol CL intensity was proportional to the concentrations of SBE7M-β-CD from 25 to 1750 μmol L-1 with the linear equation of ΔI = 1.31CSBE-β-CD + 3.39, R2 = 0.9949, RSD < 4% (n = 5). Considering the consumption of SBE7M-β-CD and stable strong CL intensity, 2.5 × 10-4 mol proportional to the logarithm of rutin concentrations over the range 0.1 to 100.0 nmol L-1, giving the regression equation AI = 32.90lgCrutin + 16.26 (R2 = 0.9952) with a detection limit of 0.03 nmol L-1 (3σ). Operational Stability of the FI-CL System 100 μL luminol solution was injected into the system in the absence and in the presence of 0.1, 100 nmol L-1 rutin and the CL intensity was recorded to test the stability of the luminol/SBE7M-β-CD system. The experiment lasted for 3 days and the flow system was regularly used over 8 h per day. The results of these replicate experiments are listed in Table 1. Each result is the average of five separate determinations, and the RSDs were less than 4.0%. It was found that the luminol/SBE -β-CD system exerted very L-1 SBE7M-β-CD solution and 2.5 × 10-5 mol L-1 luminol solution were chosen as the optimum concentrations. Mixing tube length and the flow rate had great effect on the CL intensity. The length of 10.0 cm mixing tube was selected regarding CL reaction was a fast process. As a compromise between reagents consumption and sensitivity, 2.0 mL min-1 of flow rate was recommended. Due to the luminal, CL reaction is more favored under alkaline condition, NaOH solution was added into the luminol solution to increase the sensitivity of the system. A series of NaOH solutions with different concentrations, ranging from 0.0025 to 0.25 mol L-1 was tested. The result 7M good stability. Interference Studies The interference of foreign species was tested by analyzing a standard solution of rutin into which increasing amounts of potential interfering substances were added. The tolerable concentrations of foreign species with respect to 10 nmol L-1 rutin for interference less than 5.0% level were less than 24.4 μmol L-1 for Mg2+, Ca2+ and starch, 2.4 μmol L-1 for glucose, 250 nmol L-1 for urea, 1 μmol L-1 for lysozyme, 120 nmol L-1 for I-, NO3-, Cl-, SO42-, Ac-, CO32-, HCO3-, respectively. Table 1. The Stability Test of FI-CL System Under Different Concentration of Rutin Time ICL Blank RSD % ICL 0.1 nmol L-1 RSD % ICL 100 nmol L-1 RSD % 1st day 630.2 1.0 624.7 1.6 508.7 1.2 2nd day 628.6 1.0 624.3 1.1 506.3 0.5 3rd day 627.0 1.2 625.0 1.1 511.0 1.4 The Possible Interaction Mechanism of Luminol/SBE7M- β-CD/Rutin SBE-β-CD + L KL-CD L*… SBE-β-CD L…SBE-β-CD + hυ (λmax 430 nm) (1) Luminol as a luminescence probe, the possible interaction mechanism of luminol/SBE7M-β-CD/rutin was studied by CL and FS methods. It was found that the fluorescence intensity of luminol (1.0 × 10-6 mol L-1) in the presence of SBE -β-CD (Ex/Em: 350/430 nm) enhanced In the presence of rutin, the decrement of the CL intensity was proportional to the logarithm of rutin concentrations over the range 0.1 to 100.0 nmol L-1. According to the modified CL model [28], the equation of SBE7M-β-CD/rutin could be obtained. R = Rutin, L…SBE- 7M -7 β-CD…R = L…SBE -β-CD/rutin complex, n was the with concentrations of SBE7M-β-CD from 1.0 × 10 to 1.0 × n 7M 10-4 mol L-1. That was possibly because β-CD added a protection of the luminescent intermediate of the reaction, 3- aminophthalate by the CD cavity from collisional quenching, which has been reported [26]. By the B-H equation [27]: 1/(FL-CD) = {(KL-CDkL-CDQL-CD CL)-1(CCD)-1} + (kL-CDQL-CDCL)- 1, where QL-CD is the quantum yield for luminol/SBE7M-β-CD stoichiometric ratio of rutin/SBE7M-β-CD. R may enter into the cavity of SBE-β-CD, and quench the light emission from the excited L*… SBE-β-CD complex. Hence, the interaction between L…SBE-β-CD and R can be described as follows: ’ CD-R complex, FL-CD is the fluorescence intensity of n R + L*…SBE-β-CD L…SBE-β-CD…Rn (2) luminol/SBE7M-β-CD complex, kL-CD is an instrumental where the association constant (K ’) of L…SBE-β-CD/R constant, and CL and CCD are the concentrations of luminol and SBE7M-β-CD, respectively. A linear equation was complex can be expressed as: CD-R obtained from a plot of 1/FL-CD versus 1/CCD with a linear K ’ = [L…SBE-β-CD…R ]/([R]n [L*…SBE-β-CD]) (3) CD-R n correlation coefficient of 0.9946 (Fig. 6), which indicated where [R], [L*…SBE-β-CD] and [L…SBE-β-CD…R ] are the 1:1 of the luminol/SBE7M-β-CD stoichiometric ratio with the formation constant (KL-CD) of 3.3 × 106 L mol-1. From the CL concentrations of the R, L*… n SBE-β-CD complex and method, we knew that SBE7M-β-CD could accelerate the L…SBE-β-CD…Rn complex, respectively; [L*…SBE-β-CD]o is the total concentration of the L*…SBE-β-CD complex in electrons transferring rate of excited 3-aminophthalate, giving an augmented and more lasting CL intensity of luminol. Here, L = luminol, SBE-β-CD = SBE7M-β-CD, L…SBE-β-CD = luminol/SBE7M-β-CD complex, when 100 μL 2.5 × 10-5 mol L-1 L was injected into the carrier stream the absence of R, which equals [L*…SBE-β-CD] + [L…SBE- β-CD…Rn]. Hence: [L…SBE-β-CD…Rn] = [L*…SBE-β-CD]o - [L*…SBE-β-CD] (4) and mixed with 2.5 × 10-4 mol L-1 SBE-β-CD in the FI-CL system, SBE-β-CD was in excess and L mainly existed in the form of excited L*…SBE-β-CD complex which produced the CL signal. The process was expressed as below: Io and Is are the CL intensity of luminol/SBE7M-β-CD system in the absence and in the presence of R, then we know: Is/Io = [L*…SBE-β-CD]/ [L*…SBE-β-CD]o (5) Eqn (3) can change to the form of logarithm, eqns (4) and (5) can be substituted, and then we get: lg[(Io – Is)/Is] = lgK ’ + nlg[R] (6) The binding constant (K ’) and the stoichiometric ratio CD-R (n) could be obtained from the plot of lg[(Io – Is)/Is] against lg[D] (Fig. 7), viz. pKCD-R’ = 3.11 and n = 0.5 (stoichiometric ratio of L*…SBE7M-β-CD/rutin = 2:1). eqn (2) can be written as: KCD-R R + 2 L*…SBE-β-CD (L…SBE-β-CD) …R

The binding constant (KCD-R) can be obtained as:



’ 2 = 1.6 × 106 L2 mol-2 (8)

Fig. (6). Double reciprocal plot for luminol/SBE -β-CD complex


The plot is linear, indicating 1:l complex throughout the concentration range of SBE7M-β-CD: 1.0 × 10-7 to 1.0 × 10-4 mol L-1; luminol: 1.0
× 10-6 mol L-1.

Therefore, the possible mechanism of luminol/SBE7M-β- CD/rutin interaction was showed in Fig. (8):

The Stoichiometric Ratio of SBE7M-β-CD/Rutin by CL Method
With ICL vs. stoichiometric ratio plot, the stoichiometric ratio of SBE7M-β-CD : rutin was obtained by FI-CL method.

As Fig. (9) shows, the SBE7M-β-CD with rutin was formed of 2:1 complex, the measured CL intensity (AI) at inflexion was compared to theoretical (AIo), derived by extrapolation of linear parts of the curve to their intersection, and the values of AI and AIo were 97.9 and 98.3, respectively. The stoichiometric ratio of SBE7M-β-CD : rutin was 2:1 complex.

-10 -9.5 -9 -8.5 -8 -7.5 -7

Fig. (7). Double logarithmic plot for SBE7M-β-CD/rutin complex from the proposed CL model The concentration range of rutin: 0.1 to 100 nmol L-1, SBE7M-β-CD: 2.5 × 10-4 mol L-1; luminol: 2.5 × 10-5 mol L-1.





hυ (λmax 430 nm)

Fig. (8). Schematic diagram of luminol/SBE7M-β-CD/rutin interaction process








0 0.5 1 1.5 2 2.5 3

Fig. (9). The stoichiometric ratio of SBE7M-β-CD: rutin by proposed CL method Rutin: 50 nmol L-1; SBE7M-β-CD: 25, 50, 75, 100, 150 nmol L-1.

Table 2. Results of Determination of Rutin in Tablets a

Sample No. Added
( nmol L-1) Found
( nmol L-1) RSD
(%) Recovery
(%) Content of Rutin b
( mg per Tablet)

1 0 1.1 1.3
0.3 1.3 2.9

2 0 1.0 1.1
0.5 1.5 2.8

3 0 0.9 1.2
1 1.9 1.3

4 0 1.2 2.9
3 4.1 2.4

5 0 1.1 0.5
5 5.7 3.5
a Averaged from five determinations.
b Declared content: 20 mg per tablet.

Table 3. Results of Determination of Rutin in Spiked Serum and Urine Samples a

Sample b
No. Added (nmol L-1) Found (nmol L-1) RSD (%) Recovery (%) Content of Rutin (μmol L-1)
Proposed Method Spiked

1-1 0 29.6 1.1
40 40.0 1.6

1-2 0 29.6 1.0
80 81.4 1.1

1-3 0 42.7 0.9
60 52.2 0.6

1-4 0 41.4 0.9
80 69.5 0.8

1-5 0 49.2 3.6
100 95.4 1.2

2-1 0 9.6 1.1
20 20.1 3.0

2-2 0 30.8 2.4
40 40.6 2.1

2-3 0 28.8 1.9
60 60.7 3.8

2-4 0 31.8 1.1
80 79.8 2.3

2-5 0 52.4 0.7
60 61.7 1.8
a Averaged from five determinations.
b No. 1-1 to 1-5, serum samples; No. 2-1 to 2-5, urine samples.

The quotient of these values represented the degree of association (1 – a): AI/AIo = 1 – a, where a (the degree of dissociation) was 0.004.

Determination of Rutin in Tablets
The proposed method was applied for to the determination of rutin in tablets (Shanxi Taiyuan Pharma-

ceutical Co. Ltd.) purchased at the local market. Ten tablets were weighed and ground to fine powder. The powder was dissolved in water. The resulting solution was filtered through an ordinary filter paper and diluted to a 100 mL brown glass calibrated flask. Suitable concentrations which covered the working range of this solution were taken for the determination of rutin. In order to validate the proposed method, recovery studies were carried out using the samples

Table 4. Results of Rutin in Human Urine Samples

Content (mg L-1) Half-Life Time t1/2 (h) The Total Elimination Rate Constant k Metabolism Ratio (%)
rutin/SBE7M-β-CD 13.75/1.35 1.5 0.464 68.8
rutin 16.79/1.17 1.1 0.604 84.0








0 1 2 3 4 5 6 7 8

Time /h
Fig. (10). The Time courses of changes in concentrations of rutin in human urine after oral administration of a single dose of tablet a: rutin/SBE7M-β-CD complex; b: rutin.

spiked with the known amounts of rutin, the results are summarized in Table 2.

Determination of Rutin in Human Serum and Urine
The proposed CL method was applied for the determination of rutin in spiked human serum and urine. Serum samples were supplied by the Hospital of Northwest University and urine samples were collected from healthy volunteer. To prepare the spiked samples, known quantities of rutin were spiked into serum or urine, and were then diluted 5.0 × 105 and 2.0 × 105 folds, respectively. The influence of foreign species existing in serum could be eliminated. The contents of rutin in the spiked samples were quantified by applying the standard addition method and the results were listed in Table 3.

Monitoring the Excretion of Rutin and SBE7M-β- CD/Rutin Complex in Human Urine
Rutin and CDs with 1:2 molar ratio were accurately weighed. A homogenous paste was prepared by mixing CD and a small amount of water in a mortar, then rutin was added. The paste was further ground for 2 h. The obtained masses were dried at 40 °C in an oven for 1 h, and the dried complex was ground to fine powder. A healthy volunteer took 191.8mg SBE7M-β-CD/rutin complex (about 20 mg rutin) on an empty stomach in the morning. From then on, the urine samples were collected in glass bottles after 0.5, 1.0, 1.5, 2.0, 3.0, 4.0, 6.0 and 8.0 h. Without any pre- treatment procedures, urinary rutin could be determined

relatively simply by the proposed method after dilution. For a control experiment, the healthy volunteer ingested a tablet containing 2 mg rutin and the urine samples were collected and determined with the same procedure. The amount of excreted rutin in SBE7M-β-CD/rutin complex reached maximum within 1.5 h from its oral administration, then it dropped slowly within the following few hours. For the control experiment, the amount of excreted rutin reached maximum within 1.0 h and dropped sharply. Fig. (10) showed the difference of the two excretion profiles and Table 4 summarized some of the results obtained. The difference of these two excretion profiles may be due to the fact that the drug molecule can be included in the SBE7M-β- CD cavity and the drug release rate reduced consequently.

Luminol as a CL probe, the interaction of SBE7M-β- CD/rutin was discussed and a new assay method of rutin was established. Compared with other methods, the luminol/SBE7M-β-CD CL system offers advantages of higher sensitivities, simplicity of apparatus and less reagent consumption. The method was successfully applied to monitor rutin in human urine samples after ingesting SBE7M- β-CD/rutin complex, with a total excretion of 68.8% within
8.0 h.

The authors gratefully acknowledge the financial support from Shaanxi Province Nature Science Foundation (No.

2006B05), the Foundation of Ministry of Education (No. 07JK395), the NWU Graduate Innovation and Creativity Funds (No. 09YZZ45 and 10YZZ29) and NWU Graduate Experimental Research Funds (No. 09YSY18), China.

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Received: June 27, 2011 Revised: October 28, 2011 Accepted: October 28, 2011