Interaction of Jatrorrhizine with Human Gamma Globulin in membrane mimetic environments: Probing of the binding mechanism and binding site by spectroscopic and molecular modeling methods

Journal of Molecular Structure 980 (2010) 108–113

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Interaction of Jatrorrhizine with Human Gamma Globulin in membrane mimetic environments: Probing of the binding mechanism and binding site by spectroscopic and molecular modeling methods Li Ying, Wang Chao *, Lu Guanghua Ministry of Education Key Laboratory of Integrated Regulation and Resource Development on Shallow Lakes, College of Environment, HoHai University, NanJing, JiangSu Province 210098, China

a r t i c l e

i n f o

Article history: Received 7 March 2010 Received in revised form 28 June 2010 Accepted 29 June 2010 Available online 6 July 2010 Keywords: Microemulsion Human Gamma Globulin Jatrorrhizine Spectrum

a b s t r a c t The interaction between Jatrorrhizine and Human Gamma Globulin (HGG) in AOT/isooctane/water microemulsions was studied by using fluorescence quenching, UV absorption spectroscopy, circular dischroism (CD) spectroscopy and dynamic light scattering (DLS). Fluorescence data in water-surfactant molar ratio (x0) 25 microemulsions revealed the presence of the binding site of Jatrorrhizine on HGG and its binding constants at four temperatures were obtained. The affinities in microemulsions were similar to that in buffer solution. The alterations of YGG secondary structure in the microemulsions in the absence and presence of Jatrorrhizine compared with the free form of HGG in buffer were analyzed by CD spectroscopy. In addition, the DLS data suggested that HGG may locate inside the microemulsion and Jatrorrhizine could interact with them. Furthermore, the thermodynamic functions, i.e. standard enthalpy (DH0 ) and standard entropy (DS0 ) for the reaction were also calculated, according to Van’t Hoff equation. These data showed that hydrophobic and electrostatic interaction played the main role in the binding of Jatrorrhizine to HGG. Ó 2010 Elsevier B.V. All rights reserved.

1. Introduction Reverse micelles are comprised of a polar solvent (water) sequestered by a surfactant in an organic non polar phase (oil). The resulting solution is optically clear and contains nanodroplets of water ranging in intramicellar diameter of 0.3–20 nm [1,2]. Reversed micelles are simple yet interesting models of biological membranes. The water solubilisation capacity is conventionally described in term of the water-surfactant molar ratio x0 (x0 = [H2O]/[S]) [3]. The aggregates containing a large amount of water molecules (above x0 = 15) are usually called microemulsions whereas reverse micelles correspond to droplets containing a small amount of water (below x0 = 15) [4]. These microheterogeneous systems can act as hosts for molecules such as proteins and drugs by providing them with unique microvicinity. In fact, pharmaceuticals in these systems have shown enhanced structural and conformational stability and slow diffusivity, under certain conditions [5]. In view of the easy preparation, stability and ability to solubilize a large amount of water, Aerosol-OT (AOT, sodium 1,4-bis(2-ethylhexyl)sulfosuccinate) is the most widely used surfactant in the study of reverse micelles. Water added to the AOT systems is solubilized in the polar core, forming a water pool sur* Corresponding author. E-mail address: [email protected] (W. Chao). 0022-2860/$ - see front matter Ó 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.molstruc.2010.06.044

rounded by a layer of surfactant molecules. AOT has the remarkable ability to solubilize a large amount of water with values of x0 (x0 = [H2O]/[AOT]), as large as 40–60, depending on the surrounding nonpolar medium, the solute and the temperature [6]. AOT microemulsion is optically transparent and the change in the system could be studied by different spectrophotometric techniques [7]. Human Gamma Globulin is a preparation of the proteins of liquid human serum, containing the antibodies (primarily IgG) of normal adults. It is obtained from pooled liquid human serum from several donors and may be prepared by precipitation under controlled conditions of pH, ionic strength, and temperature. Human Gamma Globulin is capable of binding an extraordinarily diverse range of metabolites, drug, organic compounds and relevant antigens [8]. With the remarkable binding properties, HGG can serve as one of important role in the discovery of novel drug delivery system and targeted drug therapy. The binding of drugs to HGG also has important role in therapeutic drug monitoring as the binding was affected by several drug and patient-associated factors, resulting in altered free drug concentration and thus drug’s efficacy and toxicity may be altered [9]. Jatrorrhizine (structure shown in Fig. 1), one of the major bioactive components isolated from tinospora cordifolia’s roots, has antibacterial, antifungal and parasite fighting abilities. The medication of Jatrorrhizine is also same with berberine. Because of its

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2.2. AOT reverses micelle preparation Microemulsions solutions of desired x0 were obtained by adding concentrated protein solutions, drug solutions or plain buffer (pH 7.40) to a 0.1 M AOT solution in isooctane. Volume of additivity was assumed in calculating AOT concentration and x0 values. The samples were gently shaken until complete clarification. The final sample concentration was calculated according to the total volume of the microemulsion. 2.3. Apparatus and methods

Fig. 1. The chemical structure of Jatrorrhizine.

pharmacological activity, it is necessary to study the interaction between Jatrorrhizine and protein for understanding the mechanism of drug action at molecular level. In previous works, the molecular interactions between protein and many drugs in aqueous solution under physiological conditions have been investigated successfully [10–13]. Zhang Yaheng and co-workers studied the interaction of several drugs with human serum albumin in AOT/water/isooctane reverse micelles [14,15]; Suzana M. Andrade et al. studied the interaction of two water-soluble freebase por-phyrins with two drug–carrier proteins in AOT/isooctane/water reverse micelles in virtue of steady state and transient-state fluorescence spectroscopy [16]; Daniel M. Davis and colleagues probed the behavior of HSA in AOT reverse micelles by fluorescence quenching and CD [17]. Yet the detailed investigation on the confined reaction of drug and protein in membrane mimetic environments monitored by diversiform analytical methods have rarely been reported. In this paper, we extended the earlier work on protein–drug complexation in aqueous solution to microemulsion, which provided ‘‘microreactors” for diversified chemical and biochemical reactions. Jatrorrhizine entering the ‘‘waterpool” of AOT/water/isooctane microemulsions and interacting with the encapsulated HGG could provide a useful model to mimic the case that drugs transfer from outsides of cells to inside and react with proteins. Using fluorescence spectroscopy, UV absorption spectroscopy, circular dichroism (CD) and dynamic light scattering (DLS) approaches, the interaction of Jatrorrhizine with HGG in microemulsions was clearly demonstrated. Attempts were made to investigate the binding constant (K), thermodynamic parameters for the reaction and the effect of Jatrorrhizine on the protein secondary structure in the membrane mimetic environments. Furthermore, the molecular modeling was studied with GLIDE docking program. 2. Experimental 2.1. Materials Sodium bis(2-ethylhexyl) sulfosuccinate (AOT) (Fluka, P96% purity) was used without further purification. Human Gamma Globulin was purchased from Lanzhou Biotechnology Company (China). All HGG solutions were prepared in the pH 7.40 buffer solution and the HGG stock solution was kept in the dark at 277 K. Jatrorrhizine chloride was of analytical grade, and purchased from the National Institute for Control of Pharmaceutical and Bioproducts (China), and the stock solutions were prepared in absolute ethanol. NaCl (1.0 M) solution was used to maintain the ionic strength at 0.1. Buffer (pH 7.40) consists of Tris (0.2 M) and HCl (0.1 M), and the pH was adjusted to 7.40. Isooctane and other reagents were of analytical grade and deionized water was used throughout all the experiments.

The absorption and Fluorescence spectra were measured with a CARY-100 UV–visible spectrophotometer (Varian, USA) and a RF5301PC spectrofluorophotometer (Shimadzu). The fluorescence excitation wavelength was 280 nm, and the emission was read at 300–500 nm. The intrinsic fluorescence of HGG was obtained at 330 nm when excited at 280 nm. A quantitative analysis of the potential interaction between Jatrorrhizine and HGG was performed by fluoremetric titration. A 2 mL solution containing 1.56  106 M HGG was titrated by successive additions of a 4.50  105 M of corresponding microemulsions of Jatrorrhizine, and the fluorescence intensity was measured (excitation at 280 nm and emission at 330 nm). All experiments were measured at four different temperatures (289, 296, 303, and 310 K). Using the fluorescence decrease, the association constants K for the complex of Jatrorrhizine with HGG at different temperatures were calculated. The binding parameters have been calculated using the Scatchard’s procedure [18]

r=Df ¼ nK  rK This method is based on the general equation, where r is the moles of drug bound per mole of protein, Df is the molar concentration of free drug, n is binding site multiplicity per class of binding sites, and K is the equilibrium binding constant. Steady state anisotropy (r) is defined by [19]:

r ¼ ðIVV  GIVH Þ=ðIVV þ 2GIVH Þ where IVV and IVH are the intensities obtained with the excitation polarizer oriented vertically and the emission polarizer oriented in vertical and horizontal orientation, respectively. The G factor is defined as:

G ¼ IHV =IHH Refer to the similar guidelines as mentioned above for the horizontal position of the excitation polarizer. Circular dichroism (CD) measurements were made on an Olis DMS1000CD (USA), using a 1 mm cell at 298 K. The spectra were recorded in the range of 200–250 nm and the scan rate is 30 nm/ min. The induced ellipticity was the protein alone defined as the ellipticity of the drug–HGG mixture minus the ellipticity of drug alone at the same wavelength. Dynamic light scattering experiments were performed to determine hydrodynamic radius of AOT reversed micelles at room temperature. The measurements were done using a BI–200SM Static and Dynamic laser light scattering system (Brookhaven, America) coupled with a 4 W laser. The crystal structure of HGG was obtained from the protein data bank at www.pdb.org (entry codes 1AJ7).The potential of 3D structure of HGG was assigned according to Protein Preparation Wizard [20]. The Hydrogens were added and the structures were energy minimized with the OLPS-2001 force field. The 3D structure of drug was processed using the Ligprep program [21] to assign protonation states appropriate for pH 7.0. The GLIDE program [22] was used to build the interaction modes between the drug and HGG.

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3. Results and discussion 3.1. Determination of x0 value for HGG microemulsion A series of microemulsions from 15 to 35 were prepared to determine an appropriate x0 value for HGG microemulsion, in which microemulsion HGG has a better stability. When x0 equaled to 35, the clear microemulsions were prone to turbid. When the x0 varied from15 to 25, the microemulsios maintained pellucid, which were more stable than 30. According to the result of Susana Andrade [23], the radius of spherical protein’s sphericity rp(Angsrom) = 0.7  Mr1/3, an empirical equation for radius of AOT micelle inner core is given by rm(Angstrom) = 4 + 1.5  x0, when rp = rm, the optimal x0 value for HGG is expected to approximate to 23. The fluorescence data of HGG in AOT microemulsions with varions x0 were recorded when kex was 280 nm and the results were reflected in Fig. 2. It is evident that HGG had a higher fluorescence intensity and anisotropy in x0 25 microemulsions. So we chose x0 25 microemulsions for the spectroscopic measurements and the main fluorometric titration experiments. 3.2. Spectrum data of HGG in microemulsions of different xo The fluorescence data of HGG in various xo microemulsions were recorded. There was no distinct shift in the fluorescence emission maximum (kmax) along with the change of xo. The kmax were all about 330 nm when xo increased from 15 to 35. And the change of the relative fluorescence intensity was not evident in different xo microemulsions. It is noticed that the properties of reverse micelle solubilized water are differ from those of bulk water, even at higher xo values, and its apparent microviscosity is 6–9 times greater than that of free aqueous solution [24]. The fluorescence anisotropy measurements of HGG in microemulsions against xo were also shown in Fig. 2. It can be shown that with the water pool increase, a significant change in the anisotropy (r) was observed, suggesting that there was appreciable additional change in the average mobility of HGG molecules. The UV–via spectra of HGG in different xo microemulsions were discussed in Fig. 3. In aqueous solutions there was an absorbance intensity band of HGG with a maximum at around 213 nm. But in AOT microemulsions the absorbance band tended towards a bathochromic shift from 213 nm to 239 nm. It suggested that the microenvironment polarity of chromophore decreased. The relative absorption intensities of peaks at 278 nm which character the conjugated double bond of tryptophan, tyrosine and phenylalanine residue to peaks at 239 nm were stronger than that in buffer solution. It means that microemulsions and water solutions pro-

Fig. 2. The fluorescence intensity and anisotropy of HGG (1.56  106 M) in microemulsions with different x0.

Fig. 3. UV absorption spectra of HGG (1.56  106 M) in microemulsions with different x0.

vide different microenvironments for HGG. Variations of water quantity in microemulsions could affect the absorbance of HGG but have no influence the peak positions. Fig. 4 was the UV absorption spectra of free HGG and Jatrorrhizine bound form of HGG in xo 25 microemulsion. It was shown in Fig. 4, with increasing amount of Jatrorrhizine in xo 25 microemulsion the absorbance of HGG at 239, 278 nm increased. This result clearly indicated the interaction between HGG and Jatrorrhizine. 3.3. Analysis of fluorescence quenching of HGG by Jatrorrhizine in microemulsion The aim of the present work was to investigate whether Jatrorrhizine interacts with HGG and changes the conformation of HGG in xo 25 microemulsion. The fluorescence spectra of HGG in microemulsion before and after addition of Jatrorrhizine compared with the native HGG in buffer were measured with the excitation wavelength at 280 nm and their representative spectra were shown in Fig. 5. The conformation changes in protein were evaluated by measuring the intrinsic fluorescence intensity of protein tryptophan residues [25] before and after addition of Jatrorrhizine. The addition of Jatrorrhizine caused a dramatic decrease in the fluorescence emission intensity of HGG with a conspicuous change in the emission spectra. It can be seen that a higher excess of Jatrorrhizine led to more effective quenching of the chromophore molecules fluorescence. The strong quenching of the fluorescence clearly indicated that the binding of the drug to HGG changed the microenvironment of tryptophan residue and the tertiary

Fig. 4. UV absorption spectra of Jatrorrhizine–HGG system in x0 25 microemulsion (a) 1.56  106M HGG; (b)–(g) 1.56  106M HGG in the presence of 2.25  106, 4.5  106, 6.75  106, 9.0  106, 1.13  105, 1.35  105 M Jatrorrhizine.

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L. Ying et al. / Journal of Molecular Structure 980 (2010) 108–113 Table 1 Binding parameters microemulsions.

and

thermodynamic

of

Jatrorrhizine–HGG

x0 25

in

T (K)

K (104 M1)

Regression coefficient

4G0 (kJ/ mol)

4H0 (kJ/ mol)

4S0 (J/ mol k)

289 296 303 310

4.60 4.48 4.46 4.07

0.9989 0.9981 0.9981 0.9955

25.82 26.35 26.88 27.41

3.94

75.71

Table 2 Binding constants of Jatrorrhizin–HGG in microemulsions of x0 different and buffer solution at 296 K.

Fig. 5. The fluorescence spectra of Jatrorrhizine–HGG system in x0 25 microemulsion. (a) 1.56  106 M HGG; (b)–(g) 1.56  106 M HGG in the presence of 2.25  106, 4.5  106, 6.75  106, 9.0  106, 1.13  105, 1.35  105 M Jatrorrhizine; (h) 1.56  106 M HGG in buffer.

structure of HGG in microemulsion. From Fig. 5, it can be also seen that HGG has a strong fluorescence emission in microemulsion with a peak at 330 nm, a large shift of emission to shorter wavelength from 335 nm of HGG in buffer. It suggested perturbations of the amino acid residue microenvironment. The quantitative analysis of the binding of Jatrorrhizine to HGG in microemulsion was carried out using the Scatchard equation as shown in Fig. 6. The linearity of Scatchard plots indicated that Jatrorrhizine binds to a single class of binding site on HGG. The binding constants were summarized in Table 1. The static quenching and dynamic quenching were differentiated by the results at different temperatures. The quenching constants decrease with increasing temperature for the static quenching, but the reversed effect was observed for the dynamic quenching. It can be found from Table 1 that the binding constants decreased with the increasing temperature in microemulsions. This indicates the static quenching interaction between Jatrorrhizine and HGG in microemulsions. The binding constants for the Jatrorrhizine–HGG in various xo microemulsions and in buffer solution at 296 K were listed in Table 2. The binding of Jatrorrhizine and HGG was relatively stronger in microemulsions with xo 25 and xo 35. It was possible that the activity of HGG in xo 25 and xo 35 microemulsions was higher. In buffer solution, the binding constant was lower to some degree than the microemulsions’s, which could indicate greater accessibility of Jatrorrhizine toward HGG when HGG was encapsulated in microemulsion. It may imply that HGG associates with the inter-

Fig. 6. The Scatchard curves of quenching of HGG (1.56  10 zine in x0 25 microemulsion. kex = 280 nm, kem = 330 nm.

6

M) with Jatrorrhi-

x0

15

25

35

Buffer

K (104 M1)

2.97

4.48

4.43

3.14

face of micrioemulsion and an intersting conformational chang is induced in the protein’s structure. 3.4. Binding studies between Jatrorrhizine and HGG in microemulsion using CD spectrum and DLS data In order to gain a better understanding in physicochemical properties of Jatrorrhizine governing its spectral behavior and to draw relevant conclusions on the Jatrorrhizine–HGG binding mechanism, CD spectroscopic measurement was performed on HGG in the absence and presence of Jatrorrhizine in xo 25 microemulsion and compared them with spectra in buffer. If the change of protein structure included the transforming of protein secondary structure in the drug–HGG complex, it can be reflected in the CD spectra. The CD spectra of HGG and Jatrorrhizine–HGG complex were shown in Fig. 7. The CD spectra of protein consist of two negative bands in the ultraviolet region at 209 and 220 nm, which is typical characterization of a-helix structure of class protein [26]. The reasonable explanation is that the negative peaks between 208 and 209 nm and 222 and 223 nm are both contributed to n ? p transfer for the peptide bond of a-helical [27]. The value in CD spectrum observed around 215 nm is the characteristic of b-sheet structure [28]. In Fig. 7, the CD spectrum of free HGG in buffer solution had a characteristic of the typical b-sheet structure. After encapsulating protein in microemulsion, the band intensity of negative Cotton effect of HGG increased, indicating the considerable changes in the protein secondary structure with the increase

Fig. 7. CD Spectra of the HGG–Jatrorrhizine System in x0 25 microemulsion and Tris buffer. j3.0  106 M HGG in x0 25 microemulsion; s3.0  106 M HGG in the presence of 1.5  105 M Jatrorrhizine in x0 25 microemulsion; w3.0  106 M HGG in Tris buffer.

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Table 3 Hydrodynamic diameter (DH) of AOT microemulsions without HGG and with HGG at x0 = 25, T = 296 (K).

DH (nm)

No HGG

HGG

HGG + Jatrorrhizine

5.89

7.34

28.57

of the b-sheet content in HGG. This could mean that AOT microemulsion provided different chiral environments for HGG. Upon Jatrorrhizine complexation, the CD spectra of bound HGG were observed to be different in shape with that in the absence of Jatrorrhizine in microemulsion and free HGG in buffer. It clearly indicated an increase of the disorder structure content in the protein. From the above results, it may be result of formation of complex between drug and protein. At same time, the intramolecular forces responsible for maintaining the secondary and tertiary structures can be altered, resulting a conformational change of the protein with the loss of some amount of b-sheet structure and turns into a-helical when drug binds to HGG. This suggested that, in spite of the impact of the environment of microemulsion on the secondary structure of HGG, Jatrorrhizine could interact with the protein in microemulsion. Dynamic light scattering (DLS) has been thoroughly applied to provide additional evidence to have insight on the localization of HGG and its binding with Jatrorrhizine which occurred after encapsulation in AOT microemulsions. Hydrodynamic diameters (DH) of xo 25 AOT microemulsions with and without HGG, HGG–Jatrorrhizine were measured in Table 3. In Table 3 the DH of xo 25 microemulsion was 5.89 nm. When introduced HGG in microemulsion, the DH value increased to 7.34 nm. In terms of those data, HGG may locate at the inside inducing an increase in the water pool, which may have relation to the conformational changes in HGG.

The adding Jatrorrhizine result in the change of DH, it could be caused by the interaction of HGG with Jatrorrhizine in the water pool. 3.5. Binding mode Generally, small molecules are bound to macromolecules through four binding modes: hydrogen bond, van derWaals force, electrostatic and hydrophobic interaction, etc. [29]. The thermodynamic parameters, enthalpy (DH0 ) and entropy (DS0 ) of reaction are important for confirming the acting force. For this reason, the temperature dependence of the binding constant of microemulsion (xo = 25) was studied. The temperatures chosen were 289 K, 296 K, 303 K, 310 K so that HGG does not undergo and structural degradation. The thermodynamic parameters can be determined from Van’t Hoff equation. The thermodynamic parameters were obtained from linear Van’t Hoff plot and are presented in Table 1. Many references have reported the characteristic sign of the thermodynamic parameter associated with the various individual kinds of interaction that may take place in the protein association process [30,31]. As shown in Table 1, 4H0 and 4S0 for the binding reaction between Jatrorrhizine and HGG were found to be 3.90 kJ/mol and 75.71 J/mol K. Thus, the negative sign for 4G0 means that the binding process was spontaneous and the formation of Jatrorrhizine–HGG coordination compound was an exothermic reaction accompanied by positive 4S0 value. For typical hydrophobic interactions, both 4H0 and 4S0 are positive, while negative 4H0 and 4S0 changes arise from van derWaals force and hydrogen bonding formation in low dielectric media. However, negative 4H0 might play a role in electrostatic interactions. Therefore, it is not possible to account for the thermodynamic parameters of Jatrorrhizine–HGG coordination compound on the basis of a single intermolecular force model. It is more likely that

Fig. 8. The best binding mode for HGG–Jatrorrhizine System according to GLIDE docking. The molecule of Jatrorrhizine is located in CDR of Fab of HGG. The secondary structure of the protein is shown and the important neighboring amino acids are labeled. Ligand structures are shown in a ‘‘ball and stick” format. The hydrogen bonds are indicated by yellow dash. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

L. Ying et al. / Journal of Molecular Structure 980 (2010) 108–113

hydrophobic, electrostatic interactions are involved in its binding process in the microemulsions. 3.6. Molecular modeling of the complex of HGG and Jatrorrhizine HGG has two heavy chains consisting of about 450 amino acid residues and two light chains consisting of about 210–230 amino acid residues. The investigation of the 3D structure of crystalline IgG1have proved that the antigen-binding site distributes over a ‘‘shallow cavity” (cleft) with the size of 1.5 nm  0.6 nm  0.6 nm, which is comprised of about 10–12 amino acid residues of the complement-determinant region (CDR) of Fab, the semiantigen can be accessible to the cleft and associate with the amino acid residues of CDR with complementary structure by hydrogen bond, Van der Waals force, electrostatic interaction and hydrophobic interaction, etc. [32]. The binding of Jatrorrhizine–HGG might involve hydrophobic interaction strongly by the thermodynamic parameters studied in this paper. However, the interaction between Jatrorrhizine and HGG via hydrogen bonds can also not be excluded and indicated by illustration. Here, partial binding parameters of the HGG–Jatrorrhizine system were calculated through GLIDE docking protocols (Fig. 8). GLIDE is one of the newest programs for docking studied. Fig. 8 showed clearly that there are interaction of hydrogen bond between Jatrorrhizine and the amino acid residues of HGG. As shown, Jatrorrhizine is mainly hydrophobic and appears to form hydrogen bonds with residues His35 and Tyr99. The interaction between Jatrorrhizine and HGG is not exclusively hydrophobic in nature since the several ionic and polar residue in the proximity of the ligand playing important role in stabilizing the drug molecule via hydrogen bond and electrostatic interaction. 4. Conclusions In the present work, we chose AOT/isooctane/water microemulsions as membrane mimetic environments to investigate the interaction of Jatrorrhizine and HGG. The binding constant is calculated from the fluorescence data in microemulsions. The results presented have clearly indicated that Jatrorrhizine is a strong quencher and binds to HGG with higher affinities in microemulsions than in buffer solution. The qualitative CD analytical data indicated that the secondary structure of HGG was influenced after introducing the protein into microemulsions and the interaction of Jatrorrhizine with HGG in microemulsions kept on changing the conformation of HGG. HGG may locate at the inside of the microemulsion and interact with Jatrorrhizine according to the DLS data.

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