extfieldsofstudy:
Medicine
Materials Science

text:
Organo-Clay Nanomaterials Based on Halloysite and Cyclodextrin as Carriers for Polyphenolic Compounds

Hybrid material based on halloysite covalently linked to a hyper-reticulated cyclodextrin network was investigated as a potential carrier for polyphenolic compounds. The absorption ability of the hybrid system was studied in different pH conditions as well as the kinetic release of curcumin, chosen as a drug model. A preliminary study was performed to assess the antioxidant capacity of the obtained carrier. The obtained results highlighted that the curcumin molecule can have sustained release from the carrier over the time, retaining its antioxidant properties due to the combination of two different host systems that give rise to an hyper-reticulated structure, allowing an increase in the drug loading and stabilization. Therefore, this work puts forward an efficient strategy to prepare organic-inorganic hybrids with three different cavities that could encapsulate two or more drug molecules with different physico-chemical properties.

Introduction
Halloysite nanotubes (HNTs) are an aluminosilicate clay with a predominantly hollow tubular structure and chemically similar to the platy kaolinite (Al 2 Si 2 O 5 (OH) 4 ). Being a natural materials, HNT is available at low cost and it has been shown to have excellent bio- [1] and eco-compatibility [2]. Generally, the inner and outer diameters of the tubes are in the ranges of 10-30 nm and 40-70 nm, respectively, while their length is in the range of 0.2-1.5 µm [3]. The halloysite tubes are positive in the inner lumen, where are located mostly of the aluminum hydroxide, whereas the external surface is negative, due to the presence of silicon dioxide [4]. Due to these features, halloysite nanotubes can be selectively functionalized at the outer and/or inner surfaces leading to the synthesis of several interesting nanomaterials [5,6]. HNTs are indeed widely used as drug carrier [7][8][9][10][11] and delivery systems [12], enzyme immobilization [13], pollutant removal [14][15][16][17], catalyst [18][19][20], and so on.
Cyclodextrins (CDs), formed by six (α-CD), seven (β-CD), or eight (γ-CD) α-(D)-glucopyranose units, are very promising materials for their ability to act as molecular [21,22] and chiral selector [23]. Studies on CDs are the object of an immense literature that is periodically reviewed [24,25]. In this context, different studies have shown that their complexation ability with, for example, some α-amino acids or small peptides could be dependent on the presence of substituents on the primary or secondary rim [23]. The complexation processes that are involved, between CD and guest molecules, are highly important in several fields especially regarding the development of drug delivery systems.
In the last years, organo-clay hybrid nanomaterials based on halloysite covalently linked to modified-cyclodextrin units were reported as carriers for polyphenolic compounds. These nanomaterials showed simultaneously carrier ability for different natural drugs [26,27].
In the last years, organo-clay hybrid nanomaterials based on halloysite covalently linked to modified-cyclodextrin units were reported as carriers for polyphenolic compounds. These nanomaterials showed simultaneously carrier ability for different natural drugs [26,27].
Recently, nanosponges [28,29], obtained by combining supramolecular hosts such as cyclodextrins, have attracted great attention since they allow one to increase the loading ratio of biological active molecules into the carrier [30].
Herein, we present a preliminary investigation on the development a potential drug carrier system for biologically active compounds, based on halloysite functionalized with a hyper-reticulated cyclodextrin network (HNT-CD) ( Figure 1). The HNT-CD hybrid, which possesses "three" different cavities, namely the HNT lumen, the β-CD, and additional nanochannels between the cross-linked hosts, could lead to an increase in the loading of drugs with respect to the non-reticulated one. Furthermore, it could offer the remarkable possibility of achieving a controlled and sustained release of one or more drugs in agreement with the interacting cavity.

Results and Discussion
The synthesis of the organic-inorganic hybrid was accomplished, by a one step thiol-ene reaction, as reported in Scheme 1. In particular, modified halloysite, possessing a degree of functionalization of 0.2 mmol·g −1 in thiol groups, was mixed with heptakis-6-(tert-butyldi-methylsilyl)-2-allyloxy-β-cyclodextrin in a 1:1.5 HNT:CD ratio. In this way, a hybrid with 40 wt % CD covalently grafted on halloysite external surface was obtained [31]. The reaction was performed by microwave irradiation and in solvent-free conditions. The HNT-CDs hybrid showed a surface area of 19.9 m 2 ·g −1 , slightly smaller than that of starting pristine halloysite (22.1 m 2 ·g −1 ). Furthermore, the physico-chemical characterization of this nanomaterial showed the presence of a hyper-reticulated structure with different "cavities"; i.e., the halloysite lumen, the β-CD cavity and some nanochannels due to the crosslinking through the hybrid structure. Furthermore, swelling studies showed that the hybrid possesses a swelling ratio of 91.2 ± 1.6% [31]. All these features are crucial in determining the interactions of the hybrid with guest molecules, so they promote the synthesized nanomaterial as a potential efficient drug carrier system for one or more different drugs. Preliminary investigations were performed choosing as a drug model three different polyphenolic compounds, i.e., curcumin, silibinin, and quercetin.

Results and Discussion
The synthesis of the organic-inorganic hybrid was accomplished, by a one step thiol-ene reaction, as reported in Scheme 1. In particular, modified halloysite, possessing a degree of functionalization of 0.2 mmol·g −1 in thiol groups, was mixed with heptakis-6-(tert-butyldi-methylsilyl) -2-allyloxy-β-cyclodextrin in a 1:1.5 HNT:CD ratio. In this way, a hybrid with 40 wt % CD covalently grafted on halloysite external surface was obtained [31]. The reaction was performed by microwave irradiation and in solvent-free conditions. The HNT-CDs hybrid showed a surface area of 19.9 m 2 ·g −1 , slightly smaller than that of starting pristine halloysite (22.1 m 2 ·g −1 ). Furthermore, the physico-chemical characterization of this nanomaterial showed the presence of a hyper-reticulated structure with different "cavities"; i.e., the halloysite lumen, the β-CD cavity and some nanochannels due to the crosslinking through the hybrid structure. Furthermore, swelling studies showed that the hybrid possesses a swelling ratio of 91.2 ± 1.6% [31]. All these features are crucial in determining the interactions of the hybrid with guest molecules, so they promote the synthesized nanomaterial as a potential efficient drug carrier system for one or more different drugs. Preliminary investigations were performed choosing as a drug model three different polyphenolic compounds, i.e., curcumin, silibinin, and quercetin.
Previous investigations have shown that native β-CD and the modified one can positively interact with both quercetin and curcumin, forming 1:1 and 1:2 supramolecular complexes [26,27]. Furthermore, the two molecules can also interact with the HNT lumen by means of electrostatic interactions and hydrogen bonding. Conversely, fluorescence and UV-vis studies have shown that silibinin does not interact with the β-CD cavity, but only with the HNT internal surface [26].
HNT-CD hybrid Scheme 1. Synthetic route for the synthesis of HNT-CD hybrid.
Previous investigations have shown that native β-CD and the modified one can positively interact with both quercetin and curcumin, forming 1:1 and 1:2 supramolecular complexes [26,27]. Furthermore, the two molecules can also interact with the HNT lumen by means of electrostatic interactions and hydrogen bonding. Conversely, fluorescence and UV-vis studies have shown that silibinin does not interact with the β-CD cavity, but only with the HNT internal surface [26].
Based on these findings a preliminary study was performed in order to evaluate the binding abilities of a hyper-reticulated HNT-CD hybrid toward quercetin, curcumin, and silibinin.
The adsorption capacity of the hybrid for the selected biological molecules was evaluated under two different pH values, namely, 1.0 and 6.0. These values were chosen according to the pKa values of the drugs (for quercetin, pKa1 = 7, pKa2 = 9, and pKa3 = 11; for curcumin, pKa1 = 8.38, pKa2 = 9.88, and pKa3 = 10.51; for silibinin, pKa1 = 7.87 and pKa2 = 9.57, respectively), so it was possible to study the interaction with the HNT hybrid in the anionic, neutral, or cationic form of the molecules.
The obtained results, calculated by using equation (1), are shown in Figure 2. As can be easily seen, the hybrid can absorb quercetin and curcumin in both the pH investigated, conversely silibinin is barely absorbed both at pH 1 and pH 6. In the last case, a slight Based on these findings a preliminary study was performed in order to evaluate the binding abilities of a hyper-reticulated HNT-CD hybrid toward quercetin, curcumin, and silibinin.
The adsorption capacity of the hybrid for the selected biological molecules was evaluated under two different pH values, namely, 1.0 and 6.0. These values were chosen according to the pK a values of the drugs (for quercetin, pK a1 = 7, pK a2 = 9, and pK a3 = 11; for curcumin, pK a1 = 8.38, pK a2 = 9.88, and pK a3 = 10.51; for silibinin, pK a1 = 7.87 and pK a2 = 9.57, respectively), so it was possible to study the interaction with the HNT hybrid in the anionic, neutral, or cationic form of the molecules.
The obtained results, calculated by using Equation (1), are shown in Figure 2. Previous investigations have shown that native β-CD and the modified one can positively interact with both quercetin and curcumin, forming 1:1 and 1:2 supramolecular complexes [26,27]. Furthermore, the two molecules can also interact with the HNT lumen by means of electrostatic interactions and hydrogen bonding. Conversely, fluorescence and UV-vis studies have shown that silibinin does not interact with the β-CD cavity, but only with the HNT internal surface [26].
Based on these findings a preliminary study was performed in order to evaluate the binding abilities of a hyper-reticulated HNT-CD hybrid toward quercetin, curcumin, and silibinin.
The adsorption capacity of the hybrid for the selected biological molecules was evaluated under two different pH values, namely, 1.0 and 6.0. These values were chosen according to the pKa values of the drugs (for quercetin, pKa1 = 7, pKa2 = 9, and pKa3 = 11; for curcumin, pKa1 = 8.38, pKa2 = 9.88, and pKa3 = 10.51; for silibinin, pKa1 = 7.87 and pKa2 = 9.57, respectively), so it was possible to study the interaction with the HNT hybrid in the anionic, neutral, or cationic form of the molecules.
The obtained results, calculated by using equation (1), are shown in Figure 2. As can be easily seen, the hybrid can absorb quercetin and curcumin in both the pH investigated, conversely silibinin is barely absorbed both at pH 1 and pH 6. In the last case, a slight increase in the As can be easily seen, the hybrid can absorb quercetin and curcumin in both the pH investigated, conversely silibinin is barely absorbed both at pH 1 and pH 6. In the last case, a slight increase in the absorption rate of the HNT-CD hybrid toward silibinin is observed. These results can be explained as follows. In acidic solution, where the silibinin molecule is positively charged as well as the halloysite lumen, electrostatic repulsions are present, and the adsorption capacity of the system is therefore limited. By increasing the pH, the silibinin molecule could be partially dissociated, so it is less soluble in aqueous medium and can therefore be loaded into HNT as a consequence of hydrophobic effects.
As for quercetin, its adsorption ratio onto the HNT-CD hybrid reflects the retention efficiency of Que on β-CD, which depends on the pH of the medium [26]. It is, indeed, reported that, in acidic solution, strong interactions exist between quercetin and the CD cavity.
On the basis of the above findings, a solid complex between the obtained hybrid and curcumin (chosen as drug model) was synthesized.
The loading of curcumin into the HNT-CD hybrid was carried out by mixing the hybrid compound with a methanolic highly concentrated curcumin solution following a procedure reported elsewhere [32]. After loading, the HNT-CD/Cur complex was washed with water to remove free drug. Cur-loaded pristine HNT, HNT/Cur, was similarly prepared as a reference material. The drug loading of HNT/Cur and HNT-CD/Cur was estimated by UV-vis measurements. The amounts of Cur loaded in the HNT nanomaterials, expressed as the percent amount of drug in the final composite, were ca. 1.5 wt % and 7.7 wt % for HNT/Cur and HNT-CD/Cur, respectively, with encapsulation efficiencies of 41% and 94% for pristine HNT and HNT-CD hybrid, respectively (Equations (2) and (3)).
The different percent loadings of drug in the different carriers can be explained, as already reported for similar carrier systems [32], by the specific functionalization of HNT external surface that promotes the curcumin adsorption because of the introduction of CD moieties and the formation of a polymeric network, so the drug can be efficiently loaded on three different cavities, the HNT lumen, the CD one, and the channels between the cross-linked CD and HNT units.
To evaluate the performances of the HNT-CD/Cur hybrid complex for application in the drug carrier field, preliminary investigations were performed; in particular, the kinetic release of the drug from the hybrid system and the antioxidant properties of the organic-inorganic complex were evaluated.
The release of curcumin loaded in HNT-CD hybrid was evaluated by the dialysis bag method using conditions designed to mimic physiological conditions (phosphate buffer pH 7.4) and the obtained kinetic data (Equation (4)) are shown in Figure 3. As it is possible to note, in these conditions the HNT-CD hybrid retains almost the total amount of the loaded curcumin for at least 96 h. These results are very different from the curcumin kinetic release from a carrier system where to HNT, is covalently grafted a single cyclodextrin unit [33], in this latter case indeed, about 15 wt % of curcumin was released after 24 h (Figure 3), which followed an initial burst release. Therefore, the introduction of a hyper-reticulated cyclodextrin core onto the HNT surface was crucial to slow down the drug release in physiological conditions, obtaining a sustained release of the biological molecule within time.
consequence of hydrophobic effects.
As for quercetin, its adsorption ratio onto the HNT-CD hybrid reflects the retention efficiency of Que on β-CD, which depends on the pH of the medium [26]. It is, indeed, reported that, in acidic solution, strong interactions exist between quercetin and the CD cavity.
On the basis of the above findings, a solid complex between the obtained hybrid and curcumin (chosen as drug model) was synthesized.
The loading of curcumin into the HNT-CD hybrid was carried out by mixing the hybrid compound with a methanolic highly concentrated curcumin solution following a procedure reported elsewhere [32]. After loading, the HNT-CD/Cur complex was washed with water to remove free drug. Cur-loaded pristine HNT, HNT/Cur, was similarly prepared as a reference material. The drug loading of HNT/Cur and HNT-CD/Cur was estimated by UV-vis measurements. The amounts of Cur loaded in the HNT nanomaterials, expressed as the percent amount of drug in the final composite, were ca. 1.5 wt % and 7.7 wt % for HNT/Cur and HNT-CD/Cur, respectively, with encapsulation efficiencies of 41% and 94% for pristine HNT and HNT-CD hybrid, respectively (Equations (2) and (3)).
The different percent loadings of drug in the different carriers can be explained, as already reported for similar carrier systems [32], by the specific functionalization of HNT external surface that promotes the curcumin adsorption because of the introduction of CD moieties and the formation of a polymeric network, so the drug can be efficiently loaded on three different cavities, the HNT lumen, the CD one, and the channels between the cross-linked CD and HNT units.
To evaluate the performances of the HNT-CD/Cur hybrid complex for application in the drug carrier field, preliminary investigations were performed; in particular, the kinetic release of the drug from the hybrid system and the antioxidant properties of the organic-inorganic complex were evaluated.
The release of curcumin loaded in HNT-CD hybrid was evaluated by the dialysis bag method using conditions designed to mimic physiological conditions (phosphate buffer pH 7.4) and the obtained kinetic data (Equation (4)) are shown in Figure 3. As it is possible to note, in these conditions the HNT-CD hybrid retains almost the total amount of the loaded curcumin for at least 96 h. These results are very different from the curcumin kinetic release from a carrier system where to HNT, is covalently grafted a single cyclodextrin unit [33], in this latter case indeed, about 15 wt % of curcumin was released after 24 h (Figure 3), which followed an initial burst release. Therefore, the introduction of a hyper-reticulated cyclodextrin core onto the HNT surface was crucial to slow down the drug release in physiological conditions, obtaining a sustained release of the biological molecule within time. The kinetic data obtained were analyzed by several models to deep investigate the release behavior of curcumin from such a complex system ( Table 1). The results showed that the data are better fitted by a double exponential model (DEM) (R 2 = 0.9824) with k 1 = 0.2 ± 0.1 min −1 and k 2 = 0.0022 ± 0.0003 min −1 . According to the literature, the DEM describes a mechanism consisting of two parallel reactions involving two distinguishable species [34]. Therefore, in our case, we observed the release of curcumin from both the cyclodextrin cavity and the HNT lumen.
As reported in literature, the curcumin molecule possesses an intrinsic antioxidant activity due to its peculiar chemical structure. Due to this activity, curcumin has been shown to inhibit lipid peroxidation and to manifest free radical-scavenging activity, in particular toward oxygen and nitrogen-centered free radicals [35]. To evaluate if the molecule retains its antioxidant ability once encapsulated into the HNT-CD hybrid system, we assessed the radical trapping ability of the HNT-CD/Cur complex by studying the reaction with 2,2-diphenyl-1-picrylhydrazyl radical (DPPH).
The antioxidant capacity of the HNT-CD/Cur complex in MeOH was evaluated by plotting the percentage of inhibition of the free radical scavenging capacity of DPPH (Equation (5)) against the concentrations of different dispersions of HNT-CD/Cur (from 0.7 to 2.56 mg·mL −1 ) (Figure 4).
The purple DPPH solution quickly faded to yellow with the addition of a small amount of the HNT-CD/Cur complex indicating an excellent antioxidant activity, of the overall system, with an IC 50 lower than 0.7 mg·mL −1 (Figure 4). In the same experimental condition, pristine HNT did not exhibit any radical scavenging activity [36]. Therefore, the curcumin molecule loaded into the HNT-CD hybrid system preserves its antioxidant properties.
Undoubtedly, the results presented herein have gone one step further for curcumin delivery for the development of efficient carrier as alternative route for the drug administration to the already reported systems. Nanosponges for the delivery of curcumin have been already reported, but these systems do not ensure the sustained release that we have reached by combining the properties of halloysite and β-CD [37,38]. Furthermore, by comparing our preliminary results with the ones obtained using other hybrid systems for the delivery of curcumin [39], one can conclude, in a first attempt, that the as-prepared hyper-reticulated network could effectively improve the stability and release profile of curcumin in simulated physiological conditions, indicating their promising potential for oral delivery.  The kinetic data obtained were analyzed by several models to deep investigate the release behavior of curcumin from such a complex system ( Table 1). The results showed that the data are better fitted by a double exponential model (DEM) (R 2 = 0.9824) with k1 = 0.2 ± 0.1 min −1 and k2 = 0.0022 ± 0.0003 min −1 . According to the literature, the DEM describes a mechanism consisting of two parallel reactions involving two distinguishable species [34]. Therefore, in our case, we observed the release of curcumin from both the cyclodextrin cavity and the HNT lumen.
As reported in literature, the curcumin molecule possesses an intrinsic antioxidant activity due to its peculiar chemical structure. Due to this activity, curcumin has been shown to inhibit lipid peroxidation and to manifest free radical-scavenging activity, in particular toward oxygen and nitrogen-centered free radicals [35]. To evaluate if the molecule retains its antioxidant ability once encapsulated into the HNT-CD hybrid system, we assessed the radical trapping ability of the HNT-CD/Cur complex by studying the reaction with 2,2-diphenyl-1-picrylhydrazyl radical (DPPH).
The antioxidant capacity of the HNT-CD/Cur complex in MeOH was evaluated by plotting the percentage of inhibition of the free radical scavenging capacity of DPPH (Equation (5)) against the concentrations of different dispersions of HNT-CD/Cur (from 0.7 to 2.56 mg·mL −1 ) (Figure 4).
The purple DPPH solution quickly faded to yellow with the addition of a small amount of the HNT-CD/Cur complex indicating an excellent antioxidant activity, of the overall system, with an IC50 lower than 0.7 mg·mL −1 (Figure 4). In the same experimental condition, pristine HNT did not exhibit any radical scavenging activity [36]. Therefore, the curcumin molecule loaded into the HNT-CD hybrid system preserves its antioxidant properties.
Undoubtedly, the results presented herein have gone one step further for curcumin delivery for the development of efficient carrier as alternative route for the drug administration to the already reported systems. Nanosponges for the delivery of curcumin have been already reported, but these systems do not ensure the sustained release that we have reached by combining the properties of halloysite and β-CD [37,38]. Furthermore, by comparing our preliminary results with the ones obtained using other hybrid systems for the delivery of curcumin [39], one can conclude, in a first attempt, that the as-prepared hyper-reticulated network could effectively improve the stability and release profile of curcumin in simulated physiological conditions, indicating their promising potential for oral delivery.

Materials and Methods
HNT-CD hybrid was synthesized as reported elsewhere [31]. UV-vis measurements were performed using a Beckmann DU 650 spectrometer (Beckmann Instruments, Inc., Fullerton, CA, USA).
The specific surface area and pore site distribution was made by N 2 adsorption/desorption Nova 2200 Quantachrome Instruments analyzer (Quantachrome Instruments, Boynton Beach, FL, USA).

Synthesis of the HNT-CD Hybrid
Thiol-functionalized HNTs (200 mg) and heptakis-6-(tert-butyldimethylsilyl) 2-allyloxy-βcyclodextrin (300 mg) were weighed in an MW test tube provided with a cap and a catalytic amount of AIBN was added. The mixture was inserted in the MW apparatus at 100 • C, under constant stirring, for 1 h, with an initial set power of 80 W. Successively, the solid was filtered off, rinsed several times with CH 2 Cl 2 and CH 3 OH (until the unreacted reagents were not detected by TLC), and dried at 80 • C under vacuum.

Batch Adsorption Experiments
All adsorption experiments were conducted on sealed vessels containing 2.5 mg of HNT-CD hybrid and 1 mL of drug solution (2 × 10 −5 M) in HCl 0.1 N and phosphate buffer pH 6.0 at 298 K, respectively. The obtained dispersions were vigorously vortexed and shaken in a thermostatic shaker with a shaking of 200 rpm at 298 K overnight. The supernatants were obtained by centrifugation for detecting drug concentration via UV-vis spectrophotometer at the maximum absorption wavelength of each drug (280, 420, and 360 nm for silibinin, curcumin, and quercetin, respectively).
The loading efficiency (LE) was estimated by using the following equation: where C 0 and C e are initial and equilibrium concentrations of the drugs (M), respectively.

Loading of Curcumin in the HNT-CD Hybrid
To a dispersion of HNT-CD hybrid (100 mg) in water (5 mL), 1 mL of a solution 10 −2 M of curcumin in MeOH was added. The suspension was sonicated for 5 min, at an ultrasound power of 200 W and at 25 • C and then was evacuated for 3 cycles. The suspension was left under stirring for 24 h at room temperature. After this time, the powder was washed with water and then dried at 40 • C.
The amount of Cur in the supernatant solution was assessed by UV-vis investigations. The drug loading and encapsulation efficiencies (LD% and EE%, respectively) were calculated according to the following equations: where Cur HNT-CD and Cur TOT are the amount of Cur loaded into HNT nanomaterials and the total feed Cur, respectively, and m HNT-CD is the amount of HNT-CD hybrid.

Drug Release Study
The release of Cur from the HNT-CD hybrid was done as follows: 20 mg of the sample were dispersed in 1 mL of dissolution medium and transferred into a dialysis membrane (Medicell International Ltd., London, UK; MWCO 12-14000 with a diameter of 21.5 mm). Phosphate buffer (0.05 M, pH 7.4, (fresh prepared)) was used as the release medium. Subsequently, the membrane was put in a round bottom flask containing 10 mL of the release medium at 37 • C and stirred. At fixed time, 1 mL of the release medium has been withdrawn and analyzed by UV-vis measurements. To keep constant the volume of the release medium, 1 mL of fresh solution has been used to replace the collected one. Total amounts of drug released (F t ) were calculated as follows: where V m and C t are the volume and the concentration of the drug at time t. V a is the volume of the sample withdrawn, and C i is the drug concentration at time i (i < t).
To determine the release mechanism, the amount of released molecules vs. time was studied using the first-order, Korsemeyer-Peppas, Higuchi, Hixon, and Crowell and the double exponential (DEM) mathematical models where M t is the drug release fraction at time t, M 0 is the initial amount of drug in the HNT-CD hybrid, M HNT-CD is the amount of drug remaining into HNT-CD at time t, k is the kinetic release constant of the respective equations, t is the release time, and n is the characteristic diffusion exponent, depending on the release mechanism and the geometry of device. If Fickian diffusion occurred, n decreased to 0.5 for slab/cylinder/sphere. If non-Fickian (anomalous) diffusion dominated, the n value was between the above value corresponding to the polymer chain relaxation (n < 1) for slab/cylinder/sphere. Thus, through determination of the n value, the drugs release mechanism could be identified.

Antioxidant Activity
The antioxidant properties were evaluated using the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH•) method. This radical is commonly used to evaluate the antioxidant properties of natural products, as the purple free radical is transformed by reductants to the yellow hydrazine through a formal hydrogen atom transfer reaction. The interaction of HNT-CD/Cur with DPPH radicals resulted in fast decoloration of the DPPH solution. Radical scavenging activity was determined following as follows. The HNT-CD/Cur hybrid (concentration ranging from 0.7 to 2.65 mg·mL −1 ) was dispersed in 1 mL of MeOH. One milliliter of DPPH solution (1.5 × 10 −4 M) in MeOH was then added to the tested sample. The mixture was then vigorously mixed and allowed to stand at room temperature in the dark for 60 min. The inhibition of free radical DPPH in percent (I%) was calculated as: where A c is the absorbance of the DDPH solution without HNT-CD/Cur (λ = 523 nm), and A HNT-CD/Cur is the absorbance of the test sample. All tests were carried out in duplicate, and inhibition percentages were reported as mean ± SD.

Conclusions
We investigated the ability of an inorganic-organic hybrid based on halloysite and hyper-reticulated cyclodextrin as a carrier for polyphenolic compounds. Preliminary studies highlighted that the carrier is promising for the delivery of quercetin and curcumin and, in the last case, can act as reservoir for the prolonged release of the drug over 96 h. Antioxidant measurements showed that the curcumin in the hybrid retains its properties, which could be beneficial for the treatment of several pathologies. Current work is devoted to assessing the feasibility of the hybrid system antioxidant/antitumoral agent by means of in vitro and in vivo studies. In this context, the hybrid system developed, still showing unreacted groups available for further functionalization, could be a promising agent for the delivery of polyphenolic compounds by improving their cellular uptake and consequently their therapeutic efficacy.


extfieldsofstudy: Chemistry Medicine

s2fieldsofstudy: Biology Chemistry

text:
Oxidative DNA damage induced by ROS-modulating agents with the ability to target DNA: A comparison of the biological characteristics of citrus pectin and apple pectin

DNA targeting anticancer agents have been very successful in clinic, especially, when used in combinatorial therapy. But unfortunately, they often exhibit high levels of toxicity towards normal cells. Hence, much effort has been put into finding agents with more selectivity, and less toxicity. Pectins are natural polysaccharides, and beneficial nutritional fibers that have attracted attentions due to their antitumor properties. However, their molecular targets, and mechanism of action are widely unknown. Here, we have reported that citrus pectin (CP) and apple pectin (AP) selectively suppress viability in MDA-MB-231, MCF-7 and T47D human Breast cancer cells, while non-toxic to L929 normal cells. Upon CP, and AP treatments, cancer cells’ ROS content increased rapidly, and led to the collapse of the mitochondrial transmembrane potential which functions upstream of the caspase-dependent apoptosis. CP and AP treated cancer cells were also arrested at the S and G1 or G2/M phases of the cell cycle, respectively. Furthermore, mRNA expression of Galectin-3 (a multi-functional lectin involved in cell adhesion, cell cycle, and apoptosis) reduced in both CP and AP treated cells. Growth inhibition of MDA-MB-231 cells by CP, and AP was concomitant with DNA damage (oxidation, and strand breaks). In this context, in an effort to clarify the mechanism of action, we showed that CP, and AP are able to interact with DNA. The strength and mode of DNA binding were established by spectroscopy techniques. We demonstrated that CP, and AP bind to dsDNA by intercalation, and groove binding/partial intercalation, respectively. In conclusion, our findings suggest that CP, and AP induce apoptosis in MDA-MB-231 cells by increasing the release of ROS, which may be related to the mitochondrial apoptosis pathway, and direct interactions with DNA. Our data indicate that these compounds may be potentially useful in cancer treatment.

CP and AP inhibited Breast cancer cells growth. Growth inhibition induced by CP, and AP after 48,
and 72 h treatments in three human Breast cancer cell lines including MDA-MB-231 (ER−/PR−/HER-2-), MCF-7, and T47D (ER+/PR+, HER-2-), and non-tumorigenic fibroblast L929 cells was evaluated. As it is shown in Fig. 1, treated samples exhibited a dose, and time dependent decline in viability compared to control groups. However, both pectins had much less toxicity against non-tumorigenic L929 cells. As indicated in Fig. 1, CP has reduced viability in T47D, MCF-7, and MDA-MB-231 in 72 h treatment, while no significant inhibition was observed at 48 h, as it is corroborated by their high IC50 values. In addition, incubating cells with 0 to 500 μg/ml of AP for 48 and 72 h exerted high cytotoxicity on MDA-MB-231 and T47D cells, while it did not induce significant cytotoxicity against MCF-7 cells (Fig. 1). As MTT assay results revealed both pectins were cytotoxic toward human Breast cancer cells especially in 72 h treatment, and did not affect non-tumorigenic L929 cells. Furthermore, AP (IC50 = 80.9 ± 1.57 μg/ml) exhibited the highest anti-proliferative activity against MDA-MB-231 cells in 72 h treatment, when compared with MCF-7, and T47D. Since among the examined Breast cancer cell lines, MDA-MB-231 cells are the most invasive cells with the highest proliferation, and metastasis power, we preferred to use this cell line to continue our studies.
CP and AP cause apoptosis, and DNA fragmentation in MDA-MB-231 cells. As it was speculated from MTT assay, the decrease in cell viability could be related to the apoptotic effect of the compounds. Therefore, SCIenTIfIC REPORtS | (2018) 8:13902 | DOI: 10.1038/s41598-018-32308-2 all three Breast cancer cell lines were treated with IC50 concentrations of CP, and AP for 24 h. Initial apoptosis assessment by using invert microscope exhibited vivid morphological alterations associated with apoptosis in treated cells, such as cell shrinkage, irregular morphology, roundness, reduced cell density, and cell membrane blebbing compared with the control ( Fig. 2A) 29 . Fluorescent microscope images also showed various extents of apoptosis specific morphological alterations, such as fragmentation and membrane disruption in the nucleus of CP, and AP treated cells as well as chromatin condensation, and increased fluorescence intensity of the nucleus (Fig. 2B), which altogether indicated that both compounds might have promoted apoptotic cell death in all three Breast cancer cell lines. Phosphatidylserine (PS) translocation into the exterior layer of the plasma membrane is one of the first signs of early apoptosis, and due to its excellent binding affinity to Annexin V protein, its detection is a good indicator in estimating apoptosis 30 . To further confirm apoptosis, MDA-MB-231 cells treated with 800 μg/ml (2x IC50) CP, and 200 μg/ml (2x IC50) AP were subjected to Annexin V-FITC apoptosis detection kit. In flow cytometry analysis of untreated cells, the percentages of viable, early apoptotic, late apoptotic, and necrotic cells were 99.55%, 0.38%, 0.00%, and 0.07%, respectively. However, after 4 h of treatment with CP, and AP, the population of early apoptotic cells (Annexin V-positive, PI-negative) increased drastically to 33.18%, and 37.37%, respectively. As it is shown in Fig. 3A, the late apoptotic cells (Annexin V and PI positive) population did not significantly change (0.00% in untreated cells to 0.4% in CP treated, and 0.97% in AP treated cells), While, there was a trivial increase in necrotic cells (Annexin V-negative, PI-positive), ranging from 2.98% in CP treated to 11.02% in AP treated cells. DNA degradation in to oligonucleosome-size fragments is another typical characteristic of cell death, and can be detected by TUNEL assay. After treating MDA-MB-231 cells with 800 μg/ml CP, and 200 μg/ml AP for 4 h, DNA fragmentation was quantified by flow cytometry, and Compared to untreated cells up to 43.8%, and 22.3% apoptosis was observed in treated cells respectively (Fig. 2C).
AP treated MDA-MB-231 cells inhibit cell cycle at G1, and G2/M phases, but CP at the S phase. CP and AP interventions in the cell cycle were inspected in MDA-MB-231 cells by measuring DNA content via propidium iodide (PI) staining. As Flow cytometry FACS analysis showed, treatment with 400 and 800 μg/ml of CP for 24 h increased cells population in the S phase fraction from 8.3% in controls to 13.2%, and 25.4% in the treated samples, respectively. However, treatment with AP led to the increase of the cell population in the G0/G1 phase from 64.2% to 69.4% (100 μg/ml), and 73.7% (200 μg/ml), furthermore, as it is shown in Fig. 3B, the MDA-MB-231 cells population in the G2/M phase has increased from 8.2% in controls to 10.3%, and 12.6% in the cells treated with 100 and 200 μg/ml of AP, respectively. CP and AP decrease Nitric Oxide (NO) production. The amount of NO production in the supernatant of MDA-MB-231 cells after 12 h treatment with CP and AP was determined with Griess reagent. During this period, the NO level decreased remarkably in the cells supernatant compared to the control group (Fig. 4A). Treating cells with 800 μg/ml CP, and 200 μg/ml AP respectively released 3.57 ± 0.15, and 2.53 ± 0.55 μM of NO, respectively, which were significantly lower than the control (7.99 ± 0.6 μM). Intracellular ROS overproduction and mitochondrial membrane depolarization are involved in CP and AP induced apoptosis. ROS has a pivotal role in activating cell death pathways. To study the impact of CP, and AP on oxidative stress, intracellular ROS production in CP, and AP treated MDA-MB-231 cells was measured by DAFH-DA probe. As illustrated in Fig. 4B, the ROS levels generated in response to (400, 800 μg/ml) CP, and (100, 200 μg/ml) AP treatments were significantly higher than untreated cells, which indicates that apoptosis is mediated by ROS production, and may have altered the cellular redox status. Excessive ROS production can influence mitochondrial functions, and initiate mitochondrial mediated cell death via disruption of mitochondrial membrane potential (MMP or ΔΨm) in cancer cells [30][31][32] . ΔΨm was monitored by Rhodamine123 fluorescent probe. Unlike viable cells, mitochondria in apoptotic and necrotic cells cannot sequester Rhodamine123 into aggregate, and diffuse it out in monomeric form, causing a shift in its fluorescence emission. Fig. 4C shows FL-1 plots of Rhodamine123 stained MDA-MB-231 cells, and the percentages of apoptotic cells analyzed by flow cytometer in different treated groups. Results obtained from stained cells, showed a significant dose dependent reduction of MMP in both CP, and AP treated cells. Furthermore, the observed loss of MMP induced by CP was much higher compared to AP. A parallel change in ROS production and MMP value suggests that mitochondria are the major intracellular sources and primary targets of ROS, and CP or AP-mediated ROS overproduction may have caused mitochondrial dysfunction. Galectin-3 mRNA expression. As the data obtained from real-time PCR analysis indicated, MDA-MB-231 cells treatment with 800 μg/ml of CP, and 200 μg/ml of AP for 4 h have reduced galectin-3 mRNA expression 0.48 (CI, 0.43-0.55) and 0.78 (CI, 0.73-0.85) folds compared to untreated control, respectively. However, the reduction in CP treated cells was more pronounced, and has reduces galectine-3 expression more than 50%.

DNA oxidation and DNA strand break in MDA-MB-231 cells induced by CP and AP. DNA
oxidation is an ongoing event in the everyday life of a cell, and leads to the formation of different lesions including oxidized bases, apurinic/apyrimidinic (AP) sites, and DNA single-and/or double-strand breaks. Compared to other DNA bases, guanine is more prone to DNA oxidation due to its low oxidation potential. 8-hydroxy-2′-deoxyguanosine (8-oxo-dG) is the most common form of oxidized guanine, which is produced by the addition of the hydroxyl radical to the C8 position of the guanine ring, which can then be either oxidized to 8-oxo-dG 33,34 . Increased levels of ROS in CP, and AP treated MDA-MB-231 cells may cause DNA damage. Hence, to explore the effect of both compounds on DNA oxidation, production of 8-oxo-dG was evaluated. As shown in (Fig. 5A), treating MDA-MB-231 cells for 2 h with 400, and 800 μg/ml of CP, and 100 and 200 μg/ml of AP have resulted in increased formation of 8-oxo (362, 470 pg/ml), and (189, 245 pg/ml) respectively in the genomic DNA. Comet assay was used to quantify the extent of DNA damage. CP and AP compounds have induced DNA strand breaks in MDA-MB-231 cells compared to the control. Analysis of approximately 100 cells after 2 h treatment with 2x IC50 concentrations of CP, and AP revealed a significant increase in DNA strand breaks in response to CP, and AP treatments in MDA-MB-231 cells compared to untreated cells. However, CP treatment has caused significantly greater DNA damage to the cells. As shown in Fig. 5B, the migrated cell nucleus with DNA strand breaks in the CP and AP treated cells and the fluorescence intensities of the migrated damaged DNA in the broom-shaped tails of the comets were stronger than in the controls. The comet assay analysis results are presented in Box and whiskers plots (Fig. 5C). These results indicate that the apoptosis induced by CP, and AP is tightly correlated with DNA damage.
CP inhibits MDA-MB-231 cells via apoptotic cell death at higher concentrations in multicellular spheroids. Monolayer or two-dimensional (2D) cell culture is conventionally used in cytotoxicity assessments. However, this model is unable to reproduce the physiological conditions, and biological barriers that a cytotoxic compound faces to reach cancer cells in a tumor. Therefore, to partially overcome such limitations, three-dimensional (3D) cell cultures or multicellular spheroids, have been devised to better mimic the physiological environment of tumor, and the drugs' effects on cancer cells 35,36 .
In this study, large spheroids (starting from about 500 μm in diameter as determined by analysis of spheroid images by ImageJ software), formed by Hanging Drop and Liquid Overlay techniques, were considered as the in vitro 3D models of solid tumors. Three distinct zones were observable in these spheroids, a proliferative zone in the outer layer, a quiescent zone in the middle created due to restricted access to oxygen and nutrients, and a necrotic core, which all together resemble the cellular heterogeneity of a solid tumor in vivo (Fig. 6A). The percentage of cell death in MDA-MB-231 cell spheroids after CP treatment was 16%, 44%, 50%, and 52% for the wells containing 250, 500, 1000 and 2000 μg/ml CP, respectively (Fig. 6C). Our results indicated that MDA-MB-231 cells 3D cell culture was 2.5 times more resistant to CP treatment (IC50 = 1200 μg/ml) than its corresponding 2D cell culture. The expected increase in the dose response in MDA-MB-231 cells in 3D cell culture reflected the in vivo-like microenvironment created in the cell spheroids, which is generally more resistant to anti-cancer drugs than 2D cell cultures. As analysis of cell spheroid images by ImageJ software revealed, a significant 15 ± 3.2 percent reduction in the volume of the CP treated spheroids compared to controls was observed. Apoptosis was also estimated in CP treated spheroids via EB/AO, and AnnexinV-FITC/PI staining. In EB/AO staining, microscopic observations revealed a clear alteration in the dead cells population of the external proliferating, and the internal quiescent zones of the CP treated cell spheroids (Fig. 6B). To further strengthen the data obtained from EB/AO staining, AnnexinV-FITC/PI staining was also employed. As flow cytometry analysis revealed (Fig. 6D), CP in its IC50 concentration has strongly inhibited the proliferation of MDA-MB-231 cells in the treated spheroids. The percentage of apoptotic cells rose from 1.9% in untreated controls to 18% in CP treated spheroids. This data suggests that CP has inhibited MDA-MB-231 cells growth in spheroids through the induction of apoptosis. The cell cycle distribution was also evaluated by FACS analysis and showed that upon treatment of MDA-MB-231 cell spheroids with CP IC50 for 24 h, cells population in the S and G2 phases of the cell cycle has increased significantly. The population of S phase arrested cells was 5.37% and 18.65% respectively in untreated controls, and CP treated spheroids, which showed a 3.5 folds increase in CP treated spheroids (Fig. 6E). G2/M phase arrested cells have also increased from 11.4% in controls to 17.35% in CP treated spheroids.
Absorption spectra of the Interaction between DNA and CP/AP. Absorption spectroscopy is one of the most useful and effective techniques in DNA-drug interaction studies. This technique allows recognizing the existence of interactions between molecules and DNA, as well as specifying the mode of interaction by utilizing the perturbations in the spectral observation 37 . Binding of different compounds to DNA may create a strong stacking interaction between the compounds, and the base pairs of the DNA, and may cause the absorbance spectrum shows some degrees of hypo-or hyperchromism. Fig. 7 shows the spectra of unbound DNA, CP, AP, and the DNA-CP/AP complexes in phosphate buffer (0.1 M, pH = 7.4). In the UV region DNA displays two peaks, one at 260 nm (indole groups), and the other at 210 nm (sugar-phosphate groups), while, CP, and AP have no absorption in these areas. As shown in Fig. 7B, upon addition of increasing amounts of CP or AP to the DNA solution, a remarkable enhancement in the absorption of DNA was observed, and hyperchromism with small shift (red shift) in the position of 260 maximum absorption peak, and a significant shift in the 210 nm (red shift) absorption peak also took place, which altogether suggest that a complex between CP/AP, and DNA has been formed.

DNA and CP/AP interaction characterization by Ethidium Bromide displacement assay.
The heterocyclic moieties of ethidium bromide (EB) molecules are able to stack between the base pairs of the DNA double helix, and act as fluorescent tags, which make the DNA-EB complex highly fluorescent. This complex fluoresce upon excitation at 500 nm, and can be used as a reliable system to characterize the interaction between small molecules and DNA 37,38 . Following the addition of DNA-interactive molecules to the DNA-EB system, EB is forced to be replaced by the molecules, and a decrease in the fluorescence occurs. The degree of dye displacement,  Figure 8A-C, summarizes the binding of CP, and AP to the rat hepatocyte DNA at pH = 7.4. Upon addition of increasing amounts CP, and AP to the DNA-EB solution EB rapidly displaced in the complex until reaching a saturation point. Consequently, the fluorescence intensity of DNA-EB at around 600 nm gradually decreased, however, No significant shift in the maximum emission wavelength of DNA-EB was observed. The results indicated that, both compounds could quench the fluorescence of DNA-EB. Quenching processes are often categorized into dynamic and static quenching. Dynamic or collisional quenching is when the excited fluorophore makes contact with the quencher, in which non-radiative transition to the ground state took place. Whereas, in static quenching a stable non-fluorescence complex forms between the fluorophore and the quencher. Dynamic and static quenching can be distinguished by the Stern-Volmer equation (Fig. 8B1 and B2). As it is illustrated in Fig. 8B1 and B2, Stern-Volmer plots of both compounds are linear, suggesting that the dynamic quenching process might have occurred. Therefore, in order to calculate the binding constant (Ka), and the number of binding sites (n) modified Stern-Volmer equation was used ( Fig. 8C1 and C2). As it is shown in Fig. 8C1 and C2, the plots were linear, and the binding constant (Ka) for the interaction between DNA and CP was 5.1 × 10 4 M −1 , which indicated that CP had a high affinity towards dsDNA, and the binding mode should be intercalation. The calculated number of binding sites (n) for the CP-DNA complex was also 0.87. In the case of DNA-AP interaction the calculated Ka and n value were 1.7 × 10 3 M −1 , and 0.62, respectively. The Circular Dichroism (CD) Studies. CD spectroscopy is able to detect DNA conformational changes in the presence of DNA binding molecules 39 . In the CD spectroscopy, the unbound B-DNA is characterized by a negative band at 245 nm due to its right handed helicity, and a positive band at 275 nm due to base stacking. Small molecules electrostatic interactions, or groove binding do not perturb base stacking, and helicity bands significantly. In contrast, intercalation drastically alters the intensities of these two bands in favor of more stabilizing the B-DNA conformation 11,40 . The CD spectra of CP/AP-DNA complexes are shown in Fig. 6C and D. The free hepatocyte DNA exhibited the two conservative CD bands, however, at the presence of CP (75 μg/ml) the intensities of both negative and positive bands increased significantly, and accompanied by a vivid blue shift in the positive band (Fig. 7C). This indicates that CP predominantly intercalates between the base pairs of the DNA, and enhances the base stacking, and helicity. On the other hand, upon treating with 75 μg/ml of AP, the CD spectrum of the DNA exhibited an increase in the negative band intensity, and a low perturbation on the positive band (base stacking) without any significant shift (Fig. 7D), which reveals that the interaction between AP, and DNA is via partial intercalation, and groove binding.

Discussion
Diet, and nutrition have been considered as effective strategies in the prevention of Breast cancer 41 . Moreover, numerous studies indicate that a quite diverse category of dietary natural components can slow down Breast cancer progression at different stages through the induction of apoptosis, proliferation, angiogenesis, and metastasis inhibition as well as sensitization of Breast cancer cells to chemotherapy. Hence, natural dietary ingredients can serve not only in the prevention of Breast cancer, but also their bioactive components can be considered as possible antitumor drugs 42 .
One of the major obstacles in cancer therapy is multidrug resistance which causes the repeated reoccurrence of tumor at the primary site of incidence. To tackle this problem, recently various studies reported the anticancer potential of herbal extracts with minimal toxicity to normal cells. Taking these facts into consideration, two plant products (CP and AP) were selected based on their therapeutic potentials.
Pectin is a heteropolysaccharide found naturally in the cell wall of fruits, and vegetables with beneficial effects on human health 43 . In industrial scale, the main sources of pectin are apple and citrus fruits. Pectin's biological properties depends largely on its molecular weight, degree of esterification, and viscosity 20,44 . Pectin's function as an anticancer agent depends on its ability to induce apoptosis in cancer cells, and in different studies CP, and AP have been modified in different conditions to improve this ability. Previous studies have shown that CP, and AP can induce apoptosis in cancer cells, however, their mechanism of action has not been understood very well. In this study, we have investigated whether CP and AP in their intact forms are able to induce apoptosis in MDA-MB-231 cells, blocking galectin-3, and interact with genomic DNA. To examine this, the cytotoxicity of CP, and AP at different concentrations was assessed on T47D, MDA-MB-231, and MCF-7 cancer cells. As MTT assay results indicated, both compounds were capable of increasing cell death in Breast cancer cells in a dose, and time-dependent manner. A higher cytotoxicity was obtained when MDA-MB-231 cells were treated with AP (IC50 = 80 μg/ml), and T47D cells with CP (IC50 = 95 μg/ml) for 72 h, which also caused a dose-dependent reduction in the cells viability. Furthermore, apoptosis specific morphological alterations, and DNA fragmentation, were observed in treated cells as light and fluorescent microscopy, and tunnel assay indicated, which supported previous studies showing the antiproliferative, and apoptotic effects of pectin in Breast cancer cells 22 . It worth noting that, treating L929 normal cells with CP, and AP did not induce any cytotoxicity. The anti-proliferative effect of both pectins on malignant cells has been attributed to various mechanisms.  anti apoptotic mechanism of Gal-3 has remained to be elucidated, it is suggested that it functions via maintaining mitochondrial integrity, and caspase inhibition 51,52 . Previous studies suggested that galectin-3 may be a potent inhibitor of mitochondrial mediated intrinsic apoptosis in Breast epithelial cells 26 . Among the galectin-3 inhibitors, many researchers have focused on pectins due to their low toxicity. Furthermore, it has been demonstrated that the pectins antiproliferative activity is in direct relation with the amount of galectin-3 expression on the surface of the cancer cells 20 . The galactose residues in AP, and particularly CP, similar to many tumor-associated antigens (TAAs) are responsible for interaction with Gal-3; however, in contrast to TAAs their binding to Gal-3 exerts antiproliferative effects and could be one of the possible mechanisms that both compounds inhibit tumorigenesis. Many previous studies have focused on Modified Citrus Pectin (MCP) as Gal-3 inhibitor with the ability of binding to Gal-3 as a ligand and claiming that due to limited solubility in water CP and AP in their naive form are unable to interact with Gal-3. However, the latest studies have reported that Pectin is a water-soluble dietary fiber 53 , and exhibit biological activities, ranging from anti-inflammatory, and anti-microbial activities to cardiovascular protective role 54 . A representative compound GCS-100, is a modified polysaccharide extracted from the peel and pulp of citrus fruits with high pH and temperature treatment, and is a Gal-3 antagonist that inhibits myeloma cell growth in vitro 55 . In addition, RN1 is another homogeneous neutral polysaccharide, purified from the flower of Panax notoginseng binds to Gal-3 and significantly suppresses its mRNA expression and downregulates the protein expression of Gal-3 in a time-dependent manner 56 . In the current study, we have demonstrated that galectin-3 mRNA expression was reduced in MDA-MB-231 cells treated with CP, and AP in their naive form. Kolatsi-Joannou et al. have also reported that MCP did not alter galectin-3 mRNA levels initially (at 2 days), But there was a statistically significant reduction at day 14 57 . CP binding to gal-3 could be one of the possible mechanisms of CP inhibiting tumorigenesis because many tumor associated antigens bind to gal-3 through their galactose residues to promote the growth and progression of tumor.
Previous studies have suggested that nitric oxide overproduction mediates pectin induced apoptosis 58,59 , but quite the contrary, here we have shown that treating MDA-MB-231 cells with CP, and AP have reduced NO production significantly, and instead have caused a rapid increase in ROS production and loss of mitochondrial membrane potential, which provides new insights into the mechanism of cell death by CP, and AP. ROS plays various physiological functions, and has both pro-and anti-apoptotic roles according to its concentration. ROS overproduction has been correlated with apoptosis 60-63 , and since the ROS level has increased significantly in the presence of CP, and AP, it can be concluded that both compounds are capable of increasing the mitochondria membrane permeability in MDA-MB-231 cells, and inducing apoptosis via ROS accumulation. On the other hand, mitochondria are also responsible for intracellular ROS production and apoptosis and suggesting that ROS might be involved in the cytotoxicity induced by CP and AP in MDA-MB-231 cells. Alteration in mitochondrial membrane permeability leads to the formation of mitochondrial permeability transition pores (MPTP) which facilitates unchecked inflow of solutes and free radicals inside the cell. This uncontrolled inflow causes increase of osmotic pressure inside the inner mitochondrial complex and thus finally leads to the overall collapse of the mitochondrial membrane which is considered as the initial step for the activation of the intrinsic cell death pathways 64 .
Consequent analysis of cell cycle arrest, nuclear condensation staining and early apoptotic observation via Annexin/PI staining, DNA damage analysis through fragmentation and comet assay confirmed the apoptotic cell death process. ROS overload also prompts oxidative damage to the cell's biomacromolecules, which culminates in the cells dysfunction and death. ROS overproduction causes DNA damage via oxidation and DNA strand brakes 61,65 . The level of 8-oxo-dG which is the major product of DNA oxidation by reactive oxygen species, increased significantly after CP, and AP treatment in MDA-MB-231 cells. AP, and in particular CP have also increased DNA strand breaks as it was observed in the comet assay. The formation of DNA breaks clearly indicated the role of CP in causing structural damage to the DNA double helical structure by increased DNA oxidation in the CP treated cells with respect to untreated group.
According to our findings, both CP and AP had considerable cytotoxicity towards the examined Breast cancer cell lines. Initially, as MTT assay results indicated AP exhibited more cytotoxicity against MDA-MB-231 cells, however, in the course of our study, we concluded that CP is more efficient in inducing apoptosis in MDA-MB-231 cells. Some of very evident features of apoptosis such as DNA fragmentation, and mitochondrial membrane depolarization were more pronounced in CP treated MDA-MB-231 cells as TUNEL assay, and ΔΨm analysis showed, respectively. More importantly, the extent of DNA damage in the form of DNA oxidation, and DNA strand breaks was significantly higher in CP treated cells. In addition as it will be discussed below, the mode of CP-DNA interaction was intercalation, which in terms of DNA-drug binding is more favorable. Regarding these results, in order to assess the cytotoxicity of CP in physiological conditions that is more similar to tumor environment, we created compact MDA-MB-231 multicellular spheroids in the cell culture. Our data indicated that MDA-MB-231 cells were still sensitive, but, more resistant to CP treatment when grown in 3D (IC50 = 1200 μg/ml). Our data indicates that CP inhibits MDA-MB-231 spheroids cells' proliferation, and induces apoptosis by arresting cell cycle progression at the S⁄G2 phase.
Biologically important small molecules like steroids and carcinogens or some effective antitumor drugs such as anthracyclines predominantly interact with DNA within the cells. Hence, searching for DNA binding small molecules has attracted attentions, since they can provide a suitable platform, and useful clues for designing more efficient DNA targeting drugs 40 . In general, noncovalent binding of small molecules to double-helix DNA is categorized into three groups: (i) electrostatic interaction (ii) groove binding, and (iii) intercalation between the base pairs. Intercalative, and groove binding agents interacts with DNA within the grooves of the double helix, whereas, the electrostatic binding can take place outside the grooves. Intercalative binding however, is the most effective mode of DNA-drug interaction in terms of the antitumor activity of the compound 9,11 . Our experiments demonstrated that CP, and AP, in their naïve forms, exhibit significant in vitro cytotoxicity against Breast cancer cells in a dose-and time-dependent manner, and for the first time in this study, we showed that CP, and AP are able to interact with double-helix DNA. There are three classes of natural or organic synthetic polymers by charge; SCIenTIfIC REPORtS | (2018) 8:13902 | DOI:10.1038/s41598-018-32308-2 cationic, anionic and neutral polymers. Cationic polymers are well documented in many studies for binding to anionic DNA to provide a controlled release of DNA polyplexes. Some neutral polymers have shown the ability to interact or bind to DNA. There are many non-ionic neutral polymers including polyvinylpyrrolidone (PVP), polyvinylpolypyrrolidone (PVPP) and poly (4-vinylpyridine-N-oxide) which are able to interact with DNA. For example, the interaction between DNA and PVP is based on hydrogen bonding with the base-pair in the DNA major groove that may lead to improved cellular uptake of DNA and enhance the transfection efficiency 66 . In the present study, we attempted to investigate CP and AP neutral polymers for their interaction with the dsDNA. Among the techniques frequently used to detect the interaction between DNA and small molecules, we employed UV-vis absorption, fluorescence, and circular dichroism (CD) spectroscopy to investigate the binding mode of CP, and AP to DNA. The UV-vis studies revealed that the absorption peak of DNA at 260 nm increases gradually with increasing concentration of both CP, and AP, which suggested that the binding between the two compounds and DNA, had occurred. Fluorescence and CD studies also indicated that CP binds DNA via intercalation, whereas, AP can binds DNA via partial intercalation and groove binding. The binding constants (Ka) of the CP, and AP with DNA were 5.1 × 10 4 , and 1.7 × 10 3 M −1 at 310 K, respectively.
In conclusion, recent studies on pectins' anti-cancer properties mostly have focused on modified pectins originated from citrus, while, our results indicated that the pectic polysaccharides from apple and citrus in their naive forms are also capable of inducing apoptotic death in cancer cells. Furthermore, we have suggested that ROS over production via increasing the mitochondria membrane permeability might be the underlying apoptosis mechanism of both compounds. ROS accumulation also has caused oxidation and strands breaks in the DNA of CP and AP treated breast cancer cells. Moreover, our drug-DNA interaction studies also indicated that CP binds DNA via intercalation, whereas, AP can binds DNA via partial intercalation and groove binding. These data suggest CP vs AP can have a potential role in prevention and treatment of human Breast cancer.

Materials and Methods
Cell cultures, treatments, and cell survival evaluations. MCF-7, T47D, and MDA-MB-231 human Breast cancer cell lines, and normal fibroblast "L929" cell line were obtained from pasture cell bank in Iran. Cells were grown in RPMI 1640 (Gibco) media supplemented with 1% streptomycin/penicillin, and 10% fetal bovine serum (Gibco). All cell lines were grown in a humidified atmosphere at 37 °C with 5% CO 2 . A stock solution of 1% (W/V) CP or AP was freshly prepared for each experiment. In detail, One gram of both pectins dissolved in 10 ml of distilled water under continuous stirring at 70 °C until a homogenous solution was obtained. The solution was sonicated using an ultrasonic system at 140 W for 5 min at 30 °C, and stored at 4 °C. After adjusting the pH to 7.4, the pectin solutions were diluted further with the culture media until achieving the desired concentration. It should be noted that CP, and AP were soluble in pure water and were stable at room temperature at neutral pH.
The cellular viability of CP and AP treated MDA-MB-231, MCF-7, T47D and L929 cells was evaluated by MTT [3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium Bromide] (Sigma-Aldrich) assay and 50% inhibitory concentration (IC50) was calculated for each cell line. Briefly, all cells were seeded in 96-well plates to a density of about 1.4 × 10 4 cells/well and supplemented with 200 μl medium per well. 24 h later, cells were treated with 0-500 μg/ml of CP and AP for 48 and 72 h at 37 °C in 5% CO 2 . After treatment, the medium was replaced with 200 μl freshly prepared MTT solution (0.5 mg/ml in phosphate-buffered saline) and incubated at 37 °C for 4 h. Subsequently, MTT solution was discarded, and 100 μl/well DMSO was added to solubilize the formazan crystals and the absorbance was measured at 492 nm using an ELISA reader (Model wave xs2, BioTek, USA). The concentration of pectins with 50% cell growth inhibition was reported as the IC50 value.

Microscopic observation of apoptosis in Breast cancer cells.
Apoptosis specific morphological alteration in treated cells was observed, and AO/EB viability staining assay was performed to evaluate the ratio of Live/ Dead cells. In brief, 2.1 × 10 6 MDA-MB-231, T47D and MCF-7 cells were seeded in 6 well plates and after 24 h they were treated with CP and AP IC50 for 24 h. After incubation, both treated and untreated cells were observed under invert light microscope and imaged. Subsequently, cells were harvested, centrifuged to collect the cells, and resuspended in PBS. An EB/AO staining solution was then added to the suspension, and the stained cells were visualized and imaged by a fluorescence microscope (Axioskop 2plus, Ziess, Germany). Green cells with intact and condensed nuclei were identified as viable and early apoptotic cells, respectively. While, fluorescently red cells were assigned as late apoptotic and necrotic cells. Twenty randomly selected areas were imaged on each slide to ensure the acquired data were representative.

Detection of apoptosis in MDA-MB-231 cells by Annexin V-FITC /PI staining and TUNEL
assay. To further confirm, and also quantify the percentage of cells undergoing apoptosis, Annexin V-FITC /PI staining and TUNEL Assay were implemented. The primary experimental procedure for both assays was to seed 3.5 × 10 5 cells/well MDA-MB-231 cells in six-well plates, and treating them 24 h later either with CP (800 μg/ ml) or AP (200 μg/ml) for 4 h. After treatment, cells were collected, washed with PBS, resuspended in binding buffer and stained with Annexin V-FITC and PI according to the manufacturer's protocol. Flow cytometry (Partec PAS, Germany) analysis was performed to identifying the distribution of differentially labeled cells, and the results were analyzed with Flomax software. DNA fragmentation in CP and AP treated MDA-MB-231 cells was detected by using In situ cell death detection kit, fluorescein (Roche) which is based on Terminal deoxyribonucleotidyl transferase dUTP nick end labeling (TUNEL) method. In brief, after treatment, cells were washed with PBS, and recollected. The pellets were resuspended in a fixing solution of 4% paraformaldehyde in PBS (pH 7.4) and incubated for 1 h at room temperature with agitation. Afterwards, the fixed cells were permeabilized with 1% Triton X100 in a 0.1% sodium citrate solution. Permeabilization solution was then discarded after centrifugation and cells were washing with PBS twice. Subsequently, the staining solution was added to the pellets and incubated for 1 h at 37 °C in the dark. The Staining solution contained TdT enzyme and its substrate and after reaching 80% confluency were treated with 800 and 200 μg/ml of CP, and AP for 4 h, respectively. Total RNA was isolated by TRIzol Reagent according to the manufacturer's protocol (LifeTechnologies Inc.), and cleaned up with RNase-free DNase I (Bio Basic). Isolated RNA was then quantified spectrophotometrically, and its integrity was verified by agarose gel electrophoresis. Approximately 1 µg of RNA was subjected to Prime Script RT reagent Kit (Takara) and converted into cDNA for real time PCR assay. PCR reaction was performed in a total volume of 20 µl including 50 ng of cDNA, 1x HOT FIREPol EvaGreen qPCR Mix Plus (Solis BioDyne), and 10 µM of each primer. Thermal cycling was performed on a Rotor-Gene Q 6plex Platform (Qiagen) with the following settings: 15 min at 95 °C for initial denaturation, followed by 35 cycles of 15 s at 95 °C, 20 s at 60 °C and 30 s at 72 °C. All reactions were performed in duplicates for each experimental condition, and results were analyzed with Rotor-Gene Q 2.1.0 software (Qiagen). Gal-3 mRNA expression was normalized by PGK (phosphoglycerate kinase) mRNA expression levels, and expressed as fold changes relative to controls using ∆∆Ct method. Primers for galectin-3 and PGK were designed with the assistance of NCBI primer designing tool, Primer-Blast. The nucleotide sequences for the primer pairs were as follows: Galectin-3: Forward 5′GTGAAGCCCAATGCAAACAGA3′, Reverse 5′AGCGTGGGTTAAAGTGGAAGG3′, PGK: Forward 5′GGCATACCTGCTGGCTGGATG3′, Reverse 5′ACAGGACCATTCCACACAATCTGC3′. DNA oxidation. In nucleic acids, 8-oxo-2′-deoxyguanosine (8-oxo-dG) is the major lesion caused by oxidation, and a good indicator for measuring DNA oxidation. Genomic DNA was extracted from MDA-MB-231 cells after 2 h treatment with CP (400, 800 μg/ml) and AP (100, 200 μg/ml) as well as untreated controls. Standard phenol/chloroform extraction procedure with a minor modification was used to extract genomic DNA. In order to prevent accidental DNA oxidation during extraction procedure, cell lysis buffer was supplemented with 1 mM ascorbic acid and 1 mM deferoxamine (iron chelator). 5 µg of extracted DNA was then digested with nuclease P1 (sigma, N8630) to release nucleotides, and then dephosphorylated by treating with alkaline phosphatase (NEB, M02903). Prepared samples were subsequently subjected to 8-Oxo detection ELISA kit (Cayman chemicals, 589320) in a total volume of 50 µl according to manufacturer's protocol. All samples were quantified in duplicates, and 8-oxo-dG concentrations were reported in pg/ml. pipetted in a 37 °C PBS solution containing 1% LMPA (low melting point agarose), and were spread over a microscope slides precoated with 1% conventional agarose under a coverslip, and chilled in 4 °C for 15 min to solidify. After removal of the coverslip, the slides were submerged for 1 h in cold lysis buffer (10 mM Tris, 2.5 M NaCl, 100 mM EDTA and 1% freshly added Triton X100, pH 10), and then immersed in denaturing buffer (1 mM EDTA and 300 mM NaOH, pH 13) at 4 °C for 30 min to unwind the DNA in the lysed cells. Subsequently, slides were electrophoresed under the same denaturing condition for 30 min at 0.6 V/cm. after electrophoresis, slides were dipped in neutralization solution (0.4 M Tris, pH 7.5) for 5 min, stained with 1x SYBRE green, and imaged by a fluorescent microscope (Axioscope 2 plus, Zeiss, Germany). Open Comet software was used to analyze the Comet images, and the presented data are the results of analysis of at least 100 comets in each sample.
Cell spheroid Generation from MDA-MB-231 breast cancer cell line. In order to generate suitable cell spheroids for screening the compounds, a combination of hanging drop (HD) and liquid overlay (LO) techniques was used as described previously 61 . In brief, 50 µl drops of a cell suspension with the concentration of about 1.0 × 10 5 cells/ml (5000 cells/drop) were deposited on the interior surface of a petri dish lid, and were incubated until the spheroids were formed. The spheroids were then transferred to a round bottom 96-well microplate precoated with 50 μl of 0.5% poly-HEMA (sigma) in 95% ethanol, to prevent the cells to adhere to the bottom of the microplate, and were supplemented with 200 µl culture medium to grow for 2 more days.
Evaluation of cell viability, apoptosis, and cell cycle distribution of the CP treated MDA-MB-231 cell spheroids. To evaluate the cellular viability, cell spheroids were treated for 48 h with 250-2000 μg/ ml of CP, and the MTT assay was employed the same as for the 2D cell culture. For estimation of apoptosis, MDA-MB-231 spheroids, pre-selected for volume and shape, were exposed to IC50 concentration of CP (1200 µg/ ml) for 24 h, stained with EB/AO, and observed under fluorescent microscope. Furthermore, in order to quantify the apoptotic cells population, CP treated spheroids were trypsinized, and the singled cell suspensions were stained with AnnexinV-FITC/PI. The percentage of apoptotic cells was determined by flow cytometry analysis. The same CP treatment and cells preparation was also used for the cycle analysis, and the test was performed the same as for the 2D cell culture. The cell cycle distribution was analyzed by flowJo software.
UV Absorption Spectra Measurements. The absorption spectra were acquired on a Cary 100 Bio-model, Australia spectrophotometer. Rat hepatocyte genomic DNA was isolated by standard phenol-chloroform procedure. Isolated DNA was quantified by measuring the absorbance at 260 nm, and its quality was evaluated by both monitoring the A260/A280 ratio ≥1.8, and agarose gel electrophoresis. DNA stock solutions were prepared in 0.1 M phosphate buffer (pH 7.4), and stored at −20 °C. CP, and AP UV Absorption Spectra studies were performed with a fixed concentration of rat hepatocyte DNA (50 μg/ml) in phosphate buffer at room temperature by spectrophotometry over a wavelength range of 190-400 nm. Initially, the absorbance of CP and AP was measured in phosphate buffer at concentrations ranging from 9 to 150 μg/ml, and 9 to 300 μg/ml respectively, the solvent was taken as reference, and then similar procedure was carried out after addition of constant amounts of DNA (50 μg/ml).
Fluorescence Spectra Measurements. The fluorescence spectra were obtained using a Carry eclipse (Australia) spectrophotometer. Ethidium bromide (EB) fluorescence quenching assay (competitive binding studies) was performed by using DNA-EB system, in which, the fluorescence of the DNA is greatly enhanced by the intercalation of EB between DNA base pairs. The fluorescence quenching was recorded by the addition of different concentrations of CP (0-7.3 μM) and AP (0-8 μM) to a solution containing both 2.6 μM EB and 50 μg/ml DNA. Measurements were done in the range of 530-700 nm upon excitation at 500 nm by applying a 1 cm path length fluorescence cuvette at 310 K. The slit width was set as 10 nm/10 nm for excitation/emission, respectively. Circular Dichroism (CD) studies. CD spectra were recorded on a Circular Dichroism 215, Aviv, USA spectrophotometer using a Quartz cuvette with a path length of 1 cm in far-UV range at wavelengths between 220 and 320 nm at room temperature, and pH = 7.4. All the reported CD data were obtained by using 75 μg/ml CP and AP in a 100 μg/ml DNA solution. The changes in CD spectra were monitored against a blank and the spectrum of the buffer solution was recorded and subtracted from the spectra of DNA and DNA-CP/AP complex. Statistical Analysis. Experimental data were processed by Microsoft Excel 2013 software. Results were obtained from three or more independent experiments, and presented as mean ± standard deviation. T-test was used to determine the significance between the means, and p-values not greater than 0.05 were considered significant.


extfieldsofstudy: Biology Medicine

text:
Developing genome-wide SNPs and constructing an ultrahigh-density linkage map in oil palm

Oil palm (Elaeis guineensis Jacq.) is the leading oil-producing crops and the most important edible oil resource worldwide. DNA markers and genetic linkage maps are essential resources for marker-assisted selection to accelerate genetic improvement. We conducted RAD-seq on an Illumina NextSeq500 to discover genome-wide SNPs, and used the SNPs to construct a linkage map for an oil palm (Tenera) population derived from a cross between a Deli Dura and an AVROS Pisifera. The RAD-seq produced 1,076 million single-end reads across the breeding population containing 155 trees. Mining this dataset detected 510,251 loci. After filtering out loci with low accuracy and more than 20% missing data, 11,394 SNPs were retained. Using these SNPs, in combination with 188 anchor SNPs and 123 microsatellites, we constructed a linkage map containing 10,023 markers covering 16 chromosomes. The map length is 2,938.2 cM with an average marker space of 0.29 cM. The large number of SNPs will supply ample choices of DNA markers in analysing the genetic diversity, population structure and evolution of oil palm. This high-density linkage map will contribute to mapping quantitative trait loci (QTL) for important traits, thus accelerating oil palm genetic improvement.

Results
Microsatellite marker analysis. A total of 123 out of 300 microsatellite markers selected were informative between the two parental palms, a Deli Dura and an AVROS Pisifera. Both parental trees were used to produce F 1 Tenera for production in Wilmar International Plantation. All the markers were used to genotype 153 F 1 palms from the F 1 production population. Finally, 123 microsatellite markers were mapped into the LGs together with the SNPs. The sequences of the microsatellite markers mapped are listed in Additional file Table S1. SNP genotyping using micro-chip assay. A set of 288 SNP markers on 16 chromosomes were used to genotype 153 palms and two parental palms by using the 96.96 Dynamic Array Chip (Fluidigm, Singapore), then 188 SNPs were mapped into the linkage groups (LGs) together with other markers in the palm population. The sequence and alleles of the SNP markers mapped are listed in Additional Table S1. Among the 188 SNPs, 39 SNPs were developed from genes associated with lipid synthesis based on the genome annotation of oil palm 33 . SNP discovery and genotyping using RAD-seq. Four RAD-seq libraries were constructed using 96 barcodes and the PstI-HF restriction endonuclease. A total of 1,076 million raw reads were obtained from the sequencing of 155 trees. After filtering out low quality and ambiguous barcodes, there remained 1,050 million reads (97.57% of total raw reads). The retained reads of the two parental palms were 11.73 and 8.89 million reads, respectively (Table 1). From the mapping population, 8 out of 153 progeny individuals were discarded from further analysis due to low coverage of sequencing reads. The average number of raw reads across all retained 145 progeny was 7.10 million.
Using the sequence data of the two parental samples, a catalogue containing 510,251 loci was built for SNP discovery. After removing the loci with >20% missing data across progeny, a total of 11,394 informative SNPs were genotyped and used for linkage map construction. Additional Table S1 lists, for each marker in the map, its alleles, flanking sequence, name of contig containing it, and its physical position in the contig.
Construction of a high-density linkage map. The 11,394 SNPs identified from RAD-seq, with another 188 SNPs from micro-chip array and 123 microsatellite markers, were used to construct the linkage map. Of these markers, 1,682 SNPs either could not be assigned to LGs or were assigned to small groups (marker number less than 50), or had segregation distortion (P < 0.001), were excluded from further analysis. The retained 10,023 markers were assigned to 16 LGs (Chromosomes) (Fig. 1). The linkage map contained 10,023 markers (9,900 SNPs and 123 microsatellites). The map covered a total length of 2,938.2 cM and had an average marker density of 0.29 cM. The lengths of LGs ranged from 53.47 cM for chromosome (Chr) 15 Table 2 and Additional Table S1.
Using the reference genome annotation resource 1 , we searched the genes associated with each SNP. Then 5,727 SNPs were annotated with gene information, with 3,975 SNPs located in intergenic regions. The number of SNPs with gene information in each of the 16 chromosomes ranged from 54 to 829 (Table 2 and Additional Table S1).

Discussion
After the RAD-seq approach was developed 34,35 , it was initially adopted in a population genomic study 36 . With the continuously decreasing cost of next-generation sequencing (NGS), RAD-seq has become a powerful approach for many research problems, from discovering thousands of SNPs for large-scale population genotyping purposes,  Table S1). The bar on left side represents the map length in cM, whereas the horizontal bars in each linkage group (chromosome) are positions of markers mapped.  to constructing the dense linkage maps for QTL mapping, scaffolding of newly sequenced genomes, and addressing a wide range of questions in molecular ecology and evolutionary biology 37 . Usually, ddRAD-seq with PstI-MspI is used for identification of SNPs in plants and animals 18,38,39 . The first SNP discovery in oil palm using ddRAD-seq with PstI-MspI was conducted with 3417 informative SNPs 9 . In a later study 32 , 2139 informative SNPs were identified using ddRAD-seq with PstI-MspI in an oil palm breeding population, and a high-density SNP genotyping array was developed 40 . In this study, we discovered more than 10,000 informative SNPs using a different approach: single digest RAD-seq with the PstI 35 , modified for a highly heterozygous breeding oil palm population. These indicate that both approaches of RAD-seq could facilitate SNP discovery and improvement of genomic resource. The significant difference in number of SNPs discovered between the two RAD-seq approaches could be caused by restriction enzyme selection, RAD-seq library preparation methods, coverage of sequences reads, as well as the heterozygosity of samples being used in the studies. The previous study showed that variance in read depth among individuals could be minimized, but the expected frequency of restriction cut was decided by a particular restriction enzyme 34 , hence ddRAD-seq with PstI-MspI could result in a lower digest efficiency of endonuclease and hence less common SNPs in the highly heterozygous oil palm samples. The previous study also indicated that the loci discovered across the genome showed the interaction between choice of restriction enzymes and the number of loci recovered from different parts of the genome 41 . Here we provide an evidence that the complexity reduction involving the restriction enzyme PstI-HF was adapted in order to avoid and lower the occurrence of repetitive regions of the high heterozygous oil palm genome. That, in combination with the 300-600 bp size selection for RAD library construction, the library single-end Illumina sequencing by Illumina NextSeq500 in our laboratory, the high throughput reads (150 bp per read) at sufficient depth of coverage which can provide sufficient contigs containing SNPs (Table 1), and reads sequenced with a mean coverage depth of 7~10× which should allow accurately genotyping at most sites across most individuals, means that a simple, highly efficient and lower cost RAD-seq platform for heterozygous oil palm samples has been constructed in this study. The entire process from DNA isolation, library preparation to sequence using Illumina NextSeq500, and SNP calling through sstacks pipeline, was relatively simple and fast. Furthermore, the overall cost of RAD-seq was economically efficient. Given the benefit of using this platform in our studies, it may be expected that utilization of this technology will become widely adopted in oil palm and other highly heterozygous samples for SNP discovery, genetic and genomic studies, and this approach has improved the tools developed, alleviating the marker limitation that researchers previously faced for studies of high heterozygous samples.
With the RAD-seq, we discovered over 10 K high-quality and informative SNP markers for oil palm. Among them, 5,727 SNPs are located in genes. These over 10 K SNPs will be useful in studies on ecology, evolution and linkage and QTL mapping for economically important traits to accelerate genetic improvement in oil palm. The genome-wide set of over 5 K SNP markers within genes could facilitate to identity causative SNPs within QTL for important traits, and can be useful in synteny analysis to understand more about genome evolution in plants 7 .
Based on SNPs discovered across the whole oil palm genome, we generated a genome-wide high-density linkage map containing 10,023 markers, distributed through all 16 chromosomes. The length is 2938.2 cM and the average marker density is 0.29 cM. Compared with previous studies, marker numbers in linkage maps have continued to increase, from 252 to 944 microsatellite-based markers 8,21-23 , to 1085 SNP-based markers 9 , and to the SNP-based ultra-high-density linkage map in this study. Also, in terms of marker density, our map is among the highest published to date, compared to the previous marker spacings of 1.26 cM 9 and 1.4 cM 8 . The high-density linkage map can enable QTL mapping for the gaining of insights into the genome-wide genetic architecture of economic traits, genomic evolutionary studies, genome assembly, revealing the patterns of chromosomal evolution, and linkage disequilibria assessing 4 . Such maps are frequently constructed using microsatellite markers, but microsatellite markers are not as abundant or easily scored as SNPs 4 . However, we noticed that in Chr 2 and 13, in a few positions, marker spaces were bigger than 20 cM. More markers should be mapped in these positions to reduce marker interval for genomic studies. Improvement of the platform of RAD-seq with high efficiency library preparation and bioinformatics procedures used to identify SNPs, as well as the use of large populations for oil palm, could be a feasible solution for this challenge.
In conclusion, we identified many SNPs using RAD-seq, supplying sufficient DNA markers for studying ecology and evolution of oil palm. Then, we constructed an ultra-high-density linkage map with 10,023 markers for African oil palm. To the best of our knowledge, this is the densest linkage map in oil palm. The linkage map will facilitate mapping QTL for economically important traits for MAS to accelerate genetic improvement in oil palm.

Materials and Methods
The palm population used for linkage mapping. A F 1 population was generated by crossing a Deli Dura (mother palm) and an AVROS Pisifera (father palm) in Wilmar International Plantation. Both parental trees were used to produce Tenera trees for production in Wilmar International Plantation. A total of 153 F 1 progenies (Tenera type) were planted into a plantation field in Indonesia in 2006. These F 1 trees were managed under the same conditions following the standard protocol of Wilmar Plantation in Indonesia (http://www.wilmar-international.com/our-business/tropical-oils/plantations/).

DNA extraction from palm leaves. DNA was isolated from young leaf samples using DNAeasy Plant Mini
Kit (Qiagen, Hilden, Germany) following the manufacturer's instruction. RNA was removed by RNAse digestion during lysis, and cell debris, precipitated proteins, and polysaccharides were removed by centrifugation through a QIAshredder spin column. Then, the lysate was loaded into the DNEasy Plant Mini spin column, in which DNA selectively binds to the silica membrane. Finally, the remaining contaminants and enzyme inhibitors were removed in one or two efficient wash steps and pure DNA was then eluted in 1xTE buffer. The quality of DNA was checked on 1.0% agarose gels. DNA quantification was conducted using Nanodrop 2000 (Nanodrop, Wilmington, DE, USA). For RAD-seq library construction, the concentration of double-strand DNA was also measured using  Additional Table S1) were developed by us 22 using a method described previously 42 Primers of each microsatellite were designed in the repeat-flanking regions using PrimerSelect (DNAstar, CA, USA). To genotype the microsartellites on the automatic DNA sequencer ABI3730xl (Applied Biosystems, Foster City, CA, USA) one primer for each marker was labelled with a fluorescent dye (i.e. either HEX or 6-FAM). The microsatellite markers were genotyped individually with PCR and electrophoresis for polymorphism and informativeness between the two parental trees. Each microsatellite marker was amplified using PCR, which contained 40 ng of genomic DNA, 2.0 units of DNA Taq Genotyping of SNPs using micro-chip based on allele-specific competitive PCR assay. From over 10,000 SNPs identified in the 17 re-sequenced palm genomes 33 , 288 SNPs were randomly selected from 16 chromosomes as anchor markers based on their genomic position from previously published genome 1 . The SNPs were genotyped using allele-specific competitive PCR assay according to our method 43 , which is a homogenous, fluorescence-based genotyping technology that is based on allele-specific oligo extension and fluorescence resonance energy transfer for signal generation. The SNP assay consisted of three parts. Firstly, the primer design: the SNP allele-specific forward primers and a common reverse primer were designed based on an approximately 120 bp long genomic sequence flanking each SNP site (http://bioinfo.biotec.or.th/WASP/). The two probes with fluoresce-nt labels (FAM and HEX, respectively) complement the tail sequence of the allele-specific forward primers. Secondly, loading the assay components: DNA sample (50 ng/µl), 2× Biotium Fast Probe qPCR Master Mix buffer, forward primer mix (0.25 µM), reverse primer (0.25 µM), probe primers mix (10 µM), ROX (50×), 20× sample loading reagent, and two probe primers were mixed together with the DNA sample and then loaded into the sample cell, and the two forward primers and the reverse primer were Construction of RAD-seq libraries and sequencing. The RAD-seq libraries were constructed according to the published method 35 with some modifications. A total of 153 trees of the breeding population and two parental trees were used for library construction. Briefly, 500 ng of genomic DNA from each individual was digested with high-fidelity PstI (New England Biolabs, Ipswich, MA, USA), and the digested fragments were then ligated to barcoded adapters. The ligation products with unique barcodes were pooled and sheared with Covaris M220 (Covaris, Woburn, MA USA) with a DNA fragment peak of 500 bp. The sheared DNA was purified with MinElute PCR purification kit (Qiagen, Hilden, Germany) and then size selected (300-600 bp) with Agencourt AMPure XP beads (Beckman coulter life sciences, Brea, CA, USA). Recovered DNA was blunt end repaired using Quick Blunting Kit (New England Biolabs, Ipswich, MA) and then A-overhangs were added with klenow fragment (New England Biolabs, Ipswich, MA, USA). Sequencing adaptor P 2 was then ligated to the DNA fragments with T4 DNA ligase (New England Biolabs, Ipswich, MA, USA). Finally, the library was amplified using Phusion ® High-Fidelity DNA Polymerase (New England Biolabs, Ipswich, MA, USA) and the PCR products were purified using Agencourt AMPure XP beads. The recovered library was quantified with Illumina library quantification kit (Kapa Biosystems, Wilmington, MA, USA) in the MyiQ Thermal Cycler (Bio-Rad, Hercules, CA, USA) and sequenced for 1 × 150 bp reads on NextSeq500 (Illumina, San Diego, CA, USA).

Genotyping of microsatellites. The microsatellites used in this study (see
Identifying of SNP from RAD-seq and genotyping. The raw sequencing reads were demultiplexed and cleaned using the program process_radtags in the software package Stacks v1.42 34 . Any uncalled base and low quality reads were removed. To minimize the sequencing errors, all the clean reads were trimmed to 130 bp. In detail, only the reads with a limit of >Q30 for Illumina sequencing were used for further analysis, the SE reads were then trimmed to 130 bp from the 3′ end and that of any uncalled base were removed. The reads with an average score within sliding window of <10 were also removed. The cleaned reads were aligned to the oil palm reference genome 1 by the use of the program GSNAP with a maximum mismatch of 5 bp. Only the alignments of unique targets were used for stacks assembly using the program pstacks (−m, 3). The assembled stacks from the parents were used to build a catalogue of loci using the program cstacks in the Stacks package. The assembled stacks from each sample were then matched to the catalogue for SNP discovery using sstacks. Finally, the program genotypes was used to call SNPs across progeny. The genotyped SNPs were filtered with a cut-off of at least 10 × sequence depth and at most 20% missing data. Then the genes associated with SNP loci were searched against the oil palm genome annotation 1 . Markers heterozygous in any one parent or both parents were selected for further analysis. There are five different allelic patterns in all called markers: type nn × np (segregating 1:1) was homozygous in the male parent and heterozygous in the female parent; lm × ll (1:1) was heterozygous in the male parent and homozygous in the female parent; hk × hk (1:2:1) was heterozygous in both parents with two shared alleles; ef × eg (1:1:1:1) was heterozygous in both parents with two sex-specific alleles and one shared allele, and type ab × cd (1:1:1:1) was heterozygous in both parents with four different alleles.
Linkage map construction. Linkage analysis was performed for markers genotyped in F 1 palms derived from crossing Dura and Pisifera. Polymorphic markers from microsatellites and anchor SNPs, and SNPs from RAD-seq, were used to construct the linkage map. The genetic linkage map was generated using software Lep-MAP2 44,45 . Before linkage map analysis, the default dataTolerance value of 0.001 (χ 2 test, P < 0.001) was used to filter out markers with segregation distortion. Then the remaining loci were grouped into linkage groups (LGs) using a LOD score threshold of 10. The order and positions of the markers within their respective LGs were determined according to the recombination probabilities between loci, with the sex-averaged option selected. And the genetic maps were drawn using the software MapChart V2.2 46 . Finally, the LGs for the mapping population were numbered according to the published linkage map (Billotte et al. 21 ) based on the anchor markers and genomic sequence 1,21 . Data availability. The RAD-seq datasets supporting the conclusions of this article are available in the DDBJ database, accession number (BioProject Accession: PRJDB5817).