Natural product; PXR

Biodistribution of Umb via ex vivo fluorescence imaging [1]
Fig. 1A shows the chemical structures of six compounds derived from A. gigas. Among these compounds, Umb, a coumarin-based compound, was selected as a tracing compound due to its strong absorbance at a wavelength of 325 nm and its fluorescence at a wavelength of 495 nm (Simkovitch et al., 2016, Vasconcelos et al., 2009). Other bioactive compounds, Cur and Dox, were also examined to monitor in vivo biodistribution. As a control dye, Eb was also administered to mice to examine its brain localization. After these single compounds and mixture of these compounds were injected into normal mice and a neuro-inflammatory mouse model, their biodistribution was determined in isolated organs. In particular, the biodistribution of active compounds (Umb, Cur, and Dox) in each organ was directly visualized using an ex vivo fluorescence imaging instrument without the need for time-consuming and complicated extraction processes, as shown in Fig. 1C. The neuro-inflammation mouse model was established by the intraperitoneal injection of LPS (Walker et al., 2013, Wang et al., 2014). To assess the anti-inflammatory effects of Cur and the Byakangelicin (Byn)/Cur mixtures, these agents were intravenously injected five times into mice (Fig. 1B). After administration, the accumulation of Cur in the brain and its anti-inflammatory responses were examined by fluorescence microscopy and by ELISA of cytokines, respectively.

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Fig. 2A shows the fluorescence images of single compounds and other compound/Umb mixtures in aqueous solution. A noticeable fluorescence signal was only observed in the presence of Umb (Fig. 2A). The normalized FIs of Umb, Byakangelicin (Byn)/Umb, Dec/Umb, and Ang/Umb were 100.9 ± 5.8, 101.3 ± 3.3, 97.3 ± 8.8, and 102.9 ± 6.0, indicating that the other compounds had negligible effects on Umb fluorescence. After intravenous injection of Umb at a dose of 160 or 320 mg/kg in mice, the organ distribution of Umb was monitored by fluorescence imaging. Strong fluorescence signals were observed in the brain, lung, and pancreas (Fig. 2B). It is well known that Umb has therapeutic effects in diabetes and neurodegenerative diseases, which are correlated to the biodistribution of Umb (Naowaboot et al., 2015, Ramu et al., 2016, Subramaniam and Ellis, 2013, Wang et al., 2015). To examine whether the biodistribution of Umb could be changed by co-administration with other compounds from A. gigas, Umb mixtures with Byakangelicin (Byn), Dec, and Ang were injected intravenously into mice. The localization of Umb in each organ was then observed by fluorescence imaging. After 2 min, much stronger Umb fluorescence signals were observed in the brain and lung following the administration of the Byn/Umb mixture than those with the other mixtures (Fig. 2C). Significantly reduced fluorescence signals in the pancreas were observed in mice treated with the Byn/Umb and Ang/Umb mixtures. However, as shown in Fig. S1A, Byakangelicin (Byn), Dec, and Ang alone showed negligible fluorescence signals in any organs after intravenous injection. The fluorescence signals in each organ were also analyzed quantitatively after extraction. The levels of Umb in the brain, lung, and pancreas were noticeably reduced following the administration of Umb with another compound (Fig. 2D). The normalized FIs of Umb alone, Byn/Umb, Dec/Umb, and Ang/Umb were 382.1 ± 69.0, 630.5 ± 104.4, 363.8 ± 104.6, and 503.2 ± 64.0 (×10−3) in the brain, and 177.4 ± 23.8, 132.3 ± 4.5, 163.4 ± 36.5, and 344.3 ± 272.9 (×10−3) in the lung. The greatest accumulation of Umb in both the brain and lung occurred after administration of Umb with Byn. As shown in Fig. S1B, FIs of Byn, Dec, and Ang were negligible after extraction from each organ. To examine whether the increased accumulation of Umb that was observed was mediated by disruption of the BBB following the administration of compound mixtures or not, Eb was also administered after compound treatment. Fig. S1C clearly shows that there was poor accumulation of Eb in the brain. This result clearly indicates that the BBB remained intact after the administration of the compound mixtures.

Accumulation of Byakangelicin (Byn)/Umb mixtures in brain [1]
The accumulation of Umb in the brain was also assessed as increasing other compound/Umb weight ratios. After intravenous injection of mice with Umb and Umb mixtures (Byakangelicin (Byn)/Umb, Dec/Umb, Del/Umb, Nod/Umb, and Ang/Umb) at an Umb dose of 4 mg/kg, the localization of Umb in each isolated organ was observed by fluorescence imaging (weight ratio of other compound/Umb of 20). As shown in Fig. 3A, Umb FI was significantly elevated in the brain and lung after treatment with the Byn/Umb mixture. However, there was no noticeable increase of FI in pancreas following the administration of Byn/Umb. Nod/Umb showed a higher accumulation of Umb in pancreas, compared with other compound mixtures. The FIs in each organ were also quantitatively analyzed. Fig. 3B shows that the normalized FIs of Umb in each organ (brain, lung, and pancreas) were 8.9 ± 4.7, 8.1 ± 9.1, and 8.2 ± 6.2 (×10−3), respectively. The FIs of Byn/Umb (at a weight ratio of other compound/Umb of 20) in the brain and lung were 37.4 ± 0.1 and 58.0 ± 29.5 (×10−3), respectively. The FIs in the brain and lung of mice treated with Byn/Umb were 4.2-fold and 7.2-fold greater, respectively than in the brain and lung of mice treated with Umb alone. To confirm whether the Umb levels in organs identified by ex vivo fluorescence monitoring correlated with conventional analytic methods, the physical amount of Umb was assessed using a fluorospectrometer after its extraction from each organ. Fig. 3C shows the relative FI in tissue lysates extracted from each organ using ethyl acetate. As expected, the brain and lung from mice injected with Byn/Umb showed a much larger amount of Umb compared with mice injected with Umb alone (Fig. 3C). However, the amount of Umb extracted from the brains and lungs of mice treated with Ang/Umb was similar to that extracted from the brains and lungs of mice injected with Umb alone, which was not consistent with the ex vivo imaging results. This could be attributed to the instability of Umb during the complicated extraction process. Mixtures of Byn and Nod with Umb showed significantly increased Umb levels in the brain and pancreas, respectively. This result was correlated well with the ex vivo monitoring in Fig. 3B. The Byn/Umb mixtures at Byn/Umb weight ratio of 20 resulted in a 2.4 ± 0.1-fold elevation of Umb accumulation in the brain, compared to Umb only.

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Biodistribution of Cur and Dox via ex vivo fluorescence imaging [1]
To investigate whether Byakangelicin (Byn) could modulate the biodistribution of different types of active compounds, Cur and Dox mixtures with Byakangelicin (Byn) were administered to mice intravenously. Cur is a well-known polyphenol, found in turmeric, for its therapeutic effect in neuro-inflammatory diseases and Alzheimer's disease (Mishra and Palanivelu, 2008). As shown in Fig. S2A, as the concentration of Cur increased, the fluorescent signal of Cur became strong accordingly. A Cur mixture with Byn (Byn/Cur) showed a similar FI to Cur alone, which indicates that the amount of Cur in tissues treated with Cur and Byn/Cur mixtures could be measured by FI of Cur in solution, respectively (Fig. S2B). Dox is a representative anticancer drug that is used to treat diverse types of tumors including neuroblastoma (MacDiarmid et al., 2016). However, poor penetration of Dox across BBB has limited its anticancer effects for brain tumors (Rousselle et al., 2000, Seol et al., 2014). Accordingly, development of promising delivery systems for Dox to brain has been greatly paid attention. Both Cur and Dox can be fluorescently excited by exterior light, which could be easily monitored after their administration in vivo (Kang et al., 2016b, Motlagh et al., 2016). Fig. 4A shows the fluorescence signals of Cur in the brain and lung after the administration of the Cur mixtures (Byn/Cur, Dec/Cur, and Ang/Cur). The Byn/Cur mixture showed a significantly strong FI in the brain and lung, compared to Cur only and other compound mixtures. The FI in each organ was also quantitatively analyzed, as shown in Fig. 4B. The normalized FIs for Cur, Byn/Cur, Dec/Cur, and Ang/Cur (weight ratio of other compound/Cur = 50) in the brain were 3.6 ± 1.2, 8.3 ± 2.1, 5.8 ± 1.7, and 7.5 ± 1.0. In addition, the normalized FIs for Cur, Byn/Cur, Dec/Cur, and Ang/Cur (weight ratio of other compound/Cur = 50) in the lung were 6.2 ± 2.7, 46.5 ± 2.0, 6.7 ± 2.7, and 18.7 ± 17.1, respectively. Interestingly, a more than 7-fold accumulation of Cur was observed in lung with the Byn/Cur mixture, compared to the Cur only. In our previous study, Cur bio-distribution determined by ex vivo fluorescence imaging was greatly correlated with that by mass spectrometry analysis of extracted Cur from tissues (Kang et al., 2016a). Accordingly, strong fluorescence intensities of Cur in tissues might indicate actual accumulation of Cur in tissues. However, it is necessary to examine Cur distribution at diverse time intervals to elucidate the maximal accumulation of Cur in different tissues (Wei et al., 2017, Zhang et al., 2019). Fig. 4C shows the accumulation of Dox in three different organs. As expected, Dox mixtures with Byn exhibited highest Dox accumulation in the brain among other compound mixtures. The normalized FIs for Dox, Byn/Dox, Dec/Dox, and Nod/Dox (weight ratio of other compound/Dox = 10) in the brain were 2.6 ± 0.7, 15.0 ± 6.8, 9.0 ± 6.5, and 2.9 ± 0.6, respectively (Fig. 4D). Taken together, Byn mixtures with Umb, Cur, and Dox greatly elevated their accumulation in the brain by 4.2-folds, 2.3-folds, and 5.7-folds, respectively, compared to their injection without Byn. To examine whether Byn could facilitate the penetration of Eb, Eb was mixed with Byn, and the mixture was used to treat mice. Fig. S3 shows that the FIs of Eb in the brain, lung, and pancreas after administration of Byn/Eb mixtures were similar to those after treatment with Eb alone. This result clearly demonstrates that Byn enhances the brain accumulation of selected active compounds without disruption of the BBB.

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Brain accumulation of Cur in of LPS-induced inflammation model [1]
To examine the localization of Cur in an LPS-induced mouse inflammation model, Cur and Byakangelicin (Byn)/Cur were administered intravenously to the mice for 5 days. After administration of Cur and Byakangelicin (Byn)/Cur on the 5th day, LPS was also injected intraperitoneally to the mice. After 24 h, the brain was isolated and fixed for the preparation of coronal sections. Accumulation of Cur in the coronal sections was seen in the cerebrum (Fig. 5A) using fluorescence microscopy and in the hippocampus (Fig. 5B) using confocal microscopy. A significant enhancement of Cur accumulation was observed in the cerebrum after Byn/Cur treatment, compared to Cur only. A strong green fluorescence signal was observed not only in the cerebrum but also in the hippocampus region. Taken together, these results indicate that Byn allowed excellent accumulation of Cur in the cerebrum and hippocampus in an LPS-induced inflammation model. Anti-inflammatory effect of Byakangelicin (Byn)/Cur in the LPS-induced inflammation model To investigate anti-inflammatory effects of Byakangelicin (Byn)/Cur in the LPS-induced inflammation model, the level of cytokines in brain homogenates and serum were measured by ELISA after administration of Cur and Byn/Cur. The amounts of TNF-α in the Control, Buffer, Cur, and Byn/Cur groups were 14.5 ± 1.4, 25.6 ± 1.6, 14.5 ± 1.3, and 11.2 ± 2.7 pg/mg, respectively (Fig. 6A). The levels of TNF-α in the brain homogenate were slightly decreased in the Byn/Cur group, compared with the Cur group. In addition, significant differences in the level of IL-1β were observed in the Cur and Byn/Cur groups. The amounts of IL-1β in the Control, Buffer, Cur, and Byn/Cur groups were 1.8 ± 1.1, 7.6 ± 1.2, 3.6 ± 0.8, and 2.3 ± 0.7 pg/mg, respectively (Fig. 6B). To compare the systemic anti-inflammatory effect of Cur and Byn/Cur, the levels of TNF-α in sera were analyzed, as shown in Fig. 6C. After 24 h of LPS administration, the level of TNF-α in sera was 30.1 ± 4.5 pg/ml. However, the levels of TNF-α in sera from the Cur and Byn/Cur groups were reduced to 23.4 ± 2.3 and 4.5 ± 4.5 pg/ml, respectively. It should be noted that TNF-α levels in the Byn/Cur group were almost similar to those in mice without LPS injection (Control). This result clearly shows that the enhanced accumulation of Byn/Cur allows for a successful anti-inflammatory effects on mouse neuro-inflammation model.

In vitro fluorescence imaging [1]
Stocks of single compounds (Umb, Byakangelicin (Byn), Dec, and Ang) in DMSO were diluted with injection buffer (PBS containing 13.5% (v/v) kolliphor) at a final concentration of 50 µg/ml. Umb mixtures (Byakangelicin (Byn)/Umb, Dec/Umb, and Ang/Umb) in DMSO were diluted with injection buffer at a constant Umb concentration of 50 µg/ml (weight ratio of other compound/Umb of 1). After the samples were loaded into 96-well black plates, the FIs were analyzed using Lago-X at an excitation and emission wavelength of 360 and 490 nm, respectively.

Determination of cytokine levels in brain lysates and sera [1]
To measure the levels of pro-inflammatory cytokines in the brain, brains were homogenized in PBS solution. After centrifugation at 13,000 rpm at 4°C for 10 min, supernatants were collected and stored at −80°C until used. To prepare serum, the isolated blood was centrifuged at 2000×g for 15 min. The amount of protein in each sample was quantified using a BCA protein assay kit according to the manufacturer's protocol. The amounts of TNF-α and IL-1β in the each brain homogenate and serum were determined using mouse ELISA kits. The absorbance was measured at 450 nm using a microplate reader.

Ex vivo fluorescence imaging of Umb [1]
For the injection of compound mixtures, ICR mice were injected intravenously with Umb and compound mixtures (weight ratio of other compound/Umb = 1) in injection buffer at an Umb dose of 80 mg/kg, and left for 2 min. After 2 min, the mice were euthanized, and organs were isolated and washed with PBS solution. Fluorescent images of each organ were analyzed using Lago-X at an excitation and emission wavelength of 360 nm and 490 nm, respectively. The total flux within a whole organ was quantitatively analyzed after subtracting background signals using the following formula: total flux in tissue from treated mice - total flux in tissue from untreated mice. A. gigas extract and single compounds (Byakangelicin (Byn), Ang, and Dec) were also injected into the mice tail vein at doses of 120 mg/kg (extract) and 40 mg/kg (single compound). After incubation for 15 min, the FI in each organ was monitored using Lago-X at an excitation/emission wavelength of 360/490 nm, respectively. To determine whether the blood-brain barrier (BBB) was disrupted by Umb or the compound mixtures, ICR mice were injected intravenously with freshly prepared Umb and Umb mixtures (other compound/Umb weight ratio of 1) at an Umb dose of 40 mg/kg. After incubation for 15 min, a 1% Eb solution in PBS was injected intravenously. After an additional 15 min of incubation, the mice were euthanized and fluorescent images of Eb in each tissue were acquired using an in vivo imaging system (IVIS) instrument at an excitation and emission wavelength of 535 and 705 nm, respectively.

Umb mixtures (Byakangelicin (Byn)/Umb, Dec/Umb, Del/Umb, Nod/Umb, and Ang/Umb) dissolved in DMSO were diluted with injection buffer at a final Umb concentration of 0.5 mg/ml, and then injected into ICR mice at an Umb dose of 4 mg/kg at different other compound/Umb weight ratios of 0, 1, and 20. After 2 min, the organs were collected and analyzed using Lago-X at an excitation and emission wavelength of 360 nm and 490 nm, respectively. The total flux in each organ was analyzed using the Lago-X software. To quantify the amount of Umb in different organs (brain, lung, and pancreas), Umb was extracted using ethyl acetate after the isolated organs were homogenized according to previous study (Kang et al., 2016a). Briefly, the organs were homogenized in PBS solution and the homogenate was then mixed with a solution of 10% SDS and ethyl acetate to give a volume ratio of homogenate:10% SDS:ethyl acetate of 1:0.5:5. After vortexing the mixture for 10 min, the sample was centrifuged at 12,000 rpm for 3 min. The supernatant was then collected and dried at room temperature. After the dried tissue extracts were re-dissolved in DMSO, the FIs in samples were measured at an excitation and emission wavelength of 325 and 495 nm using a fluorospectrophotometer (Molecular Devices, CA, USA). The FI of Umb in DMSO (0–500 ng/ml) served as standard for quantification.

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Ex vivo fluorescence imaging of Cur and Dox in brain [1]
To measure the FI of Cur and Byakangelicin (Byn)/Cur solutions, each sample was prepared in injection buffer at a Cur concentration of 1 µg/ml (at a weight ratio of Byn/Cur of 2). After samples were loaded into 96-well black plates, the FIs were analyzed using an in vivo imaging system at an excitation and emission wavelength of 430 and 509 nm, respectively. Cur mixtures with other compounds, including Byakangelicin (Byn)/Cur, Dec/Cur, and Ang/Cur, were prepared at various weight ratios (other compound/Cur weight ratios of 0, 20, and 50). Dox mixtures with other compounds, including Byn/Dox, Dec/Dox, and Nod/Dox, were also prepared at other compound/Dox weight ratios of 0, 5, and 10, respectively. For injection, the Cur and Dox mixtures in DMSO were mixed with kolliphor and PBS at 1.5:1.5:7.0 (volume ratio of DMSO: kolliphor: PBS), according to a previous study (Zhang et al., 2015). Freshly prepared Cur (1.6 mg/kg) and Dox (5 mg/kg) mixtures were injected intravenously into ICR mice, respectively. After incubation for 2 min, the brain and lung were isolated and washed with PBS solution. The fluorescent signal in each organ was analyzed using IVIS instrument at an excitation/emission wavelength of 430/509 nm for Cur, and at an excitation/emission wavelength of 465/583 nm for Dox, respectively. As a control, Eb was mixed with Byn at Byn/Eb weight ratios of 0, 0.5, 1, 2, 20, and 50. The Byn/Eb mixtures in injection buffer were intravenously administered at an Eb dose of 1.6 mg/kg. After 2 min, the mice were perfused with saline, the organs were collected and analyzed using IVIS instrument at excitation/emission wavelength of 535/ 705 nm.

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LPS-induced neuro-inflammation model [1]
A neuro-inflammation animal model was established, as previously described (Walker et al., 2013, Wang et al., 2014). C57/BL6 mice were randomly divided into four groups: a normal group treated with injection buffer (Control); a control group pre-treated with injection buffer before LPS injection (Buffer); a Cur group pre-treated with Cur before LPS injection (Cur); and a Byakangelicin (Byn)/Cur group pre-treated with Byn/Cur before LPS injection (Byn/Cur). All solutions were prepared fresh on the day of administration. The schedule for Cur injection was slightly modified based on a previous study (Kawamoto et al., 2013, Wang et al., 2014). For pre-treatment, a solution of Cur and Byn/Cur in injection buffer was injected into the tail vein once per a day at a Cur dose of 12 mg/kg and Byakangelicin (Byn) doses of 0 and 24 mg/kg for 5 days, respectively. Thirty minutes after injection on the 5th day, the mice in groups of Buffer, Cur, and Byn/Cur were injected intraperitoneally with LPS (0.83 mg/kg). After 24 h of LPS injection, the mice were anesthetized with isoflurane and sera were collected from mice. After perfusion, the brains were collected and cut into half. The brains in left-hand side were used to prepare a homogenate, whereas brains in right-hand side were used for brain imaging. For confocal imaging, the right-hand brain sections were fixed in 3.7% formaldehyde in PBS solution for 48 h, and then dehydrated in 15 and 30% (w/v) sucrose in PBS solution. The samples were frozen in optimum cutting temperature (OCT) and a coronal section with a width of 10 µm was made using a cryostat. After the samples were stained with DAPI, the FIs of Cur and DAPI in each tissue slice were observed using fluorescence microscopy and confocal microscopy, respectively.

[1]. Byakangelicin as a modulator for improved distribution and bioactivity of natural compounds and synthetic drugs in the brain. Phytomedicine. 2019 May 17;62:152963.

[2]. Byakangelicin induces cytochrome P450 3A4 expression via transactivation of pregnane X receptors in human hepatocytes. Br J Pharmacol. 2011 Jan;162(2):441-51.

Byakangelicin is a member of psoralens.
Byakangelicin has been reported in Angelica japonica, Heracleum grandiflorum, and other organisms with data available.

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Background: The elucidation of the biological roles of individual active compounds in terms of their in vivo bio-distribution and bioactivity could provide crucial information to understand how natural compounds work together as treatments for diseases.

Purpose: We examined the functional roles of Byakangelicin (Byn) to improve the brain accumulation of active compounds, e.g., umbelliferone (Umb), curcumin (Cur), and doxorubicin (Dox), and consequently to enhance their biological activities.

Methods: Active compounds were administered intravenously to mice, with or without Byn, after which organs were isolated and visualized for their ex vivo fluorescence imaging to determine the bio-distribution of each active compound in vivo. For the in vivo bioactivity, Cur, either with or without Byn, was administered to a lipopolysaccharide (LPS)-induced neuro-inflammation model for 5 days, and its anti-inflammatory effects were examined by ELISA using a brain homogenate and serum.

Results: We successfully demonstrated that the levels of active compounds (Umb, Cur, and Dox) in the brain, lung, and pancreas were greatly elevated by the addition of Byn via direct ex vivo fluorescence monitoring. In addition, sufficient accumulation of the active compound, Cur, greatly reduced LPS-induced neuro-inflammation in vivo.

Conclusion: Byn could serve as a modulator to allow improved brain accumulation of diverse active compounds (Umb, Cur, and Dox) and enhanced therapeutic effects.[1]

C17H18O7

334.324

334.105

C, 61.07; H, 5.43; O, 33.50

482-25-7

(Rac)-Byakangelicin;19573-01-4

10211

White to yellow solid powder

1.4±0.1 g/cm3

571.5±50.0 °C at 760 mmHg

123-124℃

299.4±30.1 °C

0.0±1.7 mmHg at 25°C

1.613

1.62

2

7

5

24

503

1

CC(C)([C@@H](COC1=C2C(=C(C3=C1OC(=O)C=C3)OC)C=CO2)O)O

PKRPFNXROFUNDE-LLVKDONJSA-N

InChI=1S/C17H18O7/c1-17(2,20)11(18)8-23-16-14-10(6-7-22-14)13(21-3)9-4-5-12(19)24-15(9)16/h4-7,11,18,20H,8H2,1-3H3/t11-/m1/s1

9-[(2R)-2,3-dihydroxy-3-methylbutoxy]-4-methoxyfuro[3,2-g]chromen-7-one

Biacangelicin; Byakangelicin; Byankagelicine; Byak-angelicin; Bjacangelicin; Bjakangelicin

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~50 mg/mL (~149.56 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.48 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 2.5 mg/mL (7.48 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)