Natural product; anticancer; anthelmintic; Notch

D and E brucine, separated from B. During 0–8 hour screening, javanica seeds show a decreasing effect on blood glucose in both nondiabetic mice and diabetic rats generated by STZ at a lower dose (1 mg/kg bw)[2]. Ethyl acetate fraction (EAF) exerted good inhibitory potential for α-glucosidase and GP-α compared with other fractions. Chromatographic isolation of the EAF led to the identification of seven compounds: vanillic acid (1), bruceine D (2), bruceine E (3), parahydroxybenzoic acid (4), luteolin (5), protocatechuic acid (6), and gallic acid (7). Among them, Compound (5) was identified as the most potent inhibitor of GP-α and α-glucosidase and its GP-α inhibitory activity (IC50 = 45.08 μM) was 10-fold higher than that of caffeine (IC50 = 457.34 μM), and α-glucosidase inhibitory activity (IC50 = 26.41 μM) was 5.5-fold higher than that of acarbose (IC50 = 145.83 μM), respectively. Compounds (4), (6), and (7) inhibited GP-α activity in a concentration-dependent manner with IC50 values of 357.88, 297.37, and 214.38 μM, and their inhibitory effect was higher than that of caffeine. These compounds exhibited weak potency on α-glucosidase compared with acarbose. Compounds (1), (2), and (3) showed no inhibition on both GP-α and α-glucosidase. In vivo study showed that EAF treatment significantly reduced blood glucose level, increased insulin and glycogen contents, decreased markers of oxidative stress and inflammation, and lipid levels in T2D rats compared with untreated group. Conclusions: The EAF has potential therapeutic value for the treatment of T2D via acting as GP-α and α-glucosidase inhibitors by improving hepatic glucose and carbohydrate metabolism, suppressing oxidative stress, and preventing inflammation in T2D rats. According to the results, the efficacy of EAF could be due to the presence of luteolin along with synergistic effect of multiple compounds such as parahydroxybenzoic acid, protocatechuic acid, and gallic acid in B. javanica seeds [2].

GP-α and α-glucosidase inhibition assay [2]
The fractions from B. javanica seeds were evaluated as GP-α inhibitors and were further tested for their α-glucosidase inhibitory activity as described previously. Briefly, 50 μL of samples or standard were mixed with 100 μL of α-glucosidase (0.1 U/mL) in phosphate buffer (0.1 M, pH 6.9) and incubated at 37 °C for 10 min. The reactions were initiated by addition of p-Nitrophenyl α-D-Glucoside (p-NPG, 50 μL) in phosphate buffer (0.1 M, pH 6.9) and incubated again at 37 °C for 30 min. The reactions were terminated using NaCO3 (1 M, 50 μL). The p-nitrophenol released from p-NPG in the presence of α-glucosidase was detected at 405 nm using microplate reader. Phosphate buffer (50 μL) was used as control. Blank readings (without substrate) were subtracted from specific sample wells and the percentage of α-Glucosidase inhibition (αGI) was calculated as following formula: αGI (%) = [(Acontrol – Asample)/Acontrol] × 100.

Pharmacokinetic and bioavailability study [1]
The first group was administered through intravenous with extract dissolved in deionized water with dosage of 0.41 mg/Kg bruceine D and 1.82 mg/Kg of bruceine E through tail vein. Blood (150 uL) was drawn with a syringe at time interval of 0, 15, 30, 60, 120, 240, 360 and 720 min post administration by ventral tail vein blood collection. The second group was administered orally with extract dosage of 2.05 mg/Kg bruceine D and 9.1 mg/Kg of bruceine E and subsequently the blood was drawn at 0, 15, 30, 60, 120, 240, 360 and 720 min post administration. The blood was withdrawn into heparinized tube (BD vacutainer, UK) and centrifuged at 3000 rpm for 10 min and 100 μL of plasma was harvested and stored at −20 °C analysis until further analysis.
The pharmacokinetic parameters were analyzed through non-compartmental analysis using PKSolver which were maximum plasma concentration, (Cmax), peak time (Tmax), elimination half-life (T1/2), area under the curve from zero to last measurable concentration (AUC0-t), area under the curve from zero to infinity (AUC0-∞), mean residence time (MRT), clearance (CL) and apparent volume of distribution (Vd). The oral absolute bioavailability denoted F is calculated according to the following formula: F = [(AUC0-∞) oral × (dose) i·v·]/[(AUC0-∞) i.v. × (dose) oral].

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In vivo anti-diabetic activity [2]
Experimental animals
Total 30 SD rats (eight-week-old, 200–230 g) were dived into 5 groups (n = 6, 3 male and 3 female in each group) and were housed in cages under standard laboratory conditions with a12-h dark–light cycle and humidity-controlled environment with a room temperature of 22 ± 3 °C and relative humidity of 65 ± 5%. The rats were allowed access to Laboratory Rodent Chow and drinking water ad libitum and were received human care according to the guidelines.

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Induction of type 2 diabetes (T2D) [2]
The experimental rats were fasted overnight (16 h) and diabetes was induced by single intraperitoneal (i.p) injection of STZ (60 mg/kg b.w.) freshly prepared in 0.1 M citrate buffer (pH 4.5) 15 min after i.p injection of NA (100 mg/kg b.w.) dissolved in normal saline. Diabetes was confirmed 3 weeks after NA-STZ induction by measuring tail vein blood glucose levels using glucose meter. The rats having blood glucose levels higher than 11 mmol/L were considered as diabetic and selected for study.

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Treatment protocol [2]
The experimental rats were divided into 5 groups (n = 6; 3 M/3 F) as following. Group I: non-diabetic control (NDC) and Group II: diabetic control (DC) consisted of rats were allowed to free access of water; Group III and IV were treated orally with EAF (25 and 50 mg/kg/day b.w.) diluted in distilled water, Group V was treated orally with Glibenclamide (10 mg/kg/day b.w.) and served as standard drug. All groups except group 1 are diabetic. The selected doses (25 and 50 mg/kg/day b.w) were based on prior acute oral toxicity study.

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Determination of fasting blood glucose levels and body weights [2]
After diabetes was confirmed, rats were divided into specific groups which were mentioned in the section of treatment protocol above. Fasting blood glucose (FBG) levels and body weight of rats were measured and it was considered as 0 day. The extracts and standard drug were administered orally on a daily base in single dose for 28 days. At the end of each week, animals were fasted overnight and body weights were recorded using electronic balance. Blood samples were obtained from the tail vein of the rats by Accu-Chek FastClix lancing device and blood glucose levels were analysed using glucose meter.

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Oral glucose tolerance test (OGTT) in experimental rats [2]
On the 25th day of treatment, the OGTT was carried out according to the previously reported method. All animals were fasted overnight (16 h) before commencing the experiments. Group I (non-diabetic control) and Group II (diabetic control) weretreated with distilled water, Group III and Group IV were given EAF (25 and 50 mg/kg b.w), and Group V was given glibenclamide (10 mg/kg b.w.) using oral gavage, respectively. After 30 min, α-D-glucose (2 g/kg b.w.) was administered orally into all groups of rats. Blood samples were collected from the tail vein at 0 (immediately after glucose load), 30, 60, 90, and 120 min, and blood glucose levels were determined by glucose oxidase method using a commercial glucose meter. Total glycemic responses to OGTT were calculated from respective areas under the curve for glucose (AUC glucose) by trapezoid rule for the 120 min.

Pharmacokinetic and Bioavailability Studies [1]
The validated method has been successfully applied to pharmacokinetic studies in male rats. Due to their hypoglycemic effect, quassin compounds, namely quassin D and E, were selected for this study. Rats were orally administered extracts containing 2.05 mg/kg brusin D and 9.1 mg/kg brusin E. Rats were intravenously (iv) administered extracts containing 0.41 mg/kg brusin D and 1.82 mg/kg brusin E. Previously, we reported hypoglycemic doses of brusin D and brusin E in normoglycemic mice ranging from 0.25 mg/kg to 1 mg/kg (iv), or, according to the conversion table provided by Nair and Jacob (2016), equivalent to 0.13 mg/kg to 0.5 mg/kg in rats. The LD50 values of brusin D and brusin E after intravenous injection in mice were 3.52 mg/kg and 31.86 mg/kg, respectively. According to our records, the no-observed-adverse-effects-elevation (NOAEL) was equivalent to… In rats, the doses of brusin D and E were 2.0 mg/kg and 10.0 mg/kg, respectively. Due to the limited blood volume collected, the validity of the pharmacokinetic curves could not be guaranteed; therefore, the rat doses were chosen between the effective hypoglycemic dose and the NOAEL. The mean plasma concentration-time curves for brusin D and E are shown in Figures 3 and 4, respectively. The pharmacokinetic curves of pure brusin D previously reported by Zhang et al. (2016) are almost identical to those of the extract administered in this study. Because the intravenous dose in this study (0.41 mg/kg) was half that of the previous study (0.8 mg/kg), the AUC_0-t (0.20 ± 0.07) mg·h/L and AUC_0-∞ (0.20 ± 0.08) mg·h/L were also almost halved. In contrast, intravenous administration of a higher dose of bruxine E (1.82 mg/kg) showed higher AUC_0-t (1.65 ± 0.45) mg·h/L and AUC_0-∞ (1.70 ± 0.47) mg·h/L. Following intravenous administration, the volumes of distribution (V_z) of bruxine D and E were higher, at 1.70 ± 0.67 L/kg and 1.33 ± 0.42 L/kg, respectively, indicating their extensive distribution in tissues. The clearance rates of brusin D and E were 2.18 ± 0.62 L/h/kg and 1.17 ± 0.34 L/h/kg, respectively. The calculated oral bioavailability of brusin D and E was 5.0% and 5.9%, respectively (Table 4). The low bioavailability may be due to poor absorption or extensive metabolism. It has been reported that quassin compounds have low bioavailability in rats. The bioavailability of quassin compounds parsleyone and 13α(21)-epoxyparsleyone in rats was 9.2% and 7.5%, respectively. Another quassin compound, simalica lactone E, was not detected in mouse plasma [1].

[1]. HPLC-MS/MS method for bioavailability study of bruceines D & E in rat plasma. J Chromatogr B Analyt Technol Biomed Life Sci. 2017 Sep 15;1063:183-188.

[2]. Antidiabetic effects of Brucea javanica seeds in type 2 diabetic rats. BMC Complement Altern Med. 2017;17(1):94. Published 2017 Feb 6.

It has been reported that Brucea javanica contains matrine E, and relevant data are available. Matrine D and E are matrine-like compounds extracted from the seeds of Brucea javanica (L.) Merr., and have hypoglycemic effects. This herb is used as a traditional medicine for diabetic patients. This study aimed to understand the bioavailability and pharmacokinetics of matrine D and E. We established and validated a rapid and sensitive HPLC-MS/MS method for the quantitative analysis of matrine D and E in rat plasma. A Zorbax SBC-18 column was used with acetonitrile and deionized water containing 0.1% formic acid as the mobile phase at a flow rate of 0.5 mL/min, employing gradient elution to separate matrine D and E. Electrospray ionization was used for detection of the analytes in multiple reaction monitoring (MRM) mode. Brucin D and E, quasin derivatives, were detected with ion transitions of m/z 411.2 → 393.2 and m/z 395.2 → 377.2, respectively. Another quasin derivative, parsleyone, was used as an internal standard, with an ion transition of m/z 409.2 → 391.2. This method was validated to comply with relevant regulations. The validated method was applied to pharmacokinetic and bioavailability studies in rats. Pharmacokinetic studies showed that both brucin D and E were rapidly absorbed into the circulation, reaching peak concentrations at 0.54 ± 0.34 h and 0.66 ± 0.30 h, respectively. Compared to brucin D, brucin E was eliminated more slowly, with a half-life (t1/2) almost twice that of brucin D. In summary, we established a rapid, selective, and sensitive HPLC-MS/MS method for the simultaneous determination of brucin D and brucin E in rat plasma. Both brucin D and brucin E have low oral bioavailability. [1]

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Background: The seeds of Brucea javanica (abbreviated as B. javanica), also known as "Melada pahit" in the Indo-Malaysian region, have traditionally been used to treat diabetes. This study aimed to determine the antidiabetic, antioxidant, and anti-inflammatory effects of Brucea javanica seeds on nicotinamide (NA)-streptozotocin (STZ)-induced type 2 diabetes (T2D) rats and to analyze the chemical components related to its pharmacological activity. Methods: The hydroethanol extract of Brucea javanica seeds was fractionated using hexane, chloroform, and ethyl acetate. An active component was screened for its ability to inhibit α-glucosidase and glycogen phosphorylase α (GP-α). The fractions were separated and identified by column chromatography, nuclear magnetic resonance (NMR), and liquid chromatography-mass spectrometry (LCMS/MS). All fractions were subjected to GP-α and α-glucosidase inhibitory activity assays. The antidiabetic effect of this active component was further evaluated in a rat model of type 2 diabetes (T2D). Blood glucose and body weight were measured weekly. After 4 weeks of treatment, serum insulin, lipid profile, renal function, liver glycogen, and biomarkers of oxidative stress and inflammation were analyzed and compared with the standard drug glibenclamide. [2]

C20H28O9

426.46916

412.173

21586-90-3

5315510

White to off-white solid powder

1.60±0.1 g/cm3

262-263 ºC

1.16E-18mmHg at 25°C

4.807

6

9

0

29

814

12

CC1=C[C@@H]([C@H]([C@]2([C@H]1C[C@@H]3[C@]45[C@@H]2[C@H]([C@@H]([C@]([C@@]4([C@H](C(=O)O3)O)O)(OC5)C)O)O)C)O)O

ZBXITHPYBBXZRG-QYUWQHSUSA-N

InChI=1S/C20H28O9/c1-7-4-9(21)13(23)17(2)8(7)5-10-19-6-28-18(3,14(24)11(22)12(17)19)20(19,27)15(25)16(26)29-10/h4,8-15,21-25,27H,5-6H2,1-3H3/t8-,9-,10+,11+,12+,13+,14-,15-,17-,18+,19+,20+/m0/s1

(1R,2R,3R,6R,8S,11S,12S,13S,14R,15R,16S,17R)-2,3,11,12,15,16-hexahydroxy-9,13,17-trimethyl-5,18-dioxapentacyclo[12.5.0.01,6.02,17.08,13]nonadec-9-en-4-one

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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