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 study [1]
The validated method was successfully applied to the pharmacokinetic study using male rats. Both the quassinoids, namely bruceine D and E, were selected for this study due to its hypoglycemia effect. Rats were administered orally with extract containing 2.05 mg/Kg of bruceine D and 9.1 mg/Kg of bruceine E. The doses of intravenous (i.v.) administration in rats were extract with 0.41 mg/Kg of bruceine D and 1.82 mg/kg of bruceine E. Earlier, we have reported the hypoglycemia effect of normoglycemia mice were between 0.25 mg/Kg to 1 mg/kg (i.v.) for both bruceines D and E or equivalent to 0.13 mg/Kg to 0.5 mg/Kg in rats using the conversion table presented in Nair and Jacob, 2016. The LD50 values through i.v administration in mice for both bruceine D and E were 3.52 mg/Kg and 31.86 mg/Kg, respectively and from our records the no-observed-adverse-effect-level (NOAEL) was equivalent to 2.0 mg/Kg and 10.0 mg/Kg, respectively in rats. Since a limited volume of blood can be collected to ensure the validity of the pharmacokinetic profile, the dose administered to rats were selected to be between the effective hypoglycemia dose and below the NOAEL.
The mean plasma concentration-time profiles of bruceines D and E were presented in Fig. 3, Fig. 4, respectively. The pharmacokinetic profiles of pure bruceine D reported earlier by Zhang et al., 2016 and administered extract in the present study showed almost similar profile. Since the i.v. dosage of the present study (0.41 mg/Kg) was half of the earlier study (0.8 mg/Kg), both the AUC0-t (0.20 + 0.07) mg h/L and AUC0-∞ (0.20 + 0.08) mg h/L was also reduced by almost half. In comparison, i.v. administration of higher bruceine E dosage of 1.82 mg/Kg displayed higher AUC0-t (1.65 + 0.45) mg h/L and AUC0-∞ (1.70 + 0.47) mg h/L.
Following intravenous administration, the volume of distribution (Vz) for bruceine D and E were high, 1.70 ± 0.67 L/Kg and 1.33 ± 0.42 L/Kg respectively, suggesting extensive distribution to tissues. Clearance for bruceine D and E were 2.18 ± 0.62 L/h/Kg and 1.17 ± 0.34 L/h/Kg, respectively. The calculated oral bioavailability for bruceine D and E were 5.0 and 5.9% respectively (Table 4). The low bioavailability maybe due to poor absorption or extensive metabolism. Quassinoids were reported to displayed low bioavailability in rats. The quassinoids, eurycomanone and 13α(21)-epoxyeurycomanone displayed low bioavailability of 9.2% and 7.5%, respectively in rats. Another quassinoid, simalikalactone 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.

Bruceine E has been reported in Brucea javanica with data available.

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Bruceines D and E are quassinoids from seeds of Brucea javanica (L.) Merr. exhibiting hypoglycemia effect. The crude drug is used as a traditional medicine by diabetes patients. The aim of this study is to understand the bioavailability and pharmacokinetics of both the bruceines D & E. A rapid and sensitive HPLC–MS/MS method was developed and validated for the quantification of both quassinoids, bruceines D & E in rat plasma. Both the bruceines D & E were separated with the Zorbax SBC-18 column with gradient elution and mobile phase system of acetonitrile and deionized water with 0.1% formic acid at a flow rate of 0.5 mL/min. Analytes were detected in multiple reaction monitoring (MRM) mode with electrospray positive ionization. The quassinoids, namely bruceines D & E were detected with transitions of m/z 411.2 → 393.2 and m/z 395.2 → 377.2, respectively. Another quassinoid, eurycomanone was used as the internal standard with transition of m/z 409.2 → 391.2. The method was validated and conformed to the regulatory requirements. The validated method was applied to pharmacokinetic and bioavailability studies in rats. The pharmacokinetic study indicated both bruceine D and E were rapidly absorbed into the circulation system and reached its peak concentration at 0.54 ± 0.34 h and 0.66 ± 0.30 h, respectively. Bruceine E was eliminated slower than Bruceine D with t1/2 value almost increased two-fold compared to Bruceine D. In conclusion, a rapid, selective and sensitive HPLC–MS/MS method was developed for the simultaneous determination of both the bruceines D and E in rat plasma. Both bruceines D and E displayed poor oral bioavailability.[1]

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Background: Brucea javanica (B. javanica) seeds, also known as "Melada pahit" in Indo-Malay region are traditionally used to treat diabetes. The objective of this study was to determine antidiabetic, antioxidant and anti-inflammatory effects of B. javanica seeds on nicotinamide (NA)-streptozotocin (STZ) induced type 2 diabetic (T2D) rats and to analyze its chemical composition that correlate with their pharmacological activities. Methods: A hydroethanolic extract of B. javanica seeds was fractionated with n-hexane, chloroform and ethyl acetate. An active fraction was selected after screening for its ability to inhibit α-glucosidase and glycogen phosphorylase α (GP-α). Isolation and characterization were carried out by using column chromatography, NMR and LCMS/MS. All isolates were assayed for inhibition of GP-α and α-glucosidase. Antidiabetic effect of active fraction was further evaluated in T2D rat model. Blood glucose and body weight were measured weekly. Serum insulin, lipid profile, renal function, liver glycogen and biomarkers of oxidative stress and inflammation were analyzed after 4-week treatment and compared with 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.)