Journal of Medicinal Materials, 2014, VoL 19, No. 3 (pp.135 -139)
ANTI-HYPERGLYCEMIC EFFECT OF QUERCETIN-3-O-ARABINOFURAROSIDE FROM ETHYL ACETATE EXTRACT OF EUPHORBIA THYMIFOLIA LINN
IN STZ/NAD-INDUCED DIABETIC MICE Hoang Le Son * Nguyen Thi Tuyet Lan, Tran Van Minh International University, Vietnam National University Ho Chi Minh City
* Corresponding author: [email protected]. vn (Received April 04*, 2014)
Summary
Anti-Hyperglycemic Effect of Quercetin-3-0-Arabinofuraro$ide from Ethyl Acetate Extract oi Euphorbia thymifolia Linn in STZ/NAD-Induced Diabetic Mice This research aimed to investigate the anti-diabetic activity of quercetin-3-O-arabinofuraroside from efliyl acetate extract oi Euphorbia thymifolia Linn growing in Vietnam. The isolated compound was assessed for its hypoglycemic effect in STZ/NAD-induced diabetic mice. The result showed that quercetin-3-0- arabinofuraroside (50 mg/kg) significantly reduced blood glucose level up to 28.69% in STZ/NAD-induced diabetic mice after 4 hours of administration. This finding confirms the anti-hyperglycemic effect oi Euphorbia thymifolia in which quercetin-3-O-arabinofuraroside may play a key role in reducing blood glucose level of type 2 diabetes.
Keywords: Euphorbia thymifolia, Quercelin-3-O-arabinoJuraroside. Anti-diabetic. STZ/NAD.
1. Introduction
Diabetes mellitus is a common metabolic disorder characterized by high glucose level in the bloodstream with disturbances of carbohydrate, fat and protein metabolism resulting from defects in insulin secretion and/or insulin action. The type 2 diabetes is now very common, involving dangerous symptoms, increasing risk of stioke, obesity and heart failure. Diabetes is no longer a condition of developed countries; it is now accelerating in the developing world, with an increasing proportion of affected people in yoimger age groups. The diabetes epidemic becomes a burden on the economy and society for many countries in over the world [1]. Many synthesized drugs have been prescribed for controlling the blood glucose level, but they have too high prices and many side effects. Thus, the traditional therapy (e.g., herbal medicine) is still regarded as the beneficial choice for the primary health care [2].
Euphorbia thymifolia Linn (E. thymifolia) is small prostrate herbaceous annual weed in Vietnam. It has been traditionally used for the treatment of ring worm, bowl affection, skin disease, parasitic infection, anti-diarrhea, and amenorrhea. The ethyl acetate extract of E. thymifolia has been proven to reduce blood glucose level and could be regarded as alternative therapy for diabetes [3]. Several compounds from E. thymifolia have been isolated, such as epiteraxol, n-hexacosanol, euphorbol, 24-methylene cyloartenol, etc.
from petroleum ether extract [4]; luteolin and quercetin-3-O-arabinofuranoside from ethyl acetate extract [5].
(Juercetin-3-O-arabmofuraroside is the rare component in botany. This compound was only foimd in some plants such as peel of MalidS domestica Borkh [6], Sageretia thea Leaf [7], Prunus spinosa L. flower [8] with investigation in antioxidant and antiprolif^ative activity. The ability of inhibiting a-glucosidase Journal of Medicinal Materials, 2014, VoL 19, No. 3 135
of this compound was found to be higher than that of Acarbose [5]. In this study, we evaluate the anti-diabetic activity of quercetin-3-0- arabinofiiraroside from ethyl acetate extract of K thymifolia in STZ/NAD-induced diabetic mice.
2. Materials and methods
Collection of plant material and extraction:
Plants of E. thymifolia were collected from Vinh Long Province, Vietnam in July, 2013.
The fresh plants were harvested, rinsed with tap water to remove any type of contamination and then soaked in 70° EtOH. Washed plants were then dried in the oven at 60''C until the weight of plants was unchanged. The dried plant materials were ground into the powder by grinder. Powder was defatted by n-hexan (80°C-90°C) for 9 hours in tiie Soxhlet apparatus. After completely de-fatting, the powder was dried at 60°C in the oven for 1 day. Pretreated powder vras extracted continuously by EtOAc in the Soxhlet apparatus in 9 hours. The ethyl acetate extract was dried at
60°C in rotary evaporator to remove solvent.
Isolation ofquercetin-3-O-arabinofuranoside from ethyl acetate extract of E. thymifolia:
Thin layer chromatography and column chromatography were employed to isolate and purify quercetin-3-O-arabinofuranoside from ethyl acetate extract off. thymifolia.
Evaluating the effect of quercetin-3-0- arabinofuranoside on blood glucose level in normal mice: 16 hours fasted mice were divided into six groups of six mice per group.
Group I was given de-ionized water and served as negative control. Groups II - VI were fed with quercetin-3-O-arabinofiiranoside at doses of 10, 35, 50, 100 and 150 mg/kg body weight as described in Table 1.
Quercetin-3-O-arabinofuranoside was thoroughly suspended in de-ionized water. All substances were orally administrated. Blood samples were collected from the tail vein and the blood glucose levels were measured after 1, 2, 3 and 4 hours of administration.
Table I. Feeding method on normal mice for evaluating the effect of quercetin-3-O-arabinofiiranoside on blood glucose level
Group I 11 III IV V VI
Feeding method De-ionized water, 10 mlVkg body weight
Quercetin-3-O-arabinofiiranoside, 10 mg/kg body weight Quercetin-3-O-arabinofuranoside, 35 mg/kg body weight Quercetin-3-O-arabinofliranoside, 50 mg/kg body weight Quercetin-3-O-arabinofLiranoside, 100 mg/kg body weight Quercetin-3-O-arabinofuranoside, 150 mg/kg body weight
Eslablishment of STZ/NAD-induceil diabetic mice'. All experiments were conducted under the ethical guideline for experiment. The method of establishing type 2 diabetic mice by STZ/NAD was described by Masiello P.
(1998) with modification [10]. Forty healthy male Musculus tar Albino mice weighing about 20-22 g were used. Mice were fasted for 16 hours but allowed free to access to water. Blood samples were collected from
the tail vein and blood glucose level was measured using glucometer (Omron). Mice were then intravenously injected with 120 mg/ kg body weight of NAD dissolved in salme water 0.01 mL/g body weight. 15 min later, 45 mg/kg body weight of STZ, which was dissolved m citrate buffer pH 4.5 with concentration of 75 mg/mL, was intraperitoneally injected into mice. Mice were maintained on basal diet with supplementation of 0.2 mL 136 Journal ofMeilicinal Materials, 2014, Vol. 19, No. 3
lard/ mice and 40 mL of milk /lO mice/ day ad libitum. Fasting blood glucose levels were then measured after 4 weeks.
Evaluating the anti-f^pergfycemic effect of quercetin-3-0-ard}inofvranoside in STZ/NAD- induced diabetic mice: STZ/NAD-induced diabetic mice were chosen with the significant high fasting blood glucose level compared to that of normal mice (above 126 mg/dL) after 4 weeks of treatment. Fasted mice of each
model were groiq)ed into three groiq)s of seven mice per groiq), and the feeding method was detailed in the Table 2. Each mouse was weighed and dose adjusted accordingly prior to the administration. Quercetin - 3 - O - arabinoftiranoside was thoroughly suspended in de-ionized water. All substances were orally administrated. After 4 hours of administration, blood samples were collected from the tail vein and the blood glucose levels were measured Table 2. Feeding method on STZ/NAD-induced diabetic mice
Group Feeding method
De-ionized water, 10 mlAg body weight
i Reference dn]g(Mrt&nnin,Stada). 10 mg/kg body weight j Qucrcetin-3-O-arabinofiiranoside, 50 mg/kg l)ody wei^t
Statistical analysis: Results were expressed as the mean ± standard error of mean. Statisdcal analysis was performed by using SPSS version 16.0. P-Values less than 0.05 were considered to be statistically significant.
3 . Results a n d discussion
Isolation of quaxetiii-3-O-arabiofuranoside:
After fiuther purification, the yield of quercetin-3-O-arabiofiiranoside was 0.04%
based on the original weight of dried plant material, and its piuity degree was recorded
up to 98.87% by HPLC analysis.
Effect of quercetin-3-O-arabiitbfuranoside on blood glucose level in normal mice: As can be seen fiijm the Table 3, all groups have showed a statistically significant difference' (p < 0.05) in the reduction of blood glucose level.
Table 3. Effect of quercetin-3-O-arabiofuranoside on blood glucose level in normal mice Group
I
Blood glucose level (mg/dl) )i ± SEM Oh
88.50 ± 2 . 0 9
I h 2 l i 8 3 . 1 7 ± 4 . 3 5
(-4.59 ± 3 . 9 1 ) .1 88.83.2.89 , ' , " f f , '
(-10.91 ±4.39) III I 86.67 ±0.56
IV
V
VI
88.83 ± 2.55
86.17 ± 2 i 9
84.83 ± 3.34
75.83 ± 3 . 3 4 (-12.41 ± 4 2 0 )
70.83 ± 2 . 8 2 * (-20.15±2.9S) 80.83 ± 1 . 9 0 (-6.01 ± 2 . 3 5 ) 86.17±4.61 (1.83 ±15.30)
74.50 ± 2 . 4 9 (-11.45 ± 2 . 3 2 )
72.17±5.11 (-18.99 ± 4 . 6 2 )
66.17 ±1.85«
(-23.61 ± 2 . 3 8 ) 70.33 ± 4J22*
(-20.92 ± 3 . 6 9 )
3 l i 72.83 ± 3 . 2 6 (-15.63 ± 3 . 6 4 )
67.83 ± 3 . 1 9 * (-23.81 ± 1.24)
65.83 ±2.89«
(-24.04 ± 3.29) 65.50 ± 4.06*
(-26.35 ± 3 . 1 5 ) 76.17 ± 0 . 9 5 ' 70.50 ± 0 . 9 6 * (-11.37±2.04) i (-17.91 ± 2 . 3 5 ) 71.33 ± 4 . 4 5 1 64.67 ± 4 . 4 9 * (-15.90 ± 1 3 2 1 ) j (-23.59 ± 1422)
4 h 73.33 ± 3.34 (-15.11 ±3.49)
67.17 ± 2 . 8 2 * (-24.30 ±2.6«) 62.17 ± 3 . 0 6 * (-28.29 ±3.44)"
59.00 ± 2.82*
(-33.53 ±2.92)'' 63.50 ± 1.23*
(-25.89 ±3.18)'' 63.67 ± 2.96*
(-24.85 ± 8.69)"
Values are the mean ± SEM for 6 mice per group (all are male).
'Significant difference witfa p < 0.05 of blood glucose level compared to that of same group at 0 h.
Significant differeitce vnthp< 0.05 of blood glucose level compared to that of control group at the same time.
Journal of Medicinal Materials, 2014, VoL 19, No. 3 137
There was a slight decrease without significant difference in blood glucose level of the control group. Meanwhile, all other groups showed statistically significant difference (p < 0.05) in reducing blood glucose level after 4 hours of administration. Since the blood glucose level of mice in group received 50 mg/kg reached the highest reduction percentage (33.53%), this dose was chosen for experiments using diabetic mouse model.
Table 4. Anti-hyperglycemic effect of quercetin-3-0.
Anti-hyperglycemic effect of quercetin- 3-O-arabinoftiranoside in STZ/NAD-induced diabetic mice: As can be seen from table 4, all groups showed the reduction in blood glucose level in STZ/NAD-induced diabetic mice. After 4 h of treatment, both Metformin and quercetin-3-O-arabinofuranoside group showed significant reduction (p < 0.05) of blood glucose level with the reduction percentage of 32.25% and 28.69%, respectively, -arabinofiiranoside in STZ/NAD-mduced diabetic mice Treatment
De-ionized water IVletformin, Stada Quercetin-3-O-arabinofuranoside
(SO mg/kg body weight)
Blood glucose level (mg/dL) fi ± SEM Oh
147.14 ±2.89 150.43 ±6.69 148.29 ±4.28
4h 124.86±2.11*
(-16.91 ±2.25)%
101.00 ±3.49*
(-32.25 ±3.14)%' 104.86 ±5.04*
(-28.69 ±3.41)%' Values are the mean ± SEM for 7 mice per group (ail are male)
•Significant difference with p < 0.05 of blood glucose level compared to that of same group at 0 h
^Significant difference withp < 0.05 of blood glucose level compared to that of control group at the same time The present work has been carried out to
evaluate the anti-diabetic effect of quercetin- 3-0-arabinofiiranoside in STZ/NAD-induced diabetic mice. STZ injection caused the diabetes mellitus due to the destruction of B cells of the islets of langerhans of the pancrease [11]. NAD is an antioxidant that has protective effect on the cytotoxic action of STZ by cleaning up the free radical [12].
The combination of STZ and NAD caused the partial loss of B cells led to type 2 diabetes. The dose of 45 mg/kg body weight of STZ and 120 mg/kg body weight of NAD was experimentally found to be appropriate for inducing type 2 diabetes mice.
This in vivo study reveals that quercetin-3- 0-arabinofuranoside significantly reduced fasting blood glucose levels in both normal and diabetic mice as compared to that of control group. In the anti-hyperglycemic
activity test conducted in normal mice, blood glucose level of group received 50 mg/kg had the highest eflfect after 4 hours of administration as compared to that of other groups. In the test with STZ/NAD-induced diabetic mice, quercetin-3-O-arabinofiaranoside at a dose of 50 mg/kg significantly exhibited the potential in reducing blood glucose level as compared to that of negative control group. It should be noticed that there was no significant difference (p < 0.05) in the reduction of blood glucose level between quercetin-3-0- arabinofuranoside-treated group and the Metformin-treated group as positive control.
4. Conclusion
Quercetin-3-O-arabinofiiranoside from ethyl acetate extract of E. thymifolia has ability to reduce blood glucose level in STZ/NAD- induced diabetic mice up to 28.69 % after four hours of administration. This result 138 Journal of Medicinal Materials, 2014, VoL 19, No. 3
validates the folk medicinal use of this plant relation to the anti-diabetic action, for the treatment of diabetes. Further clinical Acknowledgement: This research was trials are necessary to investigate the supported by grants from Vietnam National molecular mechanism(s) of this compound in University Ho Chi Minh City.
References
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Journal of Medicinal Materials-Hanoi, Vol 2, No 18, 82-89. 4. Bhavna Gupta, R.S. (2007), Therapeutic Uses of Euphorbia thymifolia: A Review. 5. Hoang Le Son^ Vu Thi Le Quyen, Tran Van Minh (2013), Antioxidant and alpha-glucosidase inhibitory activity of flavonoids extracted from Euphorbia thymifolia Linn. Journal of Medicinal Materials-Hanoi, Wo] 3, t^o 18, 147-152. 6. Xiangjiu He, and Rui H., L. (2008), Phytochemicals of Apple Peels: Isolation, Structure Elucidation and Their Antiproliferative and Antioxidant Activities. Journal of Agricultural and Food Chemistry, 58(21): 9905-9910. 7. Ca-Jane Shen, C.-K.C, Shoei-Sheng Lee (2009), Polar Constituents from Sageretia thea Leaf Characterized by HPLC-SPE-NMR Assisted Approaches.
Journal of the Chinese Chemical Society, 56: 1002-1009. 8. Monika Olszewska, Maria Wolbis (2001), Flavonoids from the flowers of Prunus spinosa L. Acta Poloniac Pharmaceutica. Drug Research, 58(5): 367- 372. 9. Acute Oral Toxicity - Acute Toxic Class MeUiod (2001), OECD Guidelines for the Testing of Chemicals.http://ntp.niehs.nih.gov/iccvam/SuppDocs/FedDocs/OECD/OECD_GL423.pdf 10. Masiello P el al (1998), Experimental NIDDM: Development of a new model in adult rats administered streptozotocin and nicotinamide. Diabetes. 47: 224-229. 11. WHO (1999), Definition, Diagnosis and Classification of Diabetes Mellitus and its Complication: Report of a WHO consultation. Part I: Diagnosis and Classification of Diabetes Mellitus. Geneva, World Health Org. 2-7. 12. S.N. Manjula et al (2012), Animal model for type 2 diabetes mellitus: A review. International Journal of Pharmaceutical Research and Development, 23- 33.
Journal of Medicinal Materials, 2014, Vol. 19, No. 3 (pp. 139-144)
ACETOVANILLONE INHIBITS COX-2 EXPRESSION AND PROSTAGLANDIN E2 PRODUCTION IN RAW264.7 CELLS VIA
NF- KB A N D A P - 1 ACTIVITY
Bui Thi Binh', Tran Thi Hien\ Dao Trong Tuan^\ Do Thi Ht^", Nguyen Thi Bich Tht^
'Thaibinh Medical University; ^Lund University, Sweden; ^Vietnam National Institute of Medicinal Materials; ''University of Copenhagen, Denmark
'Corresponding author: [email protected] (Received April 4*, 2014)
Summary
Acetovanillone Inhibits COX-2 Expression and Prostaglandin E2 Production in Raw264.7 Cells Via NF- KB and AP-1 Activity
The improper productions of NO and prostaglandins following the induction of cycIooxygenase-2 (COX-2) is involved in the pathogenesis of chronic inflammation. Belamcanda chinensis has been used as an oriental medicine for treatment of inflammation. Here, we isolated acetovanillone, irisflorentin, tectoridin, iristectorin A and iristectorigenin A from B. chinensis and demonsU^ted that acetovanillone inhibits the induction of COX-2 expression in RAW264.7 macrophages stimulated with lipopolysaccharide (LPS). Reporter gene assays and immunoblot analyses using subcellular fractionation revealed that transcription factor activities of nuclear factor- AB (NF-JCB) and activator protein 1 (AP-1) were suppressed by acetovanillone. In addition, protein expression
