K. W. (2012), Potent vasodilation effect of amurensin G is mediated through the phosphorylation of endothelial nitric oxide synthase. Biochemical Pharmacology. 84(11), 1437-1450. 7. Nguyen T. N. A., Dao T T., Tung B. T., Choi H. W., Kim E H., Park J S., Lim S. I., Oh W. K. (2011), Influenza A (HlNl) neuraminidase inhibitors from Vilis amurensis. Food Chemistry 124, 437-433. 8. Bachelor M. A., Bowden G. T. (2004), UVA-medialed activation of signaling pathways involved in skin tumor promotion and progression. Seminars in Cancer Biology. 14(2), 131-138 9. Quyen B. T , Choi H. K., Kang K.
W (2013), Pinl is required for ultraviolet A-stimulated cyclooxygenase-2 induction in mouse epidermal cells, Cancer Letters. 335(1), 31-40. 10 Bowden G, T (2004), Prevention of non-melanoma skin cancer by targeting ultravioiet-B-light signaling. Nature Reviews Cancer. 4(1), 23-35. I I . de Vries E.. Amold M., Aitsitsiadis E., Trakatelli M., Hinrichs B,, Stockfleth E., Coebergh J., EPIDERM Group (2012), Potential impact of intervenfions resulting in reduced exposure to ultraviolet (UV) radiation (UVA and UVB) on skin cancer incidence in four European countries, 2010-2050. British Journal of Dermatology. 167(2), 53-62.
Journal of Medicinal Materials, 2015, Vol. 20, No. 3 (pp.165 -)
EFFECT OF PIPER BETEL L. LEAF EXTRACT ON MOUSE MODEL OF ATOPIC ECZEMA
Hoang Le Son*, Nguyen Thanh Trung, Tran Van Minh International University, Vietnam National University Ho Chi Minh City
* Correspond ing author: hlson@hcmiu.edu.vn (Received August 27'\ 2014)
Summary
Effect of Piper betel L. Leaf Extract on Mouse Model of Atopic Eczema
This research aimed to assess the anti-eczema activity of ethanol extract of Piper betel L leaves on mouse model of atopic eczema at doses of I, 2. 4, 6 and 8% (w/v). The effect of ethanol extract of Piper betel L leaves on mouse model atopic eczema was evaluated by hislopathological examination and epidermal thickness. The results showed that the ethanol extract of Piper betel L leaves at a dose of 4% w/v exhibits the most effective result against eczema in mouse model. These findings provided scientific evidences for the anti-eczema property of the ethanol extract and could be further explored for the treatment of eczema.
Keywords; Eczema. Piper betel L., Histopathological examination. Epidermal thickness. Mouse model
1. Introduction medicinal plants have been traditionally used in Atopic dermatitis (AD) or eczema is a chronic the treatment of eczema, and much research is and relapsing inflammatory skin disease relying now being devoted to scientifically evaluate the on the interplay of environmental, immunological, potential of medicinal plants against eczema, and genetic factors. The incidence of AD is Piper betel L. {P. betel) is a plant with deep continuously increasing woddwide, with a green heart-shaped leaves belonging to the prevalence rate of approximately 10-20%, and is Piperaceae family. It is one of the most famous more common among infants and children. AD is spices in Nepal, India, and China, and is cultivated a multifactorial skin disease, with complex for chewing and for traditional aesthetic uses interactions of immune innate and adaptive that is known as "betel nut" or "betel quid", immune responses based on a strong genetic Phytochemical study revealed that P. Ae/e/contains predisposition and triggered by environmental carbohydrates, alkaloids, glycosides, vitamins, factors [I], Although many synthesized drugs gums, steroids, tannins, phenols, and organic have been approved for the treatment of eczema, acids [2]. Besides, P. betel is traditionally used in the search for new drugs of natural origin that can eye and skin disease. More recently, the leaf be used as alternatives for curing eczema is still essential oil has been reputed to demonstrate of interest to scientists. In fact, numerous anthelmintic, aphrodisiac, carminative, and laxative
Journal of Medicinal Materials, 2015, Vol. 20, No. 3 165
properties [3]. The aim of this study is to assess the anti-eczema activity of ethanol extract of P.
betel leaves in mouse model of atopic eczema.
2. Materials and methods
Collection of plant material and extraction P. betel was collected from Ba Diem Commune, Hoc Mon District, Ho Chi Minh City, Vietnam in December, 2014. The plant was identified by Associate Professor Tran Van Minh. The fresh plants were harvested, rinsed with tap water to remove any type of contamination and then soaked in 70° ethanol. Washed plants were then dried in the oven at SO^C until the weight of plants was unchanged. The dried plant materials were ground into the powder by grinder. The ethanol extracts were prepared by soaking 1 kg of
the dried plant powder in 10 L (1:10) of 96%
ethanol at room temperature for 24 h. Then the extracts were filtered through a Whatmann filter paper. The filtrate was concentrated under reduced pressure at 55°C for 30 min using a rotary evaporator. The extracts were then collected, kept in Petri dish and stored in a desiccator at room temperature.
Formulation
lO.Sg of cetostearyl alcohol, 20g of vaseline and 8g of liquid paraffin were melted together to create a mixture [4]. This mixture was then removed from heat to allow cool before blending ethanol extract of P. betel leaves (1.0, 2.0, 4.0, 6.0, and 8.0%, w/v) to produce five different ointment formulations (Table I).
Table 1. Formulations of/*, ie/e/leaf extract incorporated ointment Formulation
Fl J F2
F3 F4 F5
Ingredients Leaf extract (g)
0.2 0.4 08 1.24 1 69
Cetostearyl alcohol (g) 5.4 5.4 5.4 5.4 5.4
Vaseline (g) 10 10 10 10 10
Liquid paraffin (g) 4 4 4 4 4 Animal and reagents
Male Swiss mice, weighing 18-20 g, were purchased from Pasteur Institute of Ho Chi Minh City. The mice were housed in steel cages with rice husk as bedding in a constant temperature (23 ± 2°C) and humidity environment of 50 ± 10%. Food and water were freely available. 2,4- dinitrochlorobenzene (DNCB) used as a sensitizer was purchased from Sigma-Aldrich and dissolved in acetone-olive oil in a ratio of 4:1. The acetone- olive oil was used as vehicle.
Sensitization
1 1
Sensitization and challenge
The backs of mice were removed with depilatory cream, and washed with sterilized ethanol a day before sensitization. For the sensitization process (Figure I), a 1-cm^ hair removed-skin patch, applied with O.I mL of 1%
DNCB in vehicle was attached to the removed backs of the animals for 2 days on day 0 and 3.
On day 6 and 9, 0.1 mL of 2% DNCB in vehicle were used to challenge for a day as a previous sensitization [5].
Challenge
+9 2%
Day
h
Shaving
' +3 1% 1%
+6
2%
DNCB DNCB DNCB Figure I. Schematic diagram of the study protocol for mouse model of atopic eczema
DNCB
166 Joumal of Medicinal Materials, 2015, Vol. 20, No. 3
Animal group and healing
DNCB-induced eczema mice were randomly distributed into six groups of five mice each (Table 2). Group I - V were daily administered with formula Fl, F2, F3, F4, F5, respectively.
Group VI served as positive control was given the standard cream (Tacrolimus, 0.03%). Group VII served as negative control received ointment without ethanol extract of P. betel leaves. The treatment was done daily for a week, and then the animals were sacrificed 24 h after the last application to prepare for histopathological examination.
Table 2. Treatment method on DNCB-induced eczema mice Group
Group 1 Group II Group III Group IV Group V Group VI Group VII
Treatment Ethanol extract of P betel leaves, 1 % (w/v) Ethanol extract of P. betel leaves, 2 % (w/v) Ethanol extract of P, betel leaves, 4 % (w/v) Ethanol extract af P. betel leaves, 6 % (w/v) Ethanol extract of P betel leaves, 8 % (w/v) Positive Control (Tacrolimus, 0.03%) Negative control
Histopathological examination
For histopathological examinafion [6], s^kin samples from the back of each mouse were prepared in 10% neutral buffered formalin, embedded in paraffin and cut into four- micrometer-thick sections. The sections were stained with hematoxylin-eosin (HE) to evaluate epidermal hyperplasia and infiltrafion of immune cells in the dermis.
Epidermal thickness
The distance between the dermo-epidermal borderline and the beginning of the homy layer indicated the epidermal thickness. Five measurements per animal were made in every ten scales and the mean for the individual animals was calculated [7,8].
3. Results and discussion Histopathological examination
As can be observed from Figure 2 and Table 3, the normal skin tissues [Figure 2(1)] composed of three main layers including epidermis (E), dermis (D), and hypodermis (H). There are no vesiculars and hyperplasia; in contrast, a lot of the hair follicles and adipocytes in the dermis and hypodermis.
When eczema occurred [Figure 2(2a) (2b)], it damaged entirely epidermis, part of dermis, and hypodermis layer. Epidermis was totally necrotic (pink color) while hair follicle declined dramatically and a lot of died-leukocytes appeared. Wounds were being healed with the application of formula Fl, F2, F3, F4, and F5 corresponded with [Figure 2(4a) (4b)], [Figure 2(5a) (5b)], [Figure 2(6a) (6b)], [Figure 2(7a) (7b)], [Figure 2(8a) (8b)], respectively.
In group ! containing ethanol extract of P.
betel leaves, 1% (w/v) [Figure 2(4a) (4b)], there were some particular wounds such as vesiculars and hyperplasia in the epidermis compared to the epidermis of positive. Fiber hyperplasia, lymphocytes and neutrophil were observed in dermis and hypodermis whereas the amount of adipocytes was significantly decreased in the hypodermis.
The presence of lymphocytes and neutrophil [Figure 2(4b)] showed evidence for inflammation phase which occurred in the early stage of wound repair and the recovery of tissue.
In group II containing ethanol extract of P.
betel leaves, 2% (w/v) [Figure 2(5a) (5b)], the result appeared little better to the skin ofmice as compared to that of group I when there was a drop in the fiber hyperplasia in dermis and an increase of adipocytes in hypodermis.
In group ill containing ethanol extract of P.
betel leaves, 4% (w/v) [Figure 2(6a) (6b)] and control [Figure 2(3)], a successful recovery occurred when the number of vesicular declined dramatically in epidermis while hair follicles and adipocytes rocketed significantly. Besides, hyperplasia in epidermis greatly reduced.
In group IV containing ethanol extract of P.
betel leaves, 6% (w/v) [Figure 2(7a) (7b)] and group V containing ethanol extract of P. betel leaves, 6% (w/v) [Figure 2(8a) (8b)], It was witnessed a more efficient recovery than that of group III in epidermis and hypodermis when the number of hair follicle and adipocytes more increased, but occurred a lot of vesicular in epidermis because the skin was poisoned and burned with the high concentration of ethanol extract of P. betel leaves (6% and 8%).
Journal of Medicinal Materials, 2015, Vol. 20, No. 3 167
Figure 2. Histological features of mouse skin tissues before and alter induccd-ec/cma, (1) Norma! skin tissues composed of epidermis (E), dermis (D). and hypodermis (H): (2a) and (2h) negative -eczema skin tissues of group VII (negative control), observed at x40 and xlOO, respectively . (3) control - healing skin tissues of group VI with Tacrolimus, 0,03%.
(4a) and (4b) healing skin tissues of group 1 with ethanol extract of P. betel leaves. 1 % (w/v) and observed at x40 and xlOO, respectively. (5a) and (5b) healing skin tissues of group II with ethanol extract of P betel leaves, 2 %(w/v) and observed at x40 and xlOO, respectively, (6a) and (6b) healing skin tissues of group III with ethanol extract of P. betel leaves, 4 % (w/v) and observed at x40 and xlOO, respectively. (7a) and (7b) healing skin tissues of group IV with ethanol
extract of P. betel leaves. 6 % (w/v) and observed at x40 and xlOO, respectively. (8a) and (8b) healing skin tissues of group V with ethanol extract ofP betel leaves. 8 % (w/v) and observed at x40 and xlOO, respectively, (9) and (10)
showed lymphocyte, neutrophil and fiber hyperplasia
168 Jotirnal of Medicinal Materials, 2015, Vol. 20, No. 3
No.
I.l 1.2 1.3 1.4 1.5
E p i d e r m i s V e s i c u l a r
+ 0 0 0 0
Table 3. Evaluation of H&E test D e r m i s
H a i r follicle + ++
+ + ++
Fiber Hyperplasia
• H -
• H - ++
++
+++
Inflammatory cell Lym+, Neu+
Neu+
Neu+
Neu++
Neu+
Hypodermis . . . , 1 Fiber Adipocytes ( „ j , p „ p , , , i .
+ j + + ! + + + 1 + +
+ +
+ +
I n f l a m m a t o r y cell N e u + N e u + N e u + N e u + N e u +
S t r i a t e d m u s c l e N o r m a l N o r m a l N o r m a l
2.1 2.2 2.3 2.4 2.5
0 + 0 0 +
+ + + + +
+ + + ++
+
Lym+
Lym+
Neu+
Lym+
Lym+
++
+ + ++
+ 0 + ++
+ +
L y m + L y m + N e u + L y m + N e u +
N o r m a l N o r m a l N o r m a l
3.1 3.2 3.3 3.4 3.5
+ 0 + 0 0
++
++
++
+
L +
+ + + ++
+
Lym+
Lym+
Lym+
Lym+
Lym+
+ + ' + ++
-H- -H- ++
+ + + +
L y m + L y m + L y m + L y m + L y m +
N o i m a l N o r m a l
N o r m a l
4.1 4.2 4.3 4.4 4.5
+ 0 + + +
++
+ ++
+ ++
+ + + + +
Lym+
Lym+
Lym+
Lym+
Lym+
++ 1 + -H- 1 + ++
+++
+ + + + +
L y m + L y m + L y m + 0 L y m +
N o r m a l N o r m a l
N o r m a l
5.1 5.2 5.3 5.4 5.5
+ + + + 0
+ + ++
++
-H- -•- + + + +
Lym+. Neu+
Lym+
Lym+
Lym+
Lym+
-H- ++
+++
+++
++
+ ++
+ + +
L y m + + , N e u + L y m + 0 L y m +
N o r m a l
N o r m a l L y m + 1 N o r m a l
6.1 6.2 6.3 6.4 6.5
0 0 0 + 0
+ +++
+ ++
++
+ + + + +
Lym+
Lym+
Lym+
Lym+
Lym+
++
++
++
++
++
+ 1 L y m + i N o r m a l 0 ' L y m + 1 N o m i a l -f-
+ +
L y m + j N o r m a l L y m + ! L y m + N o r m a l
7 1 N + + I N-H-. H F + . Died-Leu-H-+, N e u + | + + | + N e u + + | N o r m a l N o t e :
N o I: F l c o n t a i n i n g ethanol e x t r a c t o f P . betel leaves. I % (w/v]
N . o 2 : F 2 c o n t a i n i n g ethanol e x t r a c t o f P . betel leaves. 2 % (w/v]
N . o 3 : F 3 c o n t a i n i n g ethanol extract o f P . betel leaves, 4 % (w/v]
N . o 4: F 4 c o n t a i n i n g ethanol extract o f P . betel leaves, 6 % (w/v]
N . o 5: F5 c o n t a i n i n g ethanol extract o f P . betel leaves. 8 % (w/v;
N . o 6: positive control with T a c r o l i m u s , 0 0 3 % N . o 7 : N e g a t i v e c o n t r o l .
H F : H a i r follicle L y m : L y m p h o c y t e s N e u : N e u t r o p h i l N : N e c r o s i s + : few L e u : L e u k o c y t e s + + : m e d i u m + + + : m a n y
Journal of Medicinal Materials, 2015, VoL 20, No. 3 169
Evaluation of epidermal thickness As can be seen form Table 4, both the
VI
99.92 ± 0.74
standard and samples showed a decrease in Data are expressed as the mean i^ SE M. *p < O.OS epidermal thickness compared to that of negative compared with the control
control. The treatment in group I, II and 111 was 4. Conclusion
witnessed a steady decline in epidermal thickness j h e ethanol extract of P. betel leaves at a dose while there was a slight increase in epidermal of 4% (w/v) possesses significant anti-eczema thickness when treated with higher concentration activity due to its effect on the reduction of of ethanol extract of/*. 6e/e/leaves (6% and 8%). vesicular, fiber hyperplasia, inflammatoiy cell Table 4. Effect of ethanol extract of P. betel leaves on (lymphocyte, neutrophil), epidermal thickness
epidermal thickness _^ and increasing in adipocytes, hair follicles of i Group ! Change in epidermal thickness (%) ] mouse model of atopic eczema. Further study is , l____ _ _ 75-29 ± 1.36 needed to determine the target components
11 ; 71,39 J: 1.29 j playing a key role in the treatment of eczema.
Ill 68 74 ± 1.92* ; Acknowledgement: This research was supported i IV i 70 66 ±0.95 I by the International University, Vietnam National
72.56 ±2.51 i University-HCMC.
References
1. Kim M, J., Choung S. -Y (2012), Mixture of polyphenols and anlhocyanms from Vaccinium uUginosiim L. alleviates DNCB-induced atopic dermatitis in NC/NgaMlce, Evidence-Based Complementary and Alternative Medicine, 2012, 1-15,2, Kulkami G. T., Rao Dr, N. B.. (2010), Phytochemical studies and in vitro cytotoxicity screening of Piper betel leaf (PBL) extract, International Research Journal of Pharmacy. 1, 394-391, 3 Varier P. S. (1995), Indian Medicinal Plants a Compendium of 500 species. Orient Longman Publication, Chennai, India, 4. 279-280, 4. Christopher A. L Dawn B. (2008), Pharmaceutical Compounding and Dispensing. Pharmaceutical Press, pi 89. 5. Lee K -S. Jeong E. -S., Heo S. -H,, Seo J. - 11., Jeong D. -G., Choi Y. -K, (2010), A novel model for human atopic dermatitis- application of repeated DNCB patch in BALB/c mice, in comparison with NC/Nga mice. Laboratory Animal Research, 26(1), 95:102. 6 Michael H. R,, Kaye G, I,, Pawlina W, (2002), Histology: a Text and Alias. LippincoU Williams & Wilkins, 4th edition, p. 400 7, Muruganantham N„
Basavaraj K. H,. Dhanabal S. P., Praveen T. K., Shamasundar N. M.. Rao K. S (2011), Screening of Caesalpinia bondiic leaves for antipsorialic activity. Journal of Ethnophannacology, 133, 897-901, 8. Bosman B , Matlhiesen T,, Hess V., priderichs E. (1992), A quantitative method for measuring antipsorialic activity of drugs by the mouse tail test. Skin Pharmacology. 5( 1). 41 -48.
Journal of Medicinal Materials, 2015, Vol. 20, No. 3 (pp.170-175)
SCREENING MEDICINAL PLANTS IN CENTRAL VIETNAM FOR RADICAL SCAVENGING AND FERROUS ION CHELATION ACTIVITIES Nguyen Thi Hoai', Le Thi Bich Hien', Ho VietDuc', Le Tuan Anh^, Rawiwun Kaewamatawon^
'Hue University of Medicine and Pharmacy
Quang Tri Center of Science and Technology, Mientrung Institute for Scientific Research
^Ubon Ratchathani University, Thailand
*Corresponding author: hoai77@gmail.com (Received August 27'", 2014)
Summary
Screening Medicinal Plants in Central Vietnam for Radical Scavenging and Ferrous ion Chelation Activities Nowadays the pharmaceutical industry is interested in searching for new natural products possessing valuable bioactivilies with fewer side effects. This study aims to screen for antioxidant effect of plants collected from certain areas in 170 Journal of Medicinal Materials, 2015, Vol. 20, No. 3
