Anti-diabetic activity of Anogeissus acuminata a medicinal plant ...

Wudpecker Journal of Medicinal Plants Vol. 1(1), pp. 008 - 015, September 2012
Available online at http://www.wudpeckerresearchjournals.org
2012 Wudpecker Research Journals
Full Length Research Paper
Anti-diabetic activity of Anogeissus acuminata a
medicinal plant selected from the Thai medicinal plant
recipe database MANOSROI II
1,2 Moses Z Zaruwa*, 3 Jiredej Manosroi, 4 Toshihiro Akihisa, 5 Worapaka Manosroi, 6 Aranya
Manosroi
1 Faculty of Science, Adamawa State University, P.M.B 25, Mubi, Adamawa State Nigeria
2 Faculty of Pharmacy, Chiang Mai University, Chiang Mai 50200, Thailand.
3,6 Natural Products Research and Development Center (NPRDC), Science and Technology Research Institute, Chiang
Mai University, Chiang Mai 50200 Thailand.
4 College of Science and Technology, Nihon University, 1-8, Kanda Surugadai Chiyoda-Ku, Tokyo, 101-8308 Japan.
5 Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand.
Accepted 06 September 2012
Hypoglycemic activities of the methanolic bark extract of Anogeissus acuminata (Roxb. ex DC.) Guill. &
Perr., was investigated in normoglycemic and alloxan induced diabetic mice. A. acuminata is a widely
used traditional medicinal plant for the treatment of several ailments including diabetes mellitus. The
extract was fractionated into 9 sub-fractions (SF1 - SF9) by silica gel column chromatography and liquid
phase partitioning. Sub-fractions SF5 at the dose of 400 mg/kg bw showed the highest reduction
(p

009 Agric. Med. Plants.
Thunb (Kusirin et al., 2009) and Anogeissus acuminata
(Roxb. ex DC.) Guill. & Perr. (Zaruwa et al., 2009). All of
these plants appear in the MANOSROI II database
of the Natural Product Research and Development
Center (NPRDC). The hypoglycemic efficiency of the
aqueous extract of A. acuminata has earlier been
observed to be over 70% in diabetic mice and was
comparable to insulin with five folds free radical
scavenging activity of ascorbic acid (Manosroi et al.,
2011). Apart from DM treatment, A. acuminata has been
used in the treatment of painful inflammatory conditions
in India (Hemamalini et al., 2010), anti-HIV activity
(Rimando et al., 1994) and anti-snake venom (Dahare
and Jain, 2010). This study investigated the
hypoglycemic effect of A. acuminata sub-fractions and
the sub-fraction with the highest hypoglycemic effect was
further investigated for other biological activities and
mechanism of action.
MATERIALS
The bark of A. acuminata was collected from Mae Fa
Luang (Akha Lahu) village, Chiang Rai, Thailand at an
elevation of 975 m in February, 2009 by Mr. J. F. Maxwell
of the Chiang Mai University Herbarium. Two voucher
specimens (Voucher No. 09 to 41) were deposited at the
CMU Herbarium, Faculty of Science and at the Faculty of
Pharmacy Herbarium, Chiang Mai University. The bark of
A. acuminata was air dried under shade prior to milling
and extraction. Other materials used are Finetest
Glucometer (Infopia Co., Ltd., Korea), stainless steel mill
(Hiplan Associates, Thailand), rotary evaporator (Buchi,
Switzerland), silica gel (Merck, Darmstadt, Germany).
Drugs and chemicals Insulin (Boehringer Ingelheim,
Germany), glibenclamide (Glb) and normal saline
(Government Pharmaceutical Organization, Bangkok,
Thailand), nifedipine, isosorbide dinitrate (Berlin
Pharmaceutical Co. Ltd, Bangkok, Thailand) and alloxan
monohydrate (Sigma-Aldrich, St Louis Mo, USA) and
methanol (RCI L, Bangkok, Thailand) were used as
received. All other chemicals were of analytical grade.
Extraction and fractionation
The semi-dried bark of A. acuminata was further dried in
a hot air oven and pulverized using a stainless steel mill.
The powdered bark (650g) was extracted by soxhlet
extraction with 3,000 ml 80% v/v method. The crude
methanol filtrate was filtered and evaporated under
reduced pressure. The crude methanolic extract was
dissolved in chloroform (2,000 ml) and the chloroform
solution was filtered and evaporated under reduced
pressure, while the methanol residue was also collected
and dried by evaporation. The methanol residue was
dissolved in water and fractionated by liquid phase
partition with ethyl acetate, n-butanol and methanol to
obtain the sub-fractions SF2, SF4 and SF5, respectively.
All sub-fractions were evaporated under reduced
pressure. The chloroform extract was loaded onto
silica gel column and fractionated using chloroform,
chloroform-methanol (9:1) and methanol to obtain the
sub-fractions SF7, SF8 and SF9, respectively. The
eluates were collected according to each band and
evaporated under reduced pressure by a rotary
evaporator. The extraction scheme of A. acuminata subfractions
and their yield are shown in Figure 1.
Experimental animals
ICR mice of both sexes (22 to 28g, 9 to 12weeks) were
purchased from Animal Center, Mahidol University,
Bangkok, Thailand. The mice were fed with standard
feed, water ad libitum and maintained under standard
conditions of temperature, humidity and light (25 + 2 0 C;
70% RH and 12 h light/dark).
Ethical clearance
All methods used were ethically approved by the Chiang
Mai University’s Animal Ethics Committee. Protocol
Number: 40/2552.
Induction of diabetes mellitus
Diabetic mice were produced by intravenous injection of
10% w/v alloxan monohydrate in normal saline solution
(75 mg/kg bw) via tail vein after 18 h fasting as described
by Manosroi et al., (2011).
Hypoglycemic studies
The sub-fractions of A. acuminata were administered to
groups (n = 5) of normoglycemic or alloxan induced
diabetic ICR mice at the doses of 100, 200 and 400
mg/kg bw using a feeding tube after 18 h fasting (Moufid,
2009) and monitored over a 4 hour period as described
by Manosroi et al., (2011). Fasting blood glucose (FBG)
was assayed from the tail vein blood of the mice using
Finetest Glucometer. The effect of each extract was
compared with standard hypoglycemic drugs, insulin and
glibenclamide.
Oral glucose tolerance test (OGTT)
OGTT was performed by using the modified methods as
described by Wu et al., (2011). Briefly, groups of 5
normal mice were fasted for 18 h and fed orally with DW

Zaruwa et al. 010
Figure 1. Extraction scheme of A. acuminata fractions and sub-fractions (SF) using silica gel column chromatography (c.c.) and
liquid phase partition (LPP). SF1: methanol extract, SF6: chloroform extract; SF2: ethyl acetate, SF3: chloroform, SF4: butanol, SF5:
methanol, SF7: chloroform, SF8: chloroform-methanol, SF9: methanol sub-fractions.
or SF5, the sub-fraction which showed the highest
hypoglycemic activity. After two hours, different
carbohydrates were administered orally, namely; glucose
(2.5 g/kg), sucrose (2.5 g/kg), corn starch (6 g/kg) and
lactose (6 g/kg) as described by Wu et al., (2011). The
blood glucose levels were tested at 0, 1, 2, 3 and 4 h.
Insulin (0.5 iu) and glibenclamide (Glb) (1 mg/kg bw, oral)
was used as control.
Mechanistic studies (Co-treatment with Ca + and K +
ion channel regulators)
Normoglycemic mice were divided into four groups (n =
5) as follows: 1. Normal control (NC) (ip) group: treated
with 0.5 ml normal saline (NSS). 2. Treated with the SF5
at 250 mg/kg bw in combination with normal saline (po).
3. SF5N: the same treatment as group 2 plus nifedipine
(N) 13.6 mg/kg (po). 4. SF5I: the same treatment as
group 2 plus isosorbide dinitrate (I) 6.8 mg/kg (po). FBG
was assayed from the tail vein blood of the mice using
Finetest Glucometer at 0, 2, 4 and 6 h after treatment.
Comparison of FBG after intraperitoneal injection and
oral administration of the sub-fractions in diabetic
mice
In order to evaluate the most effective route of
administration of the SF5, diabetic mice were divided into
five groups (n = 5) and treated as follows:
1. The negative control group: given (po) distilled water
(DW).
2. The positive control group I: Injected – (ip) with 0.5
iu/kg insulin (Ins).
3. The positive control group II: given (po) with 1.0 mg/kg
bw of glibenclamide (Glb).
4. Treated group A: received (ip) with the SF5 dispersed
in DW (62.5 mg/kg bw).
5. Treated group B: given SF5 dispersed in DW (120
mg/kg bw). FBG was monitored at 0, 1, 2, 3 and 4 h post
treatment.
TLC, UV and HPLC analysis of the M SF5
Preliminary analysis of the phytochemical constituents in
the SF5 using TLC sprayed with 10% H 2 SO 4 or FeCl 3
was performed together with HPLC analysis. 10 mg of
the SF5 was dissolved in DW (1 ml) and filtered through
0.45 µm membrane filter. 0.75 ml of the filtrate was
analyzed on a UV visible spectrophotometer (Shimadzu,
Japan) to scan for a suitable spectral wave length for
further analysis. 1 ml of the filtered solution was
transferred into an amber bottle for HPLC analysis. In
HPLC analysis, Gemini– Nx 5µ C18 110A, 250 x4.60mm
column (Thermo Finnigan-Auto sampler, U.S.A) was
used. The mobile phase was 40% methanol in DW using
an isocratic program for 45 mins with the flow rate of 0.8
ml/min at 218 nm. 5 mg of Mangosteen bark (powder)
(Jujun et al., 2009; Walker, 2007) dissolved in 40%
methanol was used as a standard reference.

011 Agric. Med. Plants.
Data analysis
The results are expressed as mean ± SEM and
compared by means of student’s t test. Values were
considered statistically significant at p

Zaruwa et al. 012
Figure 2. Hypoglycemic effects in normoglycemic mice of various A. acuminata sub-fractions which
demonstrated the decrease in FBG. Values were mean ± SEM of five mice (n=5). *:Significant
reduction of FBG (p

013 Agric. Med. Plants.
Figure 4. Hypoglycemic effect of the SF5 on postprandial blood glucose (PPBG) levels of normoglycemic mice. The carbohydrates were
given 2 h after being fed with SF5 (250 mg/kg bw) or glibenclamide (1.00 gm/kg bw). *p

Zaruwa et al. 014
Figure 6. Effect of calcium (nifedipine) and potassium (isosorbide) channel regulators on hypoglycemic activity of the SF5 in
normoglycemic mice (NM). NM fed with distilled water only, SF5: fed with 250 mg/kg ip, SF5N: the SF5 at 250 mg/kg ip plus
nifedipine (13.6 mg/kg-po), SF5I: treated with the SF5 (250 mg/kg-ip) plus isosorbide dinitrate (6.8 mg/kg, po). *p

015 Agric. Med. Plants.
of Diabetic rats. Int. J. Pharmacol., 3: 143-148.
Ayodhya S, Kusum S, Anyali S (2010). Hypoglycemic
activity of different extracts of various herbal plants. Int.
J. Res. Ayurveda and Pharm., 1: 212-224.
Bnouham M, Ziyyat A, Meklifi H, Tahri A, Legsseyer A
(2006). Medicinal plants with potential antidiabetic
activity. A review of ten years of herbal medicinal
research (1990 – 2000). Int. J. Diab. Metab., 14: 1-25.
Cramer JA, Benedict A, Muszbek N, Keskinasian A, Khan
ZM (2008). The significance of compliance and
persistence in the treatment of diabetes,
hypertension and dyslipidaemia: a review. Int. J. Clin.
Pract., 62: 76-87.
Dahare DK, Jain A (2010). Ethnobotanical studies on
plant resources of Tahsil Multai, District Betul,
Pradesh, India. Ethnobot Leaflets 14: 694-705.
Hemamalini K, Naik Prasad OK, Ashok P (2010). Antiinflammatory
and analgesic effect of methanolic extract
of Anogeissus acuminata leaf. Int. J. Pharm. Biomed.
Res., 1: 98-101.
Jujun P, Pootakham K, Pongpaibul Y, Tharavichitkul P,
Ampasavate C (2009). HPLC determination of
mangostin and its application to storage stability study.
Chiang Mai Univ. J. Nat. Sci., 8: 43-53.
Kasznicki J, Glowaca A, Drzewoski J (2007). Type 2
diabetes patient compliance with drug therapy and
glycaemic control. Diabetologia, 7: 199-203.
Kusirin W, Srichairatanakool S, Lerttrakarnnon P, Lailerd
N, Suttajit M, Jaikang C, Chaiyasut C (2009).
Antioxidative activity, polyphenolic content and antiglycation
effect of some Thai medicinal plants
traditionally used in diabetic patients. Med. Chem., 5:
139-147.
Koster JC, Permutt MA, Nichols CG (2005). Diabetes and
insulin secretion: The ATP-sensitive K + channel K + -
ATP connection. Diabetes 54: 3065-3072.
Mahabusarakam W, Prodfoot J, Taylor W, Croft K
(2000). Inhibition of lipoprotein oxidation by phenylated
xanthones derived from mangostin. Free Rad. Res., 33:
643-659.
Manosroi J, Zaruwa MZ, Manosroi A (2011). Potent
Hypoglycemic Effect and Phytochemical Constituents
of Nigerian Anti-diabetic Medicinal Plants. J. Compl.
Int. Med., 8: 1-16.
Manosroi J, Zaruwa ZM, Manosroi W, Manosroi A (2011).
Hypoglycemic activity of Thai Medicinal Plants Selected
from the Thai/Lanna Medicinal Recipe Database
MANOSROI II. J. Ethnopharm. 138(1): 92-98.
Moufid A (2009). Mechanistic study of antidiabetic effect
of Chamaemelum nobile in diabetic mice. Adv. Phytoth.
Res. (Res. Signpost) 2(37): 661.
Nissen SE (2010. Setting the record straight. J. Am. Med.
Assn., 303: 1194-1195.
Nissen SE, Wolski K (2007). Effect of Rosiglitazone on
the risk of myocardial infarction and death from
cardiovascular causes. N. Eng. J. Med., 356: 2457-
2471.
Parihar MS, Madhulika C, Shetty R, Hemnani T (2004).
Susceptibility of hippocampus and cerebral cortex to
oxidative damage in streptozotocin treated mice:
prevention by extracts of Withania somnifera and Aloe
vera. J. Clin. Neurol. Sci., 11: 397-402.
Rajasekaran S, Siragnanam KK, Subramanian S (2005).
Antioxidant effect of Aloe vera gel extract in
streptozotocin induced diabetes in rats. Pharmacol.,
Report 57: 90-96.
Rimando AM, Pezzuto JM, Farnsworth NR, Santisuk T,
Reutrakul V, Kawanish K (1994). New lignans from
Anogeissus acuminata with HIV-1 reverse transcriptase
inhibitor activity. J. Nat. Prodt., 57: 896-904.
Udani JK, Singh BB, Barrett ML, Singh VJ (2009).
Evaluation of mangosteen juice blend on biomarkers
of inflammation in obese subjects: a pilot dose
finding study. Nutr. J., 8: 1-7.
Uhuegbu FO, Ogbechi KJ (2004). Effect of aqueous
extract (Crude) of leaves of Vernonia amygdalina (Del)
on blood glucose, serum albumin and cholesterol levels
in diabetic rats. Global J. Pure and Appl. Sci., 10: 189-
194.
Walker EB (2007). HPLC analysis of selected Xanthones
in Mangosteen fruit. J. Sep. Sci., 30: 1229-1234.
World Health Organization and International Diabetes
Federation (1999). Definition, diagnosis and
clarification of diabetes mellitus and its complications.
Geneva, Switzerland: World Health Organization.
Wu C, Li Y, Chen Y, Lao X, Sheng L, Dai R, Meng W,
Deng Y (2011). Hypoglycemic effect of Belacanda
chinensis leaf extract in normal and STZ-induced
diabetic rata and its potential active fraction.
Phytomed., 18: 292 -297.
Zaruwa ZM, Manosroi A, Manosroi J (2009). Free radical
scavenging activity and phytochemical constituents of
traditional medicinal plants for hypoglycemic treatment
in the north of Thailand. J. Thai Trad. Alter. Med., 7:
2(Suppl).