Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
81
EXTRACTION ,ISOLATION AND IDENTIFICATION OF
SOME ALKALOIDS COMPOUNDS FROM IRAQI MEDICINAL
PLANT HALOXYLON SALICORNICUM AND STUDY
ANTIHYPERGLYCEMIC EFFECT IN ALLOXAN –INDUCED
DIABETIC RABBITS
Jamal Harbi Hussein Alsaadi
Department of Chemistry, College of Science,University of Thi qar,Thiqar,Iraq
Keywords:Antihyperglycemic, alkaloids , alloxan
Corresponding Author e-mail : noorjamal713@yahoo.com
ABSTRACT
The present study was carried out to determine and investigate the
antihyperglycemic action of the alcoholic extract and alkaloids extracted from Iraqi
Medicinal Plant Haloxylon salicornicum on blood glucose level of Alloxan –Induced
Diabetic Rabbits (150 mg/kg intraperitoneal) . In present study were prepared are
alcoholic extract and isolated alkaloids . Preliminary qualitative tests were
carried out for all prepared extracts and identification bio active chemicals alkaloid
compounds (Acetic acid (amino carbonyl) ; 4-fluoro histamine ; hydroxyl urea; nhexyl
methyl amine ; 1-methyl dodecyl amine ; Octadomine) by TLC and GC-MS
chromatography . Alloxan –induced diabetic rabbits was prepared after injecting the
experimental rabbit with three dose of alloxan (I.V).Alloxan –induced diabetic rabbit
, the results indicated that alcoholic extract and alkaloids extracted from Haloxylon
salicornicum showed significant decreasing in glucose conc. levels. Alkaloids
isolated from Haloxylon salicornicum showed very high activity to decrease blood
glucose levels in hyperglycemic rabbits ,where significant decreasing (P<0.05)
was found at second hrs , significant decreasing (P<0.01) at fourth hr. and high
significant decreasing (P<0.001) at sex and twenty forth hrs. Both alcoholic extract
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
82
and alkaloids extracted Haloxylonsalicornicum have potential hypoglycemic effect in
hyperglycemic induced rabbits
INTRODUCTION
Diabetes Mellitus (DM) is the number one killer among all chronic diseases in the
world (1) , by an increased concentration of blood glucose due to derangement in
carbohydrates metabolism and defective secretion of insulin. The main symptom of
DM is hyperglycemia, which leads to many complications classified into “micro
vascular” and “macro vascular” , micro vascular complications, such as diabetic
nephropathy and diabetic retinopathy, result from damages to the small blood vessels,
whereas the macro vascular complications are caused by damages to arteries, leading
to coronary artery and periphery artery diseases, and stroke. Basically, hyperglycemia
is the result of relative insulin deficiency, insulin resistance or both (2, 3). diabetes
mellitus (DM) is increasing across the world , reported showed that in the year 2000,
there were about 171 million diabetes mellitus cases worldwide in patients ages 20
years or more, and in the year 2011 the World Health Organization (4) estimated that
over 346 million of people live with DM worldwide. Nearly 80% of deaths due to DM
occur in low and middle income countries Traditional herbal material plays an
important part in the treatment of diabetes. Many herbal medicines have been
recommended for the treatment of diabetes mellitus, medicinal plants have the
advantage of having no side effects , in addition high cost of conventional treatments
with synthetic drugs, traditional treatment with plants becomes an alternative option
for financially poor populations , More than thirteen thousand plants have been
studied for various pharmacological properties(5) .Therefore, the goal of
pharmacotherapy is to normalize the blood glucose levels (6). Haloxylon
salicornicum is a desert plant belongs to the family Chenopodiaceae, which has 120
genera and more than 1300 species. H. salicornicum is a branched shrub grows up to
1 m height. The new branches are green , succulent while the older ones are yellowish
white to silvery white (7 ), plant is known locally as Hamit and it is widely
distributed in Iraq specialty southern of Iraq and It is distributed from Western
Mediterranean region to Arabia, Iran, Mangolia , Burma and Southwest of China (8).
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
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The plant is reported to be used as anti-diabetic (9), antibacterial (10) and antiinflammatory
(11). Two species of the genus were recorded in the literature to have
folkloric uses. H. salicornicum is reported to be used for antiseptic and antiinflammatory
(9 , 7) .Traditional healers are using it to treat intestinal ulcers (12).In
Oman the stems of this species are used as a mordant for dyeing wool in traditional
weaving. In addition, Haloxylon scoparium {Haloxylo articulatum} is used to treat
eye disorders (13). Infusion and powder infusion of aerial part of H. scoparium are
used in Morocco for their antidiabetic effects (14,15). The qualitative phytochemical
analysis of the aerial parts of the plant revealed,the presence of alkaloids, cardiac
glycosides, anthraquinones, flavonoids, saponins, coumarins, sterols, tannins, volatile
oils and volatile bases (9). On the other hand, few species of the genus Haloxylon
(seven species) have been chemically investigated, which resulted in the isolation of
the several alkaloids belonging to mainly seven classes of alkaloids. These classes
are: aliphatic quaternary alkaloids, pyridine alkaloids, indole alkaloids, isoquinoline
alkaloids, isoquinolone alkaloids, β-carboline alkaloids and phenyl ethylamine
alkaloids (16). A piperidyl alkaloid “haloxynin” has also been isolated and
characterized form Haloxylon salicornicum by mass spectrometry, among the 80
identified alkaloids. New flavonoids quercetin 3-O-β-glucosyl [1→2]-α-
rhaminoside-7-O-α–rhaminoside and quercetin3-O-pcoumaryl [1→6]-β-glucosyl
[1→6]-β-glucoside-7-O-α rhaminoside, together with knowncompounds quercetin 3-
gentiobioside, isoquercetin, quercitrin and kaempferol were isolated from the aerial
part of O. baccatus (17).
MATERIAL AND METHODS
Plant collection and sampling
Plant Material. H. salicornicum were collected from South Nassira city , Southern
of Iraq and identified at the department of Biology, college of Sciences, University
of Thi-Qar. , Iraq. The dried aerial parts plant in 25 C were powdered using a milling
machine. The powder, weighing about 890 gm.
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
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Preparation of Alcoholic Extracts
50 gm. of dried grounded aerial part were refluxed in 250 ml of 70% ethanol for 24
hours, the precipitate was removed by filtration, through filter paper no.1, then filtrate
was concentrated und er vacuum using freeze drier to afford (6.76) gm.
Isolation of Alkaloids and nitrogen compounds
50 gm. of defatted powder were mixed with 250 ml of 10% ethanol and acetic acid
on magnetic stirrer for 24 h .Then filtered and concentrated to quarter of previous volume
by using vacuum rotator evaporator at 70ºC and the pH was adjusted to 9 with ammonium
hydroxide to precipitate the alkaloids. The mixture was put in separation funnel ,then 20 ml
of chloroform was added and the mixture was mixed well. The organic layer was collected .
This step was repeated three times then they were dried by vacuum using rotator evaporator
to yield (2.30) gm.( 18 ).
Column chromatography(CC)
column chromatography(CC). separation of Alkaloids and nitrogen compounds were
carried out size-exclusion column chromatography over Sephadex using (CH2Cl2: MeOH, 1:1)
using 10:90(Methanol :Di chloro methane ) as the mobile phase(19). .
Thin layer chromatography (TLC)
Thin layer chromatography (TLC) were used for isolation ,separation and
purification the active chemical compounds in biochemistry laboratories at chemistry
department in college of sciences at university of Thi Qar in Iraq. TLC analysis was
carried out on 0.2 mm silica gel, aluminium-backed plates (Merck Art.5554), by using
solvent system Dichloromethane:Methanol ) (50:50).The plates were developed using
(anisaldehyde: conc. H2SO4: methanol [1:2:97] spray regent and heating(19).
Gas Chromatography Mass Spectrometry (GC-MS)
GC-Mass spectra were achieved by using a Hewlett Packard G1800A GCD system in
Chemistry department , college of pure sciences education at University of Basra ,Iraq.
The alkaloid extract was dissolved in methanol depending on solubility, a 2 μl sample
was injected then volatilized at 250°C. The column, a 30 m x 0.250 m, 0.25 Å, HP-5-MS
column, was heated initially to 50°C for 3 min and the ramped up to 250°C at 10°C per
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
85
minute for 2 minutes, the temperature was then held at 250°C for the remaining 20
minutes.
Phytochemical Analysis
Phytochemical qualitative analysis was performed using the general method for
phytochemical screening of tannins, glycosides, flavonoids, alkaloids and saponins
with modification (20).
Identification of Tannins
Half a gram of extract was stirred with 10 mL of distilled water and filtered. Four
drops of a 1% ferric chloride solution were added to 2 mL of the filtrate. Blue-black,
green or blue-green precipitate
indicated the presence of tannins.
Identification of Glycosides
Half gram of extract was dissolved in 1 mL of distilled water. Aqueous NaOH 2M
was added to the solution and the resulting yellow coloration indicated the presence of
glycosides.
Identification of Flavonoids (Ferric Chloride Test)
Half gram of extract was boiled with 3 mL of distilled water. The solution was then
filtered; and 2 mL of the filtrate were mixed with 5 drops of a 10% ferric chloride
solution. The color change to
green-blue or violet indicated the presence of the phenolic hydroxyl group.
Identification of Alkaloids (Dragendorff’s Test)
Approximately 0.2 g of extract were stirred with 5 mL of 1% hydrochloric acid (HCl)
in a water bath.The solution was filtered; and 1 mL of the filtrate was added to a test
tube. Next, Dragendorff’s Reagent
was added to the filtrate. The presence of an orange color signaled the presence of
alkaloids.
Identification of Saponins
Approximately 0.2 g of extract were dissolved in 5 mL of distilled water and the
mixture was heated until boiling. The solution was then cooled and filtered. Next,
3mL of distilled water was added to the filtrate and the tube was shaken vigorously.
Formation of froth indicated the presence of saponins.
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
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Animals
Rabbits weighting 1.25-1.800 Kg were procured from local market in Basra ,
Iraq .Animals were kept in clean polypropylene cages and fed on standard antibiotic
free diet. All animals were kept in fast for 24 hr. before staring the experiments .
Diabetes Induction of Rabbits
The rabbits were induced practically using three injections of alloxane
monohydrate dissolved in sterile normal saline. After this process, rabbits became in
diabetes case. Solution of alloxane was used immediately after preparation and was
administrated at period of 48 hrs in a dose equal to 150mg/Kg body weight of rabbits
and injected via marginal ear vein under light in 1ml syringe (21), then 20% of
glucose dissolved in drinking water was given to rabbits (20% glucose) orally and
they were kept in fast for 18 hrs after seven days from last administration. Glucose
concentrations were measured in blood of alloxan-induced diabetic rabbits by using
glucose oxidase peroxidase enzymatic colorimetric GOD-PAP method (22).
Effect of alcoholic extract and alkaloids on glucose level in alloxan –induced
diabetic rabbits
Twelve fasted hyperglycemic rabbits were divided into two equal groups. The
first was given 3ml of normal saline and while the second group was given 0.3gm/kg
body weight of alcoholic extract dissolved in normal saline concentration was fitted
as 100 mg /ml ,each rabbit dose 3ml /Kg BW which was considered as treatment
group. Blood samples were collected at times (0 (as fasting), 2,4,6 and 24 hrs.). The
glucose concentrations were measured at each time point (21,22).and same methods
administration of 3 ml of 0.3gm/kg BW alkaloids extract.
RESULTS AND DISCUSSION
Yield of Haloxylon salicornicum Extracts
Percentages of H. salicornicum extracts were calculated , as illustrated in
table ( 1)
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
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Table(1). Percentages of prosopis juliflora pods extracts
No Extract type Yield (%)
1. Alcoholic Extract 13.52
2. Alkaloids 4.60
Qualitative Analysis of Alcoholic Extract and Alkaloids of Haloxylon
salicornicum
The phytochemical analysis of Haloxylon salicornicum extracts plant showed the
presence of major metabolites of saponins, alkaloids, tannins, glycosides and cardiac
glycosides .Table (2) indicates qualitative analysis results of alcoholic and alkaloids
extract of Haloxylon salicornicum . The number of positive signs indicates the
intensity of the reactions that reflects the quantity of secondary metabolism active
compounds present in the extract.
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
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Table (2). Qualitative analysis of extracts of Haloxylon salicornicum
+ positive test - negative test +++ quantitative presence
The preliminary phytochemical analysis of alcoholic extract of study plant (Table 2)
showed the presence of a high number of phytochemicals.The presence of major
metabolites of saponins, alkaloids, tannins, glycosides and cardiac glycosides. The
number of positive signs indicates the intensity of the reactions that reflects the
quantity of phytogroup present in the extract. ( 23,24,25,26 )
Reagents
Extracts
Alkaloi Results
ds
Alcoholic
Dragendroff +++ +
alkaloids are present
Wagner +++ +
Mayer +++ +
Marquis +++ +
Molish +++ + Carbohydrates
are present
Benedict - + Glycosides are present in alcoholic
extract
5% Hg Cl2 - +
Saponin is present in alcoholic extract
Silver mirror - +
Folin - + Phenols are present in alcoholic extract
Alcoholic KOH - - Flavonoids are absent in alcoholic
Mg- turnings - - extract
1% Ninhydrin - ++
Free amine groups and peptides are
present in alcoholic extract
1% lead acetate - +
Tannins are present in alcoholic extract
1 % FeCl3 - +
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
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Determination of alkaloids compounds from Haloxylon salicornicum by Thin Layer
Chromatography (TLC)
Alkaloids and alcoholic extracts from Haloxylon salicornicum were separated on
TLC and after spraying dragondorff reagent it was observed contains highest
number of alkaloid with 6 spots giving positive alkaloid test. Active spots fractions
separated by TLC showing in pictures(1and 2) .
Pictures (1and 2) indicate TLC results of alcoholic extract and alkaloids from
Haloxylon salicornicum plant. Many chemical compounds of extracts because
found many spots in TLC plates were have different Rf ,but alkaloids has six
nitrogen components , and this corresponded with extraction percentage of both
extracts.
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
Gas liquid chromatography
Extract
Gas Chromatogram analysis for alkaloids identification
identified in the alkaloids
, this results of mass spectrum showing in table (
Table(3 ) the chemical constituents of
No. Compound name
1 Acetic acid (amino carbonyl)
2 4-fluoro histamine
3 hydroxyl urea
4 n-hexyl methyl amine
5 1-methyl dodecyl amine
6 Octadomine
Fig.1 Mass Spectrum for
90
chromatography-mass spectrometry(GC-MS) of Haloxylon salicornicum
sex compounds were
extracted of Haloxylon salicornicum
3 ) and figures (1)-(
Haloxylon salicornicum by GC
Molecular formula
C3H4O4N2
C5H8N3F
C H4N2O2
C7H17N
C13H29N
ctadomine C8H19 N
Acetic acid (amino carbonyl)
(6).
GC–MS analysis
m/z [M+H]
+ 132
+ 129
+ 76
+ 115
+ 196
+ 128
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
Fig.2
Fig.
Fig.4 Mass Spectrum for
91
Mass Spectrum for 4-fluoro histamine
3 Mass Spectrum for hydroxyl urea
n-hexyl methyl amine
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
Fig.5 Mass Spectrum for
Fig.
Effect of Oral Administration
glucose level in alloxan induced diabetic rabbits
The effect of alcoholic extract
glucose conc. in hyperglycemic rabbits at
indicated in table (4 ). It was found that this
conc. after 4hrs (P<0.05) and a significant decreasing occurred
The significant decreasing was recorded
92
1-methyl dodecyl amine
6 Mass Spectrum for Octadomine
Alcoholic extract of Haloxylonsalicornicum
lcoholic of Haloxylon salicornicum on blood
different times after oral administration, is
extract decreases significantly glucose
after 6 hrs
after 24hrs (P<0.01).
on
hrs. (p<0.01).
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Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
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Table (4 ): Effect of oral administration of alcoholic extract of
Haloxylonsalicornicum on blood glucose conc. in hyperglycemic rabbits
Groups N
Blood glucose conc. (mg/dl)
0 hrs. 2 hrs. 4 hrs. 6 hrs. 24 hrs.
Control 3m l normal
saline
6
328.2±
7.41
330.4±
4.48
317.8±
2.43
312.6±
4.17
283±
4.85
300mg/kg BW (alcoholic
extract)
6
339.12
±2.24
307.1±
4.56
266.94*±
3.28
232**±
3.89
159.47**
±3.43
Blood glucose conc. were represented as mean ± S.E.M.
P *<0.05, P**< 0.01, N = number of rabbits in each group.
Effect of Oral Administration of the alkaloids extract from Haloxylon
salicornicum on glucose level in alloxan –induced diabetic rabbits
The means of blood glucose conc. in fasted hyperglycemic rabbits at different
times after oral administration of alkaloids extracted fromHaloxylon salicornicum ,
are indicated in table (5). A significantly decreasing result of glucose conc. was found
after 4 hrs. , 6hrs (P < 0.01) and also a significant decrease was recorded after 24hrs
(P<0. 001).
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
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Table (5): Effect of oral administration of alkaloid compounds extracted from
Haloxylon salicornicum on blood glucose conc. in fasted hyperglycemic
rabbits
Groups N
Blood glucose conc. (mg/dl)
0 hrs. 2 hrs. 4 hrs. 6 hrs. 24 hrs.
Control 3ml normal
saline
6
341.12±
12.51
332.22±
14.18
358.61±
2.74
316.92±
11.23
299.78±
9.88
300mg/Kg BW
(alkaloid compounds)
6
353.21±
7.40
289.85*±
8.86
220.44**±
10.72
182.36**±
6.12
134.31***±
9.10
Blood glucose conc. were represented as mean ± S.E.M.
P*<0.05, P**< 0.01, P***<0.001, N = number of rabbits in each group.
In general Alloxan has been known when ijected at different dose causes
diabetogenic and induces DNA strand breaks in isolated rat pancreatic islets in vitro
to cause Diabetes mellitus (27). This is in accordance with our data, where alloxan
injection induced significant increase in blood glucose levels versus the control (non
diabetic) group. This is compatible with many of the studies that have been used
Alloxan - induced of diabetes in rabbits and rats ( 28 , 29). Oral administration
effect of alcoholic extract and alkaloids of study plant on blood glucose
concentrations in hyperglycemic rabbits appeared decreasing effect in
hyperglycemic rabbits .The results showed that administration of alcoholic extract
group did not any significant difference after 2 hours compared to the control
groups, but decreasing blood glucose levels in rabbits induced alloxan after 2 hrs
from oral administration with alkaloids extract , because of metabolism of active
compounds begins after 2hrs from oral administration of extract (30). This agrees
with the findings of previous workers using different medicine plants (31,32 ). have
hypoglycemic activity. These plant extract contain compound like polysaccharides,
flavonoids terpenoids and tannins(33,34 ), steroid , polypeptides and alkaloids
(35,36), and these compounds are responsible for the antidiabetic activity. Phyllanthus
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
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amarus leaf extract is reported to contain compounds like lighans, alkaloids,
flavonoids, galloatnoids and glycosides (37 ). The higher blood glucose levels are
expected in alloxan induced diabetic mice, since alloxan causes a massive reduction
in insulin release, by the destruction of the β- cells of the islets of Langerhans and
inducing hyperglycemia ( 38 ) reported that there are two possible explanations for
the antidiabetic property of aloe barbadensis. It may have exerted its effect by
preventing the death of β-cells and it may permit recovery of partially destroyed β-
cells, study plant may also have initiated cell proliferation ,may be the
hypoglycemic effect come back to active nitrogen compounds in activities of β-cells
in pancreas.
CONCLUSIONS
Both alcoholic extract and alkaloids extracted Haloxylonsalicornicum have
potential hypoglycaemic effect in hyperglycaemic induced rabbits
Haloxylon استخلاص ،عزل وتشخیص بعض المرکبات القلویدیة من النبات الطبی العراقی
ودراسة تأثیرھا المخفض على مستوى کلکوز الدم فی الأرانب المصابة salicornicum
بفرط السکر المستحدث بالالوکسان
جمال حربی حسین
قسم الکیمیاء ،کلیة العلوم ، جامعة ذی قار ،ذی قار ، العراق.
الخلاصة
أجریت ھذه الدراسة لتحدید وتقصی عمل التأثیر المخفض لمستخلصات الکحولیة والقلویدیة المعزولة
لمستویات سکر الدم فی الأرانب المستحدث بھا Haloxylon salicornicum من النبات الطبی العراقی
فرط السکر بالألوکسان ( ١٥٠ ملغم / کغم من جسم الحیوان). تم تحضیر المستخلصات من نبات قید الدراسة ،
المستخلص الکحولی وعزل عائلة کیمیائیة فعالة (القلویدات ). أجریت اختبارات نوعیة أولیة لجمیع
Acetic acid ) المستخلصات المحضرة ثم تم تشخیص المرکبات الکیمیائیة الفعالة فی مستخلص القلوید
(amino carbonyl);
4-fluoro histamine ; hydroxyl urea; n-hexyl methyl amine ; 1-methyl dodecyl
ثم درس التأثیر المخفض لمستویات ، GC-MS و TLC بواسطة تقانات (amine ; Octadomine
کلوکوز الدم فی الأرانب المصابة بفرط السکر (المحدث بوساطة الالوکسان) لمعرفة فعالیة المستخلصات فی
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
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تخفیض سکر الدم فی ھذه الأرانب، إذ أشارت النتائج أن جمیع المستخلصات قد أظھرت تخفیضا جیدا لمستویات
فعالیة عالیة جدا فی Haloxylon salicornicum الکلوکوز، أظھر مستخلص القلویدات المعزولة من
فی الساعة (P< تخفیض مستویات سکر الکلوکوز فی دم الأرانب . إذ وجد انخفاض معنوی عند ( 0.05
فی الساعة الرابعة و السادسة وانخفاضا معنویا عالیا وملحوظا (P< الثانیة بعد التجریع وانخفاضا معنویا ( 0.01
فی الساعة الرابعة والعشرین فی الأرانب المصابة بفرط السکر . (P<0.001)
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