EFFECT OF AQUEOUS EXTRACT OF SAGE AND MARJORAM ON CHRONIC KIDNEY PATIENTS.

Conclusion: From the above, we have confirmed the antioxidant effect
of the aqueous extract of sage and marjoram on chronic kidney patients
treated with hemodialysis which include (flavonoids, phenolic acids and
terpenes) and the best drinks was the mix of aqueous extract of sage (5g)
195 Egypt. J. of Appl. Sci., 35 (11) 2020
and marjoram (5g), and that the remaining concentrations have a good
effect, but not to the same degree of improvement.

EFFECT OF AQUEOUS EXTRACT OF SAGE AND
MARJORAM ON CHRONIC KIDNEY PATIENTS
Neimat- Allah S. Salla1 ; Mohammed H. Haggag1 ; Naeem M. Rabeh1
and Osama M. Ahmed2
1Nutrition and Food Science Department, Faculty of Home Economics, Helwan
University
2 Faculty of Medicine in Damietta, Al-Azhar University
Key Words: hemodialysis, oxidative stress, antioxidants, sage, marjoram
superoxidedismutase, malondialdehyde, patients.
ABSTRACT
Chronic kidney disease (CKD) is a worldwide public health
problem. Oxidative stress is the causative factor for a wide variety of
diseases, including CKD. Medicinal plants used in the management of
CKD are effective in renal detoxification and reduce the effects of
dialysis treatment. This work was conducted to investigate the effect of
aqueous extract of sage (Salvia Officinal) and marjoram (Origanum
Majoranum) on End Stage of Chronic Kidney patients under dialysis.
The experiment was carried out on sixty patients (40-50 years old),
diagnosis based on detailed clinical history, clinical examination and
other relevant biochemical investigations. The patients were divided into
6 groups (each group contain 10 CKD patients treated with (hemodialysis
and regular medical treatment for 3 months) as followed: positive control
patients were treated with regular medical treatment, other patients
consumed aqueous extracts of (5g sage, 5g marjoram, 10g sage, 10g
marjoram, mixture of 5g sage + 5g marjoram and mixture of 10g sage +
10g marjoram) twice today respectively. Results illustrated that the
aqueous extract of marjoram or sage are rich in antioxidants components
(Phenolic acids, Flavonoids, Oxygenated monoterpenes, Diterpenoids
and Triterpenes), antioxidant capacity and phenolic content. Results
clarify that glomerular filtration rate (GFR) increased, while erythrocyte
sedimentation rate (ESR) and C-reactive protein (CRP) significantly
decreased (P<0.05) in all treated compared to control group.
Malondialdehyde (MDA) decreased and superoxidedismutase (SOD)
increased. The mixture of aqueous extracts of marjoram (5g) and sage
(5g) recorded the best drink. Therefore, this study recommended the use
of the aqueous extracts of marjoram (5g) and sage (5g) in decreasing the
oxidative stress and improve kidney health in hemodialysis patients.
Egypt. J. of Appl. Sci., 35 (11) 2020 186-201
INTRODUCTION
The kidneys filter plasma in the glomerulus to form a protein-free
ultrafiltrate. This ultrafiltrate passes through the various tubular segments
where reabsorption of essential constituents and secretion of unwanted
products occur (Byham-Gray et al, 2014). CKD is a worldwide public
health problem characterized by either reduced glomerular filtration rate
or the presence of kidney damage that leads to abnormal kidney function
and higher proteinuria, blood urea nitrogen, and serum creatinine levels
(Nogueiraa et al., 2020).
Reactive oxygen species (ROS) are produced by the aerobic
metabolism. The imbalance between production of ROS and antioxidant
defence in any cell compartment is associated with cell damage. ROS
play a pivotal role in the pathogenesis of acute and chronic kidney (Xu et
al., 2020). Oxidative stress is the causative factor for a wide variety of
diseases, including CKD. Antioxidants are the molecules that combat the
oxidative stress developed from an imbalance between the rate of
production and removal of produced oxidants (Amarasiria et al., 2020).
Medicinal plants are rich sources of bioactive compounds that
have been reported to exert nephron protective mechanisms, such as
antioxidant, anti-inflammation, diuretic, and immunomodulation.
Medicinal plants used in the management of CKD are effective in
diuresis and renal detoxification, and thus are beneficial to reduce the
adverse effects of dialysis treatment. Therefore, these plants are of
utmost importance to protect renal function and slow the occurrence and
the progression of CKD, thus forestalling the need for end-stage
treatment options such as renal replacement therapy (El‐Wakf et al.,
2020).
A key advantage to medicinal plants is that they are rich sources
of antioxidants, mainly present in the form of phenolic compounds
(flavonoids, phenolic acids, tocopherols, tocotrienols), ascorbic acid, and
carotenoids. These substances work to prevent renal damage by reducing
lipid peroxidation and increasing endogenous antioxidants. Therefore,
supplementation with medicinal plant-derived antioxidants is of utmost
importance in curtailing the free radical pathologies present in CKD. The
capacity of natural whole products rather than single antioxidants or their
combinations effect in preventing or managing kidney diseases
(Amarasiria et al., 2020). Among such plants, sage (Salvia officinalis L.)
and marjoram (Origanum majorana L.) have described to contain high
percentage of biologically active polyphenolic compounds which are
useful to human health and have effective therapeutic benefits (El‐Wakf
et al., 2020).
Sage is considered as one of the most popular herbs consumed
widely and traditionally as an herbaceous infusion. The incorporation of
187 Egypt. J. of Appl. Sci., 35 (11) 2020
sage infusion in the daily diet can provide considerable benefits such as
anti-mycotic, anti-carcinogenic, antidiabetic, antimicrobial, antiinflammatory,
antioxidant, and anti-proliferative. In addition to these
effects, sage infusion exhibits antiradical activity which correlates
strongly with their high level of total phenolic content. Rosmarinic acid,
salvianolic acid K, and luteolin-7-O-glucuronide were detected as the
main phenolic compounds of sage aqueous extract (Sotiropoulou et al.,
2020).
Marjoram is widely utilized as a spice and for better food flavor.
It is a strong remedy for coughs, respiratory infections, cardiovascular
disorders, skin lesions, and digestive problems. Marjoram could also
assist in managing liver and kidney diseases (El‐Wakf et al., 2020).
The aim of this work was to investigate the effect of aqueous extract
of sage (Salvia Officinal) and marjoram (Origanum Majoranum) on end
stage of chronic kidney patients under dialysis.
MATERIAL AND METHODS
Materials: Sage (Salvia officinalis) and marjoram (Origanum
majoranum L.) were obtained from Family Pharmacia Company for
Herbs, 38 Sharkia ind. Zone, Belbis – 10th of Ramadan road.
Subjects: Sixty (40-50 years old) patients suffering from chronic kidney
disease selected from Al-Azhar University Hospital, New Damietta,
Egypt. The patients were diagnosed on the basis of detailed clinical
history, clinical examination and other relevant biochemical
investigations. Patients were divided into 6 groups (each group contain
10 CKD patients treated with hemodialysis) and regular medical
treatment for 3 months as followed: + ve control contain patients treated
with regular medical treatment, other patients consumed aqueous extracts
of (5g sage, 5g marjoram, 10g sage, 10g marjoram, mixture of 5g sage
+5g marjoram and mixture of 10g sage +10g marjoram) respectively
twice today.
Methods
A. Aqueous extracts preparation:
The air parts of the analyzed plants, specially dried leaves (5
grams) were treated with double distilled water at 100 degrees Celsius,
for a period of 15 minutes. The samples were filtered. The resultant
filtrates were made off up to 50 ml with double-distilled water. Three
samples for each analyzed condiment plant were done according to (Al-
Turki et al., 2007).
B. Determination of antioxidant activity by DPPH method:
Antioxidant activity was determined using the 2, 2-diphenyl-1-
picrylhydrazyl (DPPH) radical scavenging method. This method was
taken from (Buřičova and Reblova, 2008 and Lo Scalzo, 2008) with
some modification.
Egypt. J. of Appl. Sci., 35 (11) 2020 188
C. Determination of total phenolic compounds:
The content of total phenolics was determined
spectrophotometrically at 760 nm by using Folin-Ciocalteu reagent. The
results were expressed as the content of gallic acid per unit mass of the
sample (Dorman et al., 2003 and Stratil et al., 2008).
D. Dietary methods:
:
Twenty four hour recall for 3 days before the experiment and put
on regimen diet for kidney disease. At the end of the experiment patients
were under 24 hour recall for 3 days. Daily food intake data was
analyzed into energy and other nutrients by using food composition
tables of National Nutrition Institute (NNI) of Egypt 2006.
:
Dietary history is used to estimate the patient’s usual food intake
and meal pattern over a long period of time. The components of the
dietary history include usual eating pattern, a crosscheck of frequency of
consumption of specific food items. A shorter time frame (less than 1
month) produces better validity (Gibson, 2005).
E. Biochemical Assay
Blood samples were collected at zero time and at the end of the
study. The serum was separated within 2 hours after blood withdrawal,
by centrifugation at 3000 rpm for 10 minutes. The serum samples were
kept frozen at -20°C until determination parameters in the laboratory of
Al –Azhar University Hospital in New Damietta. Hemoglobin (HGB),
red blood cell (RBC), white blood cells (WBC) and palatables (Plt) count
were measured using automated hematology analyzer (Sysmex, Kobe,
Japan). ESR measuring was performed using (Westergren, 1926)
method. Serum creatinine and uric acid were determined according to
Kaplan, (1984), Patton and crouch, (1977) and Murry, (1984),
respectively. Malondialdehyde (MDA) determined according to (Satoh,
1978 ). Superoxide dismutase was determined according to the Indirect
Method (Flohe and Ötting, 1984). Serum C-reactive protein (CRP) was
analyzed by an automated analyzer (Olympus AU400) with a latex
turbidimetric immunoassay kit (CRP-UL assay, Wako Chemicals, Neuss,
Germany). The glomerular filtration rate (GFR) was estimated according
to criteria of the National Kidney Foundation/ Kidney Disease
Outcomes Quality Initiative, using the equation proposed by (Levey et
al., 1999).
F. Statistical analysis:
Data was statistically analyzed using (SPSS) software version (18).
Analysis of variance (ANOVA) was used to show the significance
P189 Egypt. J. of Appl. Sci., 35 (11) 2020
RESULTS
Chemical composition of aqueous extract of marjoram and sage:
The results illustrated that the aqueous extract of marjoram and
sage are rich in antioxidants components as (Phenolic acids, Flavonoids,
Oxygenated monoterpenes, Diterpenoids and triterpenes). Marjoram
aqueous extract are rich in Terpinen-4-ol (20.23%), followed by vanillic
acid (14.44%), ursolic acid (13.09 %), α -terpineol (7.64%) and trans
pinene hydrate (6.63%) and other components as shown in Fig (1). The
main compounds in sage aqueous extract were, rosmarinic acid
(26.03%), luteolin-7-O-glucuronide (16.5%), caffeic acid (12.69%),
ursolic acid (11.43%) and apigenin (6.6%) as shown in Fig (2). These
results agree with (Beddows et al. 2000, 2001; Exarchou et al. 2002;
Fasseas et al. 2007; Celikel and Kavas 2008; Calikoglu et al. 2009;
Chrpova et al., 2010; Abdel-Massih et al., 2010 and Babili et al.,
2011; Miron et al., 2011 and Bina et al., 2017)) that approved the high
phenolic content and high antioxidants components as Flavonoids,
Oxygenated monoterpenes, Diterpenoids and triterpenes of sage and
marjoram and were detected that rosmarinic acid is the main phenolic
compounds of sage aqueous extract and terpinen-4-ol is the main
component in marjoram aqueous extract With varying proportions of the
other mentioned ingredients.
Fig (1): The chemical constitutions of marjoram aqueous extract.
Trans
Pinene
hydrate
7%
Vanillic
acid
15%
Terpinen-4-ol
20%
Coumarin
α-Terpineol 2%
8%
Ferulic acid
6%
Linalyl
acetate
3%
Ursolic acid
13%
Apigeninglucuronide
5%
Carnosol
5%
Trans
caryophyllen
e
6%
Catechin
6%
Not
identified
4%
Marjoram
Egypt. J. of Appl. Sci., 35 (11) 2020 190
Fig (2): The chemical constitutions of sage aqueous extract.
Antioxidant capacity and total phenolic contents of plants aqueous
extract:
Table (1) shows the strongest antioxidant capacity of the aqueous
extracts of marjoram and sage. The total antioxidant capacity of the
aqueous extracts of marjoram and sage were (42.1 and 60.6), respectively
and the phenolic content values were expressed as mg equivalent of
gallic acid (GAE) were (27.7 and 24.3), respectively. This result
exhibited the strongest antioxidant capacity of the aqueous extracts of
marjoram and sage which agreed with (Roby et al., 2013 and Dabija et
al., 2018).
Table (1): Antioxidant capacity and total phenolic contents of plants
aqueous extract
Herbs Latin name Family
DPPH
(mg AAE/g)*
Phenols
(mg GAE/g)**
Marjoram Origanum majorana L. Lamiaceae 42.1 27.7
Sage Salvia officinalis L. Lamiaceae 60.6 24.3
*AAE = ascorbic acid equivalent ** GAE = gallic acid equivalent
Effect of sage and marjoram extracts on some parameters of
hemodialysis patients:
By observing the results that shown in Table (2) and when
comparing the results after the before and after the experiment, it is clear
that the positive control group which includes the hemodialysis patients
Rosmarinic
acid
26%
luteolin-7-Oglucuronide
17%
caffeic
acid
13%
Ursolic
acid
11%
Apigenin
7%
Carnosic acid
5%
α –
Terpineol
4%
α -Terpinyl
acetate
3%
Trans
caryophyllene
3%
a-Humulene
4%
quercetin
3%
D-Limonene
2%
Not
identified
2%
Sage
191 Egypt. J. of Appl. Sci., 35 (11) 2020
had increase in ESR and CRP which indicate inflammation occurrence,
this result accepted with (Fine, 2002) with slightly improvement in GFR.
But the treated groups by the aqueous extracts of sage and marjoram had
a noticeable decrease in ESR, CRP with value differences and a
noticeable increase in GFR when comparing the groups to each other's.
Group (6) which treated with mix of the aqueous extract of marjoram (5
g) and sage (5 g) recorded the best result in GFR, it is show that the
improvement in kidney qualification. Group (2) which treated with the
aqueous extract of sage (5 g) recorded the best decrease in ESR unlike
CRP which had the best decrease with group (5) which treated with the
aqueous extract of marjoram (10 g), thus lead to the best decrease in
inflammation occurrence. This can illustrate the good effect of some
substances in the aqueous of used herbs.
Table (2): Effect of the Aqueous Extract of Sage and Marjoram on
Glomerular Filtration Rate, Erythrocyte Sedimentation
Rate and C- Reactive Protein in hemodialysis patients:
Groups
Parameters
GFR
(ml/min/1.73 m2)
ESR
(mm/hr)
CRP
(mg/L)
Group 1: (+ ve control) 8.48 ± 0.32c 48.70 ± 3.54a 9.30 ± 0.79a
Group2: (5g sage ) 12.86 ± 0.70b 12.40 ± 2.02b 7.20 ± 0.80b
Group3 : (10g sage ) 12.85 ± 1.08b 13.60 ± 2.74b 6.85 ± 0.58b
Group4 : (5g marjoram ) 11.26 ± 0.66b 19.00 ± 2.38b 6.82 ± 0.53b
Group5 : (10g marjoram ) 11.11 ± 0.59b 20.40 ± 3.02b 6.78 ± 0.58b
Group6 : (5 g marjoram +
5 g sage)
15.22 ± 0.28a 15.40 ± 1.72b 6.89 ± 0.24b
Data expressed as Means ± SE.
Means with different superscript letters in the same column are significantly
different at P< 0.05.
Fig (3 and 4) showed the good effect of herbs that enhance kidney
work clear in the results of creatinine and uric acid, where at creatinine
value increased and uric acid slightly decreased in the control comparing
to all treated groups which had decreased in creatinine and uric acid in
each group separately after the research period compared to before it. But
when compare between groups to each other find that group (6) which
consumed the mix of aqueous extract of herbs recorded the best decrease
in creatinine while all groups recorded increase in uric acid compared to
the control. This result unlike the result which appeared when compared
results before and after the research. This does not negate the effect of
herbs on lowering the level of uric acid, as the treated groups suffered
from high uric acid before the experiment compared to the control.
Egypt. J. of Appl. Sci., 35 (11) 2020 192
Fig (3) Effect of the Aqueous Extract of Sage and Marjoram on
creatinine on patients.
Fig (4) Effect of the Aqueous Extract of Sage and Marjoram on uric
acid on patients.
When looking to table (3) find the blood picture results in groups
before and after the experiment show the decrease in Hb, RBCs and
WBCs values with increase in PLTs in the control group and all values
decreased compared to the normal values. This result agreed with
(Hakim et al., 2016). When observing results in treated groups after the
0
2
4
6
8
10 8.47
6.38 6.23 6.6
5.71
4.05
Creatinine ( mg/dl )
Creatinine
0 1 2 3 4 5 6 7 8
4.7
5.53
7.04 7.12 6.92 7.29
Uric acid ( mg/dl )
Uric acid
193 Egypt. J. of Appl. Sci., 35 (11) 2020
experiment compared to before it showed the increase in Hb, RBCs,
WBCs and Plts to the normal values. When compared the results between
groups to each other show that the best increase in Hb was in group (2)
which consumed sage (5 g) aqueous extract, followed by group (3) which
consumed marjoram (5) aqueous extract. Group (6) which consumed the
mix of aqueous extract of herbs recorded the best increase in WBCs and
RBCs values. Group (3) recorded the best increase followed by group
(6). Last results indicate the improvement of hemodialysis patients' cases.
Table (3): Effect of the Aqueous Extract of Sage and Marjoram on
Complete Blood Count in hemodialysis patients
Groups
CBC
Hb
(g/dL)
RBC
(×1012/L)
WBC
(×103/μL)
PLT
(×103/μL)
Group 1: (+ ve control) 8.56 ± 0.44b 3.03 ± 0.15c 4.16 ± 0.37b 117.97 ± 2.47d
Group2: (5g sage ) 11.11 ± 0.16a 4.45 ± 0.17ab 5.56 ± 0.31a 140.10 ± 4.06bc
Group3 : (10g sage ) 11.02 ± 0.30a 3.77 ± 0.13b 5.87 ± 0.20a 160.69 ± 5.79a
Group4 : (5g marjoram ) 10.09 ± 0.41a 3.90 ± 0.25b 5.92 ± 0.66 a 131.90 ± 2.96cd
Group5 :(10g marjoram ) 10.16 ± 0.30a 4.33 ± 0.27ab 6.24 ± 0.52a 134.64 ± 5.33c
Group6 : (5 g marjoram +
5 g sage)
10.59 ± 0.54a 4.95 ± 0.28a 6.66 ± 0.67a 151.86 ± 7.88ab
Data expressed as Means ± SE.
Means with different superscript letters in the same column are significantly
different at P< 0.05.
Results in Fig (5 and 6) showed the increase in MDA values and
decrease in SOD values in the control group opposite the decrease in
MDA values and increase in SOD in all treated groups. The best result
was recorded in group (6).
These results indicate that chronic kidney patients treated by
hemodialysis suffered from oxidative stress increase which agreed with
(small et al., 2012) and antioxidants improve kidney health agreed with
(El-Ashmawy et al., 2005; 2007 and Abd El-Ghany and El-Metwally,
2010). The aqueous extracts of sage and marjoram had antioxidant effect
agreed with (Nakatani, 2000; Novak et al., 2000; Heo et al., 2002;
Kelly, 2004; Hazzit et al., 2006; Ayatollahi et al., 2009; Ahmed et al.,
2009; Misharina et al., 2009 Stanojevic et al., 2010 and Hossain et al.,
2010) and improve kidney health agreed with (Halliwell and
Gutteridge, 2007; Baradaran et al., 2014; and Nasri and Rafieian-
Kopaei, 2014) which appear in the improvement of GFR and the decline
in CRP, ESR, uric acid and creatinine and he increase in Hb, WBCs,
RBCs and palatable. It is worth noting that the groups that registered the
most improvement in most medical analyzes was group (6) which
consumed the mix of aqueous extract of sage (5g) and marjoram (5g)
Egypt. J. of Appl. Sci., 35 (11) 2020 194
with the difference in the positive effect of other doses used in the
experiment on patients (El-Ashmawy et al., 2005).
Fig (5): Effect of the Aqueous Extract of Sage and Marjoram on
MDA on hemodialysis patients.
Fig (6): Effect of the Aqueous Extract of Sage and Marjoram on
SOD on hemodialysis patients.
Conclusion: From the above, we have confirmed the antioxidant effect
of the aqueous extract of sage and marjoram on chronic kidney patients
treated with hemodialysis which include (flavonoids, phenolic acids and
terpenes) and the best drinks was the mix of aqueous extract of sage (5g)
195 Egypt. J. of Appl. Sci., 35 (11) 2020
and marjoram (5g), and that the remaining concentrations have a good
effect, but not to the same degree of improvement.
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تأثير المستخمص المائي لممريمية والبردقوش عمى مرضى الکمى المزمن
نعمت الله سعد صمى 1، محمد حمدي حجاج 1، نعيم محمد ا ربح 1 وأسامة محمد احمد 2
-1 قسم التغذية وعموم الأطعمة بکمية الاقتصاد المنزلي جامعة حموان
-2 کمية الطب بدمياط جامعة الأزهر
مرض الکمى المزمن هو مشکمة صحية عالمية. الإجهاد التأکسدي هو العامل
المسبب لمجموعة متنوعة من الأم ا رض، بما في ذلک مرض الکمى المزمن. النباتات الطبية
المستخدمة في علاج مرض الکمى المزمن فعالة في إ ا زلة السموم من الکمى وتقميل آثار علاج
الغسيل الکمى. تم إج ا رء هذا العمل لمعرفة تأثير المستخمص المائي لممريمية والبردقوش عمى
المرحمة النهائية لمرضى الکمى المزمن المعالجين بالغسيل الکمى. تم إج ا رء التجربة عمى ستين
04 سنة( مقسمة إلى 6 مجموعات )کل مجموعة تحتوي عمى 04 مرضى الکمى - مريضا ) 04
المزمن تم علاجهم بالغسيل الدموي( وعلاج طبي منتظم لمدة 3 أشهر عمى النحو التالي:
Egypt. J. of Appl. Sci., 35 (11) 2020 200
مجموعة ضابطة ايجابية تحتوي عمى مرضى تم علاجهم بعلاج طبي منتظم وتناول المرضى
الآخرون ) 0 جم مريمية ، 0 جم بردقوش ، 04 جم مريمية ، 04 جم بردقوش ومزيج من 0 جم
مريمية + 0 جم بردقوش( مستخمصات مائية مرتين في اليوم. تم تشخيص المرضى عمى أساس
التاريخ الطبي المفصل والفحص السريري التحاليل البيوکيميائية الأخرى ذات الصمة. أوضحت
النتائج أن مستخمص البردقوش المائي وخلاصة الميرمية غنية بمکونات مضادات الأکسدة مثل
)الأحماض الفينولية، الفلافونويد، أحادي التربينات المؤکسدة، الديتيربينويدات والت ا ريتيربين(.
تحتوي المستخمصات المائية من البردقوش والمريمية عمى نسبة عالية من مضادات الأکسدة
ومحتوى الفينول. أوضحت النتائج أن معدل الترشيح الکبيبي قد ا زد بشکل ممحوظ ، وانخفضت
سرعة ترسيب ک ا رت الدم الحم ا رء والبروتين المتفاعمي سي في جميع المجموعات المعالجة مقارنة
بالمجموعة الضابطة. انخفض المالونداي الدهيد و ا زد السوبر اکسيدديزموتيز کما سجل مزيج
المستخمصات المائية من البردقوش ) 0 جم( والمريمية ) 0 جم( أفضل نتائج. لذلک أوصت هذه
الد ا رسة بأن استخدام المستخمصات المائية من البردقوش ) 0 جم( والمريمية ) 0 جم( مفيدة جدًا
في تقميل الإجهاد التأکسدي وتحسين صحة الکمى لدى مرضى غسيل الکمى. هناک حاجة إلى
مزيد من الد ا رسات لتحديد تأثير مضادات الأکسدة للأعشاب والأطعمة عمى أم ا رض الکمى
المزمنة.
201 Egypt. J. of Appl. Sci., 35 (11) 2020