Table Of ContentBIOCHEMICAL STUDIES ON MULBERRY
By
HANAN ABD ELKAREEM GHANAM
B.Sc. Agric. Sci. (Biotechnology), Fac. Agric., Cairo Univ., 2004
M.Sc. Agric. Sci. (Agic. Biochemistry), Fac. Agric., Cairo Univ., 2011
Thesis
Submitted in Partial Fulfillment of the
Requirements for the Degree of
DOCTOR OF PHILOSOPHY
In
Agricultural Sciences
(Agricultural Biochemistry)
Department of Agricultural Biochemistry
Faculty of Agriculture
Cairo University
EGYPT
2017
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Name of Candidate: Hanan Abd El-Kareem Degree: Ph.D.
Title of Thesis: Biochemical studies on mulberry
Supervisors: Dr. Abdel-Moniem Mohamed Nagib
Dr. Abd El-Kader Moursy Abd El-Samad
Dr. Hala Mohamed Zaki
Department: Agricultural Biochemistry Approval: 10 / 9 /2017
ABSTRACT
Black mulberry fruits were examined for their proximate analysis (based on dry weight),
mineral, total phenolic compounds, total flavonoid, total anthocyanine and total and reducing sugar.
Results of the proximate analysis revealed that the moisture percentage was 83.29% for black
mulberry fruits. Ash content in black mulberry was 6.67%. The assigned crude protein content in
black mulberry fruits was 11.46%. The total lipid was 4.81% for black mulberry fruits. The fiber and
total carbohydrate of black mulberry were 9.93 and 67.13. Total phenolic compounds in black
mulberry was 71.51 mg/100g while, the estimated value in extracts mulberry was hexane 280.22,
ethyl acetate 942.21,methanol extracts 107.12 and aqueous extracts 374.95 mg/100g as Gallic acid,
respectively. Total flavonoid in different extract of black mulberry was black mulberry fruits 0.106,
hexane 1.97, ethyl acetate 0.25, methanol extracts 6.76 and aqueous extracts 5.43 mg/100g as
Qercetine, respectively. The reducing and total sugar in black mulberry was 28.16 and 41.56
mg/100g, respectively, while the estimated value in extracts mulberry was methanol extracts was
53.43 and 55.29 mg/100g, respectively and aqueous extract was 5.73 and 5.84 mg/100g, respectively.
Methanolic extract has an effect against all tested bacterial pathogens such as salmonella, E. coli,
Bacillus, and staphylococcus, especially low concentrations while ethyl acetate extract has an effect
against all tested bacterial pathogens as strong as antibacterial especially high concentrations. The
hexane and aqueous extract of black mulberry had low or no effect against all tested bacterial
pathogens. The biological effects of black mulberry extract glucose, lipid profile, kidney and liver
functions, some enzyme activities and enzymes in blood and histopathology. The best decrease for
lipid profile was recorded by 400mg/kg b.w in aqueous extract, while 400mg/kg b.w in methanolic
extract black mulberry expect HDL, the best increase 400mg/kg b.w,400mg/kg b.w in methanolic and
aqueous extract of mulberry, respectively. The best increased of catalase and total antioxidant in
aqueous extract in experimental protective.
Key words: black mulberry, proximate analysis, antioxidant, antimicrobial, HPLC, diabetes,
oxidative stress.
INTRODUCTION
Plants have been the most important sources of medicine since ancient
times. Traditional medicine and other literatures mention the use of plants in
treatment of various human ailments (Grover et al., 2002). Mulberry (Morus
nigra L.) is belonging to the family Moraceae, commonly known as black
mulberry. The fruits are known by the Persian-derived names toot (mulberry)
or shahtoot (King’s or “Superior” mulberry) (Imran et al., 2007).There are 24
species, 100 varieties and one subspecies of Morus genus, widely distributed in
North America, South America, Africa, and eastern, central, and southern Asia
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for silk production. There are three kind of mulberry; red, white and black.
White mulberry is grown in West Asia; red mulberry is grown in North
America and black mulberry (M. nigra), which originated from Iran, is mainly
grown in Southern Europe, Southwest Asia and in Turkey (Yaltirik, 1982 and
Tutin, 1996). Moreover, 95% of the mulberry trees grown in Turkey are white
(M. alba), whereas 3% are red (M. rubra) and 2% are black (M. nigra) (Ercisli,
2004). Mulberry is found from temperate to subtropical regions of the Northern
hemisphere to the tropics of the Southern hemisphere and it can grow in a wide
range of climatic, topographic and soil conditions (Tutin, 1996 and Machii et
al., 2007). Black mulberry has juicy fruits with extraordinary color and a
unique, slightly acidic flavor (Őzgen et al., 2009). The fruits are 2-3 cm long
(Gerasopoulos and Stavroulakis, 1997). Some mulberry species of genus are
widely cultived in many countries, in particular in China and Japan, where
mulberry is used for its foliage to feed the silkworm (Nomura, 1988).Where the
harvesting period is between June and August (Yaltirik, 1982). Black mulberry
is a fruit known not only for its nutritional qualities and its flavor, but also for
its traditional use in natural medicine as it has a high content of active
therapeutic compounds (Darias-Martin et al., 2003). Black mulberries are
commonly consumed fresh, but can also be consumed as shelf-stable processed
products, such as juices, molasses, marmalades, liquors, pestils, komes and
mulberry pulp are made using its fruits (Özan et al., 2008 and Hojjatpanah et
al., 2011). Fruits, root and stem barks of Tut plant have been used as strong
antibacterial and antifungal (Sohn et al., 2004) and used in the treatment of
jaundice and hepatitis, cancer (Abbasi et al., 2009 and Naowaratwattana et al.,
2010), inflammation (Sohn et al., 2004), diabetes, dyslipidemia, purgative,
anthelminthic, diarrhea, dyspepsia, hypertension (Kumar and Chauhan, 2008),
edema, fever, headache and wounds (Yang et al., 2010). Leaves of Tut plant
have been reported to use in the treatment of diabetes, anxiety (Jaslin et al.,
2010 and Katsube et al., 2010), depression (Mithun et al., 2011), ulcer
(Abdulla et al., 2009) and cerebral ischemia (Kang et al., 2006).
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Diabetes mellitus is one of the major endocrine disorders responsible for
renal failure, blindness or diabetic cataract, poor metabolic control, increased
risk of cardiovascular disease including atherosclerosis and AGE (advanced
glycation end) products (Burke et al., 2003), also is a chronic disease
characterized by high blood glucose levels and abnormal metabolism of
carbohydrates, proteins, and fat associated with a relative or absolute
insufficiency of insulin secretion and with various degrees of insulin resistance
(Goodman, 2003). The antihyperglycemic effects of this plant are attributed to
its ability to increase insulin output from the pancreas, or inhibit intestinal
absorption of glucose, or some other processes (Huang et al., 2005).
Aim of Work: This investigation was undertaken to study the chemical
composition of black mulberry fruit and the biological effects of Morus nigra,
which has been used in the traditional medicine for anti-diabetic activity, on the
blood glucose level in streptozotocin diabetic rats, antioxidant and
antimicrobial.
REVIEW OF LITERATURE
1. Proximate analysis of mulberry (Morus nigra)
The moisture was found to be 89.3% in mulberry (Morus nigra L.) fruits
(Wills et al., 1987), 78.03 to 82.40 g/100g in fresh weight (Imran et al., 2010).
Mulberry (Morus rubra L.) leaves were found to be 77.30 % - 84.27 % of black
mulberries fruits (Morus nigra L.) (Koyuncu et al., 2014). The ash was found
to be 0.8% in black mulberry (Morus nigra L.) fruits (Wills et al., 1987),
11.73% in Morus rubra L leaves, 8.91% Morus alba and 9.12% Morus nigra
(Iqbal et al., 2012), 0.12 % - 0.36 % in Morus nigra fruit (Koyuncu et al.,
2014). The lipid was found to be 0.2% in mulberry (Morus nigra L.) fruits
(Wills et al., 1987), 4.30% (Ou et al., 2011), 1.10% in M. alba, 0.95% in M.
nigra and 0.85% in M. rubra (Ercisli and Orhan, 2007), 4.24% of Morus
rubra L leaves, 6.57% Morus alba and 5.13% Morus nigra (Iqbal et al., 2012),
5.75 % of black mulberry fruits (Koyuncu et al., 2014), 0.4% black mulberry
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(Morus nigra Linn) fruits (Kumar and Chauhan, 2008). The protein was
found to be 2.2% in mulberry (Morus nigra L.) fruits (Wills et al., 1987), 0.96-
1.73% of different mulberry species fruits (Imran et al., 2010), 1.67% (Ou et
al., 2011), 10.25 % in fruits and 25.72 % in leaves (Koyuncu et al., 2014),
8.90% and 13.33% of dry weight of fruits in M. nigra and M. alba (Sánchez-
Salcedo et al., 2015). The crude fiber contents of mulberry (Morus nigra L.)
fruits was found to be 8.17% Morus rubra L leaves, 10.11% Morus alba and
12.32% Morus nigra (Iqbal et al., 2012), 0.8% of black mulberry fruits (Kumar
and Chauhan, 2008). total carbohydrate content and available carbohydrate
content of mulberry (Morus nigra L.) seeds 68.145%, 62.483%, respectively
(Shukla et al., 2015), 61.90 % in M. alba (Khan et al., 2009).
2. Mineral content
Koyuncu et al. (2014) indicated that K was the main mineral of fruit
followed by Na and P. Furthermore, black mulberry leaves were rich in
sodium, calcium and potassium. The mineral compositions of the fruits were
determined between 3.10 and 3.36 g /100g for N, 0.298 and 0.417 g/ 100g for
P, 1.82 and 2.12g/ 100g for K, 2.63 and 2.93g/100g for Ca, 0.593 and
0.723g/100g for Mg, 0.180 and 0.325g/100g for S, 63.7 and 71.2 mg/ kg for
Na, 114.3 and 126.3 mg/ kg for Fe, 69.8 and 76.8 mg/ kg for Mn, 29.0 and
36.2 mg/ kg for Zn, 11.2 and 17.2mg/kg for Cu during the harvest season.
There were significant differences (P<0.01) between the harvest dates in terms
of P, K, Ca, Mg, S, Na, Fe, Zn and Cu content of mulberry fruits and statistical
differences (P<0.05) were also found in Mn.
3. Secondary metabolites of Morus nigra
Aydin et al. (2011) investigated the flavonoid analyses carried out with
high performance liquid chromatography (HPLC) equipment, it was observed
that fruit extracts has polyphenolic compounds such as rutin, resveratrol,
myricetin and naringerin.
Issa and Abd-Aljabar (2013) showed that the black mulberry fruits
(Morus nigra L.) contained the highest amount of total phenolic content
followed by Eth. ext., whereas flavonoid ext. contained the highest amount of
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total flavonoid content followed by black mulberry fruits. Phenolic
compounds: flavonoids, phenolic acids, anthocyanins, stilbenes, tannins,
lignans, and lignins, are important for normal plant growth and development as
well as a defense against infection and injury. These compounds are commonly
found in both edible and non-edible plants, and they have been reported to have
multiple biological effects, including antioxidant activity (Kahkonen et al.,
1999).
The phenolic and flavonoid composition of different Morus extracts was
determined by the HPLC method. The extracts prepared from fruits, leaves and
roots of M. alba and M. nigra exhibited different characteristics. M. nigra roots
extract showed the highest total phenolic (186.30 mg CAE/g), while the highest
total flavonoids content (67.37 mg RE/g) was determined for M. nigra leaves
extracts. In addition, black mulberry leaf extracts with the highest antioxidant
activity had the highest phenolic acids contents. The dominant phenolic
components in the samples were rutin and chlorogenic acid. All investigated
mulberry dry extracts showed high content of phenolic compounds and
significant antioxidant activity. This work contributes to knowledge of the
antioxidant properties of Morus species. The obtained results may be useful in
the evaluation of new dietary supplements and food products (Radojković et
al., 2012).
The fruits of four Morus species, namely Morus alba (white mulberry),
Morus nigra (black mulberry), Morus laevigata (large white fruit), and Morus
laevigata (large black fruit). Low riboflavin (vitamin B2) and niacin (vitamin
B3) contents were recorded in all the fruits, while ascorbic acid (vitamin C)
was in the range from 15.20±1.25 to 17.03±1.71 mg/100 g fresh weight (FW)
(Imran et al., 2010).
Total anthocyanins of black mulberry genotypes were 2149 mg of gallic
acid equivalent per g and 719 mg of cyanidin 3-glucoside equivalent (CY 3-
glu) per g of fresh mass. In purple mulberry, these values were for GAE 1690
mg/g and for Cy 3-glu 109 mg/g on fresh mass basis (Ercisli et al., 2010).
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The total anthocyanin was varied significantly among clones of M.
nigra. These results are keys for the design of future dietary intervention
studies examining the role of mulberry fruits in disease risk reduction. They
can also be used for the development of mulberry derived-products rich in
phenolic compounds (Sánchez-Salcedo et al., 2015).
4. Biological activities of Morus nigra
Kim et al. (2011) found that the mulberry leaves extract
supplementation decreased plasma insulin concentrations increased by high-fat
or high-sucrose diets and improved insulin sensitivity by increasing the
expression level of insulin receptor in muscle and adipose tissue. Mulberry was
used in old Chinese herbal medicine for reducing blood serum glucose
(Andallu et al., 2001). HemoglobinA1C, a biomarker for chronic exposure to
high concentration of glucose, was also significantly decreased in the MA-
treated group (6.78%) in comparison to untreated group (9.02%). An increased
expression of glycolysis-related genes and suppression of thiobarbituric acid
reactive substances concentrations in the liver of quercetin 3-(6-
malonylglucoside) group compared to control mice were observed. The results
showed that dietary consumption of quercetin 3-(6-malonylglucoside), the
quantitatively major flavonol glycoside in mulberry leaves, improved
hyperglycemia in obese mice and reduced oxidative stress in the liver. Eighteen
N-containing sugars were isolated from the roots of M. alba (Asano et al.,
1994). Mulberry leaf extract produced significant reductions in blood glucose
increases for the initial 120 minutes of the study (Mudra et al., 2007).
SUMMARY
1. Chemical composition of black mulberry fruit
a. Proximate analysis of black mulberry fruit
The moisture, total ash, total carbohydrate, crude fiber, crude protein
and total lipid contents were found to be 83.29%, 6.67%, 67.13%, 9.93%,
11.46% and 4.81%, respectively in black mulberry fruit.
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b. The mineral content of black mulberry fruit
The ash mineral content of black mulberry fruit (as mg/100g based on
dry weight). It is clear that potassium, calcium, phosphorus and magnesium,
and represent the higher content in the fruit (1801, 617.70, 207.4 and 91.2
mg/100g), respectively.
c. The yield percent of black mulberry fruit extracts.
It is clear that the yield percentage of black mulberry fruit 5.84%,
0.67%, 49.42% and 9.47% in hexane extract, ethyl acetate extract, methanol
extract and aqueous extract, respectively.
The major compound of total phenolic in black mulberry fruit E-vanillic
(536.40), chlorogenic (164.78), pyrogallol (140.10), salicylic (72.50), benzoic
(52.75), and Epi-catechin (51.01), cinammic in hexane extract, cinammic,
pyrogallol, oleuropein, catechol, p-cumaric, iso ferulic and E-vanillic in ethyl
acetate extract, pyrogallol, iso ferulic, Epi-catechin and oleuropein in
methanolic extract; while, pyrogallol in aqueous extract (5.47 mg/10g). It was
found that the highest hespirdin compounds in ethyl acetate and where the
luteolin and naringin are the major compounds in the ethyl acetate extract
followed by methanolic extract followed by aqueous extract.
d. Antioxidant activity of black mulberry fruit extracts.
DPPH free radical scavenging activity of different extracts of Morus
nigra L. All tested extracts show appreciable free radical scavenging activities.
Methanolic extract has the strongest radical scavenging activity as well as
aqueous extract followed by ethyl acetate and hexane extract, total anthocyanin
revealed that the black mulberry, methanolic extract and aqueous extract as
578,890 and 430% respectively. The reducing and total sugar in black mulberry
was 28.16 and 41.56 mg/100g respectively, while the estimated value in
extracts mulberry was methanol extracts was 53.43 and 55.29 mg/100 g
respectively and aqueous extract was 5.73 and 5.84 mg/100g respectively.
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Total phenolic compounds in black mulberry was 71.51 mg/100g while, the
estimated value in extracts mulberry was hexane 280.22, ethyl acetate
942.21,methanol extracts 107.12 and aqueous extracts 374.95 mg/100g as
Gallic acid respectively. Total flavonoid in different extract of black mulberry
was black mulberry fruits 0.106, hexane 1.97, ethyl acetate 0.25, methanol
extracts 6.76 and aqueous extracts 5.43 mg/100g as Qercetine respectively.
2. Biological effects in experimental animal
Seventy two adult male albino rats (150-200g weight) were obtained and
housed food technology Research Institute, Agricultural Research Center, Giza,
Egypt.
Glucose, total cholesterol, triglyceride, LDL, HDL, total lipid, urea,
creatinine, Albumin, total protein contents, ALT, AST and total antioxidant
activities in serum, catalase activity in plasma, insulin hormone in serum and
HbA1C in plasma, were determined after 40 days in rats fed on normal diet
supplemented with different extracts of black mulberry fruits and different
concentrations of black mulberry extract.
a. Effect of different black mulberry fruit extracts (M. nigra) on blood
glucose level, insulin hormone and glycated hemoglobin (HbA1c) in
STZ-induced diabetic rats.
Serum glucose in protective diabetic group (G2) was significantly
higher 362.74 mg/dl compared with normal group (G1) 81.02 mg/dl after 15
days. Also, significantly higher 307.37 mg/dl compared with normal group
(G1) 71.78 mg/dl in the end experimental. It is clear that the glucose in G6 with
orally administration of aqueous extract of black mulberry (400mg/kg body
weight) have the lowest level (209.18 mg/dl after 15 days and 103.60mg/dl in
the end experimental). Moreover, the lowest aqueous extract concentration
(G6) protected recorded by the diabetic control (G2), the serum insulin in
protective experimental diabetic group G2 was significantly lower (0.53
μU/ml) compared with normal group G1 (4.37 μU/ml). It is clear that the
insulin in G6 with orally administration of aqueous extract of black mulberry
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(400mg/kg body weight) have the highest level (1.83μU/ml). Moreover, the
highest aqueous extract concentration (G6) protected recorded by the diabetic
control (G2) and the glycated hemoglobin (HbA1c) in blood protective
experimental diabetic group G2 was significantly higher (7.83g/dl) compared
with normal group G1 (5.07g/dl). It is clear that the glycated hemoglobin in G3
with orally administration of methanolic extract of black mulberry (200mg/kg
body weight) have the lowest level (4.13g/dl).
b. Effect of different black mulberry fruit extracts on serum lipid profile in
STZ-induced diabetic rats.
1. Total cholesterol of rat blood serum
The serum total cholesterol in diabetic group G2 is significantly higher
(126.83mg/dl) compared with normal group G1 (94.1mg/dl). It is clear that the
serum total cholesterol in G4 and G6 which fed on methanolic and aqueous
extracts of black mulberry (400mg/kg b.w) showed the maximum reduction in
blood serum total cholesterol compared to the other applied concentration.
2. Serum triglyceride content
The serum triglycerides content in diabetic control G2 is significantly
higher (108.62mg/dl) compared with normal control G1 (73.63mg/dl). The
used methanolic extract of black mulberry concentration (400mg/kg b.w)
showed the maximum reduction in blood serum triglyceride compared to the
other applied concentration and the best dose in reduction.
3. High density lipoprotein cholesterol (HDL) of rat blood serum
Serum HDL cholesterol in diabetic group (G2) value (67.45mg/dl) was
insignificantly lower compared with normal control (G1) (82.06mg/dl). It is
clear that the HDL-cholesterol in G4 with orally administration of methanolic
extract of black mulberry (400mg/kg body weight) have the highest level
(97.47mg/dl) followed by aqueous extract G6 (400mg/kg body weight) have
the highest level (89.21mg/dl).
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Description:HANAN ABD ELKAREEM GHANAM. B.Sc. Agric. Sci. (Biotechnology), Fac. Agric., Cairo Univ., 2004. M.Sc. Agric. Sci. (Agic. Biochemistry), Fac. Agric.
