ABSTRACT
In this study the micronutrient, phytochemicals composition, acute toxicity and antidiabetic activity of the ethanolic extracts of Alternanthera brasiliana stem, Asystesia gangetica and Abutilon mauritianum leaf were evaluated. The micronutrient and phytochemical composition were carried with standard methods of analysis. Acute toxicity study of the ethanol extracts was carried out using 30 albino mice randomly assigned into 6 groups for the treatment. For the acute and sub acute antidiabetic study, 50 adult rats were randomly divided into 5 groups and were used for each plant sample treatment. The rats were made diabetic with alloxan monohydrate. Blood glucose levels of the rats were determined. The effect of these plant samples on the hematological profile, biochemical profile, lipid profile and histopathology parameters of the rats were measured. The thiamine, riboflavin, niacin and ascorbic acid for the plant samples ranged from 0.33 to 0.07 mg/100 g, 1.96 to 0.02 mg/100 g, 0.72 to 0.01 mg/100 and 26.96 to 8.65 mg/100 g, respectively. The calcium, magnesium, phosphorous, potassium and iron content for the plant samples ranged from 14.37 to 1.94 %, 3.83 to 2.08 %, 1.74 to 0.41 mg/100 g, 5.01 to 1.15 mg/100 g and 2.10 to 0.12 mg/100 g, respectively. The saponins, steroids, tannins, alkaloids, flavonoids and hydrogen cyanide contents of the plant samples ranged from 2.88 to 6.65 %, 12.37 to 0.22 %, 6.84 to 0.01 %, 2.91 to 0.09 %, 10.86 to 1.56 % and 10.81 to 0.67 mg/g, respectively. The acute toxicity study indicated no death with and signs of toxicity in any of the groups for the 3 samples, while that of the antidiabetic study showed significant (p˂0.05) rise in the plasma glucose level in the diabetic rats when compared with the normal rats. Treatment of the diabetic rats with 400 and 800 mg/kg of the extract significantly (p˂ 0.05) reduced the blood glucose level of the diabetic rats both in acute and sub-acute treatment. The activity of the extract during acute study was incomparable to that of the reference standard drug except at 0 and 2 hour for A. brasiliana stem, 4 and 6 hours for A. gangetica sample, but was only comparable to that of the standard drug during sub-acute treatment at 7, 14 and 21 days for A. gangetica sample and at 14 day for Abutilon mauritianum sample at both doses. It was observed that the treatment with the plants extract had high % fall than the treatment done with glybenclamide. Interestingly, the treatment with 800 mg/kg had the highest value of % fall in glucose for the A. mauritianum leaf (52 %). This implied that the extract rate of lowering potentials are dose-dependent and plant type; and could replace the drug in the management of diabetes since it is safe. Also, significant (p˂0.05) differences were observed in serum lipid profile, biochemical study and hematology study while at histopathology had no significant (p˃0.05) difference when compared with diabetic control.
TABLE
OF CONTENTS
Title Page i
Declaration ii
Certification iii
Dedication iv
Acknowledgements v
Table of Contents vi-x
List of Tables xi
List of Figures xiv
List of Plates xv
Abstract xvii
CHAPTER 1
INTRODUCTION
1.1
Statement of Problem 5
1.2 Objective 6
1.3 Significance 7
CHAPTER 2
REVIEW OF
RELATED LITERATURE
2.1 Diabetes Mellitus 8
2.1.1 Types of diabetes mellitus 10
2.1.2 Predisposing factors of diabetes mellitus 13
2.1.3 Diabetes mellitus signs and symptoms 13
2.1.4 Diabetes mellitus long-term complications 14
2.1.5 Diagnosis of diabetes mellitus 15
2.1.6 Diabetes mellitus management 17
2.1.7 Diabetes mellitus prevention 22
2.2 Brazilian Joy Weed (Alternanthra brasiliana) 23
2.2.1 Nutritional composition of red flower 24
2.3 Akpu-arachi (Asystesia gangetica) 27
2.3.1 Nutritional composition and uses of Asystesia gangetica 28
2.4 Udor (Abutilon
mauritianum) 31
2.4.1 Nutritional composition and uses of udor 31
2.5 Related Review on Plant Extract in
Antidiabetic Studies 34
CHAPTER 3
MATERIALS
AND METHODS
3.1 Experimental Design 37
3.2 Collection,
Identification and Preparation of Plant Material 37
3.3 Preparation of Plant Extract 38
3.4 Animal Collection and Preparation 38
3.5 Acute and Sub –
acute Anti-diabetic Study of the Plant Extract 39
3.6 Acute Toxicity Studies 39
3.7 Chemical Composition Analysis (minerals
and vitamins) 40
3.7.1 Calcium and magnesium content determination 40
3.7.2 Iron content determination 41
3.7.3 Potassium content determination 41
3.7.4 Phosphorous content determination 42
3.7.5 Vitamin B1 (thiamine)
determination 43
3.7.6 Vitamin B2 (riboflavin)
determination 44
3.7.7 Vitamin B3 (niacin) determination 45
3.7.8 Vitamin C determination 46
3.8 Phytochemical Composition Analysis 46
3.8.1 Determination of flavonoids 46
3.8.2 Determination of alkaloids 47
3.8.3 Determination of steroids 48
3.8.4 Determination of tannins 48
3.8.5 Determination of saponnins 49
3.8.6 Determination of cyanogenic glycoside 50
3.9 Hematological
Studies 50
3.10 Biochemical Profile Test 51
3.10.1 Serum creatinine 51
3.10.2 Serum
urea 52
3.10.3 Determination of aspartate aminotransferase
(AST) activity 52
3.10.4 Determination of alanine transferase (ALT)
or glutamate pyruvate
transaminase (GPT) activity 53
3.10.5 Determination of alkaline phosphatase (ALP)
activity 53
3.10.6
Quantitative determination of serum total
bilirubin 54
3.10.7 Determination of total protein 54
3.10.8 Serum
albumin (alb) concentration 55
3.11
Lipid Profile 56
3.11.1 Serum
cholesterol determination 56
3.11.2 Triacylglycerol determination 57
3.11.3 HDL-cholesterol
determination 58
3.12 Histopathology Study 59
3.13 Data
and Statistical Analysis 60
CHAPTER 4
RESULTS AND
DISCUSSION
4.1 The Vitamin Composition of the Fresh,
Dried and Ethanol Extract
Samples of Herbal Stem and Leave 61
4.2 Mineral Composition of the Fresh, Dried
and Ethanolic Extract
Samples of Herbal Stem and Leave 65
4.3 Phytochemical Composition of the Fresh,
Dried and Ethanol Extract
Samples of Herbal Stem and Leave 70
4.4 Acute Toxicity Studies 74
4.5 Acute Anti-diabetic Study of the Herbal Stem
and Leaf Ethanolic Extract
Samples 76
4.6 Percentage Fall in Blood Glucose Level in Acute Antidiabetic
Study of Alternanthera
brasiliana Stem, Asystesia gangetica
and
Abutilon
mauritianum Ethanolic
Extract 80
4.7 Sub-acute Anti-diabetic Study of
Ethanolic Extract Samples of
Alternanthera brasiliana Stem, Asystesia
gangetica Leaf and
Abutilon mauritianum Leaf 84
4.8 Percentage Fall in Blood Glucose Level after 21 days for
Alternanthera
brasiliana Stem, Asystesia gangetica and Abutilon
mauritianum
Ethanolic Extract 89
4.9 Biochemical Profile of the Anti-diabetic
Study of Ethanolic Extracts 91
4.10 Lipid Profile of the Anti-diabetic Study of
Ethanolic Extracts 98
4.11 Hematology Profile Blood Test of
Anti-diabetic Study of Ethanolic
Extract Samples 102
4.12 Histopathological Examination of the Liver
Tissue 107
4.13 Histopathological Examination of Pancreatic
Tissue 118
CHAPTER 5
CONCLUSION AND
RECOMMENDATIONS
5.1 Conclusion 130
5.2 Recommendations 130
References
LIST OF
TABLES
2.1 Diagnostic criteria of diabetes mellitus 17
2.2 Phytochemical constituents of A. brasiliana 28
2.3 The nutritional composition of Asystasia gangetica leaves
per
100 g edible portion 32
4.1 Vitamin composition of fresh, dried and
ethanolic extract
samples of Alternanthera
brasiliana stem in mg/100 g 62
4.2 Vitamin composition of fresh, dried and
ethanolic extract
samples of
Asystesia gangetica leaf in mg/100 g 63
4.3 Vitamin composition of fresh, dried and
ethanolic extract
samples of Abutilon
mauritianum leaf in mg/100 g 64
4.4 Mineral composition of fresh, dried and
ethanolic extract
samples of Alternanthera
brasiliana stem (mg/100 g) 66
4.5 Mineral composition of fresh, dried and
ethanolic extract
samples of Asystesia
gangetica leaf (mg/100 g) 67
4.6 Mineral composition of fresh, dried and
ethanolic extract
samples of Abutilon
mauritianum leaf (mg/100 g) 68
4.7 Phytochemical composition of fresh, dried
and ethanolic extract
samples of Alternanthera
brasiliana stem 71
4.8 Phytochemical composition of fresh, dried
and ethanolic extract
samples of Asystesia
gangetica leaf 72
4.9 Phytochemical composition of fresh, dried
and ethanolic extract
samples of Abutilon
mauritianum leaf 73
4.10 Acute toxicity
studies of the ethanolic
extract samples of
Alternanthera brasiliana stem, Asystesia
gangetica leaf and
Abutilon mauritianum leaf 75
4.11 Acute antidiabetic study of Alternanthera brasiliana stem ethanolic
extract 77
4.12 Acute hypoglycemic study of Asystesia gangetica leaf ethanolic
extract 78
4.13 Acute hypoglycemic study of Abutilon mauritianum leaf ethanolic
extract 79
4.14 Sub-acute antidiabetic study of Alternanthera brasiliana stem
ethanolic extract 85
4.15 Sub-acute antidiabetic study of
Asystesia gangetica leaf ethanolic
extract 86
4.16 Sub-acute antidiabetic study of Abutilon mauritianum leaf ethanolic
Extract 87
4.17 Percentage fall in glucose level after 21
days for Alternanthera
brasiliana stem, Asystesia
gangetica and Abutilon mauritianum
leaf samples respectively 90
4.18 Biochemical profile of sub-acute study of Alternanthera brasiliana
stem ethanolic extract 92
4.19 Biochemical profile of sub-acute study of Asystesia gangetica leaf
ethanolic extract 93
4.20 Biochemical profile of sub-acute study of Abutilon mauritianum leaf
ethanolic extract 94
4.21 Lipid profile of sub-acute study of Alternanthera brasiliana stem
ethanolic extract in mg/dl 99
4.22 Lipid profile of sub-acute study of Asystesia gangetica leaf ethanolic
extract in mg/dl 100
4.23 Lipid profile of sub-acute study of Abutilon mauritianum leaf ethanolic
extract in mg/dl 101
4.24 Hematology profile blood test of
anti-diabetic sub-acute study
of Alternanthera
brasiliana stem ethanolic extract 104
4.25 Hematology profile blood test of
anti-diabetic sub-acute study of
Asystesia gangetica leaf ethanolic extract 105
4.26 Hematology profile blood test of
anti-diabetic sub-acute study of
Abutilon mauritianum leaf ethanolic extract 106
LIST OF
FIGURES
4.1 Percentage fall in blood glucose level in
acute antidiabetic study of
Alternanthera brasiliana stem ethanolic extract 81
4.2 Percentage fall in blood glucose level in
the acute antidiabetic study
of Asystesia
gangetica ethanolic extract 82
4.3 Percentage fall in
blood glucose level in the acute antidiabetic study
of Abutilon
mauritianum ethanolic
extract 83
LIST OF PLATES
2.1 Red flower (Alternanthera brasiliana) 25
2.2 Akpu-arachi (Asystesia gangetica) 29
2.3 Udor (Abutilon
mauritianum) 333
4.1 Liver slide for normal control rats 109
4.2 Liver
slide for diabetic untreated 110
4.3 Liver
slide for diabetic rats treated with 5 mg/kg glybenclamide
111
4.4 Liver slide for diabetic rats treated with
400 mg/kg Alternanthera
Brasiliana 112
4.5 Liver
slide for diabetic rats treated with 800 mg/kg Alternanthera
Brasiliana 113
4.6 Liver
slide for diabetic rats treated with 400 mg/kg Asystesia gangetica 114
4.7 Liver
slide for diabetic rats treated with 800 mg/kg Asystesia gangetica 115
4.8 Liver
slide for diabetic rats treated with 400 mg/kg Abutilon mauritianum 116
4.9 Liver
slide for diabetic rats treated with 800 mg/kg Abutilon mauritianum
117
4.10 Pancreatic
slide for normal control rats 119
4.11 Pancreatic
slide for diabetic untreated 120
4.12 Pancreatic
slide for diabetic rats treated with 5 mg/kg glibenclamide 121
4.13 Pancreatic
slide for diabetic rats treated with 400 mg/kg Alternanthera
brasiliana 122
4.14 Pancreatic
slide for diabetic rats treated with 800 mg/kg Alternanthera
brasiliana 123
4.15 Pancreatic
slide for diabetic rats treated with 400 mg/kg Asystesia
gangetica 124
4.16 Pancreatic
slide for diabetic rats treated with 800 mg/kg Asystesia
gangetica 125
4.17 Pancreatic
slide for diabetic rats treated with 400 mg/kg Abutilon
mauritianum 126
4.18 Pancreatic
slide for diabetic rats treated with 800 mg/kg Abutilon
mauritianum 127
CHAPTER 1
INTRODUCTION
Diabetes mellitus is a chronic metabolic disease with life-threatening
complications. It was estimated by International
Diabetes Federation (IDF) that 285 million people are living with diabetes, in
2010 showing that about 6.4 % of the world populace had diabetes and by 2030,
the prevalence rate will increase to 439 million people amounting to about 7.7 %
of the world population (Shaw et al., 2010). Over 90 % of the cases of diabetes are diabetes mellitus type 2 (T2D)
(Boyle et al., 1999; Attele et al., 2002).
The
management of diabetes mellitus is associated with a huge economic cost for the
afflicted people and countries. In 2007, approximately 17.5 million people
living with diabetes mellitus were reported to have spent about US 174
billion dollars in the management of diabetes mellitus (Cashen et al., 2008). However, in Nigeria, about 1-7 % of the populace
is afflicted with diabetes mellitus (Wokoma, 2002; Fabiyi et al., 2002).
Despite
considerable progress in scientific studies on T2D and research and development
of anti-diabetics agents, yet the cause is not completely
understood. Though, mounting facts
from epidemiological research finding suggests that the primary causes of T2D
remain environmental and genetic factors. Both factors are the contributing factor to insulin resistance and
loss of β-cells function that result in impairment in insulin action, insulin production, or both. The occurrence
of hyperglycemia in diabetes mellitus is due to impairment in insulin
action (Laakso, 2001). Such hyperglycemia results to glucotoxicity which
affects the cells and peripheral tissues, which are clinically important in the
cause of diabetes-related complications such as cardiovascular disease, nephropathy, retinal
blindness, neuropathy, and peripheral gangrene (Clement and Bell, 1985). Therefore,
the most effective therapy for people living with diabetes mellitus is the maintenance
of glycemic homeostasis.
Moreover, the formation of lipotoxicity due to abnormal adipose lipid metabolism
promotes complications in diabetes. The pancreatic cells play essential role in glycemic homeostasis. Leahy et al. (2010) reported that the β-cells
function and survival are modulated by inflammatory mediators, glucotoxicity, oxidative stress, Lipotoxicity
and incretin. Currently, there
are no clinically proven anti-diabetic drugs effective in the prevention
of β-cells degeneration.
Though thiazolidinediones (TZDs) and glucagon-like peptides-1 (GLP-1) analogues
have been proved to be effective
in preventing β-cells atrophy in animals but not in humans yet
(Gastaldelli et al., 2007).
Therefore,
when the β-cell function is maintained and improved,
it has the ability to delay, stabilize and reverse diabetes mellitus type 2 (Leahy et al., 2010). It has been proposed that β-cell homeostasis is
regulated by nuclear factors, specific growth factors and cell cycle
mediators (Ackermann and Gannon, 2007 and Szabat et al., 2012). The occurrence of unwanted and harmful
effect in diabetes type 2 patients is due to loss of glycemic control. Consequently, reduction of kidney and gut glucose
absorption is required for the normalization of blood sugar in other to
maintain glycemic homeostasis in diabetic patients. Additionally,
blood sugar level can be reduced by inhibiting intestinal carbohydrate
breakdown by α-glucosidases.
Contrarily, about 90 % of urine glucose is recovered by the renal tubule
which helps the body to maintain glucose homeostasis.
Glycemic
control is the most accepted approach for diabetes mellitus type 2 treatments in
an attempt to lessen complications and death. Apart
from drugs, lifestyle and diet are very significant approach in the management
of diabetes mellitus and should not be neglected. The lack of efficiency and undesirable side effect make the current anti-diabetic
drugs unsuitable and require alternative (Howlett and Bailey, 1999). An insulin
secretagogues that cause hypoglycemia, weight gain and unable to prevent death
of cell are good examples of
negative side effect of antidiabetic drug (Krentz and Bailey, 2005; Purnell and
Weyer, 2003). More so, an incretin-based drugs cause vomiting, sour stomach,
diarrhea, belching, nausea and indigestion (Egan et al., 2003). There is
an inability for daily insulin injection to match the natural insulin dosage
and timing from the pancreas with response to hyperglycemia arising from severe
complications in well-managed patients. Besides, the above stated side effect of drugs, it also has
major limitation of not being able to cure the diabetes mellitus disease but to
alleviate it. In other to ensure the safety of the people living with diabetes
mellitus, it becomes a necessity to develop alternative antidiabetic medicine
that will be devoid of the above stated challenges with suitable efficiency.
Plants have
a key role in health care. WHO reported that about 80 % of the world’s populations
are dependent on traditional medicine, particularly plant drugs for primary
health care. Herbs were used to treat different types of disease conditions
before the birth of traditional Western medicine (Basch et al.,
2003). A number of plants have shown varying degrees of hypoglycemic and
anti-diabetic activities (Onoagbe et al.,
1999a; Onoagbe et al., 1999b),
and hundreds of such plants are in common use. However, so many of them have
not been scientifically proven to have anti-diabetic activities or either been checked
on its dosage-dependent effect.
Akpu-arachi (Asystesia
gangetica) commonly known as
ganges primrose, Chinese violet from family of Acanthaceae is a herb (Adeyemi et
al., 2011). It is called Ganges because it is obtained from the river name
called Gange where it is assumed that the species exist. The Marathi name
Lavana valli (another common name) means salt resistant creeper (also called
akpu-arachi by the Igbos). Ganges is a herbaceous
ground cover plant that grows to the height of 30-60 cm. it has a cream colored
flower with tessellated purple mark on the palate that makes the plant very
attractive. It also grows fast and spreads easily with dark green leaves. Over
a long period of time the flowers are produced followed by brown seeds capsules
(Ramesar et al., 2008). In tropical areas it can grow rampantly. This plant
is widely distributed across South Asia, tropical America, Sub-Saharan Africa,
Oceania, etc. The edible parts include the tender leaves and stems which are
eaten fresh, stir-fried or boiled. Akpu-arach (Astystasia gangetica) is reported to contain biologically active
substances such as carbohydrates, proteins, alkaloids, tannins, steroidal aglycones,
saponins, flavonoids and triterpenoids (Akah et al., 2003). And also, has been claimed by a known local
herbalist in Nsulu Oloko village in Ikwuano Local Government Area of Abia State
Nigeria to have anti-diabetic effect.
Udor (Abutilon mauritianum) is
of the family of Malvaceae. It is
widely found in the drier parts of the Senegal, Mozambique, Ethiopia, Somalia, South
Africa, Angola, Zambia and Zimbabwe
(Burkill, 1985) and Nigeria. The plants vernacular names are Bush Mallow and
country mallow in English, Mauves des champs in French, Furu and Kawo in
Nigerian traditional languages (locally called Udor in Igbo land). It is a
perennial shrub with stem that turns woody. It grows from 0.5-2 meters tall
with occasional specimens reaching a height of 3.5 meters. It is found in
riverine forest; river-banks; roadsides, etc; very common in red soils at
forest edges and on the coast in bush land on coral (Kenya) (Bernard, 2003);
rice-fields (Nigeria); from sea level to 2,300 meters. The leaves are edible
and broadly utilized in African traditional medicine. Modern research has
demonstrated the presence of bioactive compounds in the plant – saponins,
flavonoids, tannins and alkaloids have all been reported in the leaf
(Rajalakshmi and Kalaiselvi, 2009).
Brazilian
Joyweed (Alternanthera brasiliana)
from the family of Amaranthacecae, is
a common weed that grows wild and abundantly in Mexico, Brazil and West Indies.
It is usually used in homes and offices as a
decorative plant (Aaawan et al., 2011). From ethno medical
sources (Brazilian folk medicine), it is alleged to be used to treat variety of
ailments and researches have been undertaken to prove its wound healing
properties (Baru et al., 2012) and
its antimicrobial activities (Macedo et
al., 1999). However, no available record has indicated any research findings
on its anti-diabetic activity.
The
scientific mind will not be satisfied by mere claims no matter from whatever
source they originate, unless corroborated by experimental and clinical
evidences. As it is evident that plants are treasure house for many potent
medicines, it is important to scientifically evaluate the traditional practices
as well as upgrade the existing knowledge and make it accessible
to the universal public. A good
number of popularly used Nigerian plants have not been
scientifically-scrutinized for their efficacy and anti-diabetic properties (Onoagbe
et al., 1999). This present
investigation is an extension of the continuing research efforts at providing the requisite scientific
information on the therapeutic value of some medicinal plants and its anti-diabetic
claims.
1.1
STATEMENT OF THE PROBLEM
Diabetes is one of the commonest and leading
causes of death in Nigeria and the globe. Its prevalence is based on the fact
that it affects both old and young, and equally rich and poor alike (Adodo,
2009). It is estimated that about 150 million of the world population is
diabetic and this number is likely to increase to 300 million or more by the
year 2025 (King et al., 1998). Its
greatest occurrence is in Africa and Asia where diabetes mellitus rates could
rise to 2-3 folds than the present rates (ADA, 1997). In Nigeria, about 1-7 %
of the population is affected (Wokoma, 2002; Fabiyi et al., 2002). According to a Nigerian Non-communicable disease
survey, a prevalence rate of about 2.2 % for diabetes mellitus is reported
(Akinkugbe, 1997). Diabetes can lead to blindness, kidney failure, and nerve
damage (Wokoma, 2002). These types of damage are the result of damage to small
vessels, referred to as micro vascular disease. Diabetes is also an important
factor in accelerating the hardening and narrowing of the arteries
(atherosclerosis), leading to strokes, coronary heart disease, and other large
blood vessel diseases (Wokoma, 2002). This is referred to as macro vascular
diseases. Diabetic patients are at increased risk of developing specific
complications including: nephropathy, retinopathy, neuropathy and
atherosclerosis (Rahman et al.,
2005). Traditional use of anti-diabetic plants in the form of herbal remedies
for management of diabetes mellitus is usually faced with the challenge of poor
acceptance. This is because they are often served as concentrated extracts
which are usually associated with bitter, sour or astringent taste as well as
leafy flavor, dull appearance which make them unacceptable and general belief
that herbs are toxic to the liver. Examining the micronutrient, phytochemical
components, toxicity and the anti-diabetic effect of these plants will validate
the claims by the local healers and also serve as an encouragement on their
usage for diabetes mellitus management.
1.2
AIMS AND OBJECTIVES
The aim
of this study is to investigate micronutrient, phytochemical components, acute
toxicity and the anti-diabetic effects of the ethanolic extracts of Alternanthra brasiliana stem, Asystesia gangetica and Abutilion mauritianum leaves in albino mice and Alloxan-induced
diabetic rats, respectively.
Specific
objectives:
1.
To extract and concentrate the herbal plant leaves and stem into semi-solid
form.
2.
To determine the chemical and phytochemical composition of the fresh and
dried herbal powder, and herbal ethanolic extract.
3.
To determine the acute toxicity of the herbal products.
4.
To determine the anti-diabetic properties of the ethanolic plant
extracts on the alloxan-induced diabetic rats.
1.3 SIGNIFICANCE
OF STUDY
There is
a growing interest in the use of plant extracts to treat chronic metabolic
diseases. More so, in Africa, drugs for diabetic therapy and management are
quite expensive and unaffordable to the larger population; this underscores the
use of locally available medicinal plants as a remedy. This research provided scientific
evidence to the effectiveness of these herbs in management of diabetes mellitus,
and also provided information with respect to their safety based on their
dosage. Furthermore, the findings may proffer sustainable alternative to the
use of synthetic drugs used for management of diabetes mellitus.
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