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Guava Leaf Extract Diminishes Hyperglycemia and Oxidative Stress, Prevents β -Cell Death, Inhibits Inflammation, and Regulates NF-kB Signaling Pathway in STZ Induced Diabetic Rats

Authors:
  • Vignan`s Foundation for Science Technology and Research (Deemed to be University)

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Traditional Chinese medication has been utilized by Chinese medical practitioners to treat the varied symptoms of diabetes mellitus (DM). Notably, guava leaf has been used to treat diabetes in Asia. Our present study has been designed to analyze the action of guava leaf extract (GLE) at the molecular level in treating DM. A low dose of streptozotocin (STZ) was used to induce experimental diabetes in animals. Rats were treated with GLE at different concentrations (100, 200, and 400 mg/kg b.w.). The standard drug glibenclamide (GB) (600 μ g/kg b.w.) was used for comparison. The diabetic rats showed a reduced level of insulin, accompanied by exaggerated levels of blood glucose, lipid peroxidation product, and augmented expressions of inflammatory cytokines, and showed reduced levels of antioxidants compared to the control rats. Supplementation with GLE counteracted the consequences of STZ. It suppresses the oxidative stress and inhibits the state of inflammation and the results are almost similar to that of standard drug group (GB group 5). Our present research, therefore, provides useful data concerning guava leaf extract by a thorough assessment in diabetes management. Being a natural product, additional analysis on GLE can shed light on finding effective phytochemicals within the field of diabetes mellitus.
(a) Total phenolic and flavonoid contents of guava leaf (dry weight basis) by using different solvent systems. (b) Quantification of phenolic compounds in guava leaf extracts by high performance liquid chromatography (HPLC). GA, gallic acid; PA, protocatechuic acid, THBA, 2,3,4-trihydroxybenzoic acid; DHBA, 3,4-dihydroxybenzaldehyde; HBA, 4-hydroxy benzoic acid; CA, chlorogenic acid; HDDBA, 4-hydroxy-3,5-dimethoxybenzoic acid; VNN, vanillin; PCA + SA, p-coumaric acid plus syringaldehyde; FA, ferulic acid; and SNA, sinapic acid. (c) HPLC chromatographs of phenolic compound standards (A) and guava leaf extracts (B): (1) gallic acid (GA), (2) protocatechuic acid (PA), (3) 2,3,4-trihydroxybenzoic acid (THBA), (4) 3,4-dihydroxybenzaldehyde (DHBA), (5) 4-hydroxybenzoic acid (HBA), (6) gentisic acid, (7) chlorogenic acid (CA), (8) vanillic acid (VA) plus caffeic acid (CAA), (9) 4-hydroxy-3,5-dimethoxybenzoic acid (HDDBA), (10) vanillin (VNN), (11) p-coumaric acid (PCA) + syringaldehyde (SA), (12) ferulic acid (FA), (13) sinapic acid (SNA), and (14) salicylic acid (SAL). (d) Possible phenolic compounds identified in guava leaf extract. (1) Gallic acid (GA), (2) protocatechuic acid (PA), (3) 2,3,4-trihydroxybenzoic acid (THBA); (4) 3,4-dihydroxybenzaldehyde (DHBA), (5) 4-hydroxybenzoic acid (HBA), (6) gentisic acid, chlorogenic acid (CA), (7) 4-hydroxy-3,5-dimethoxybenzoic acid (HDDBA), (8) vanillin (VNN), (9) and (10) p-coumaric acid (PCA) + syringaldehyde (SA), (11) ferulic acid (FA), and (12) sinapic acid (SNA).
… 
(a) Total phenolic and flavonoid contents of guava leaf (dry weight basis) by using different solvent systems. (b) Quantification of phenolic compounds in guava leaf extracts by high performance liquid chromatography (HPLC). GA, gallic acid; PA, protocatechuic acid, THBA, 2,3,4-trihydroxybenzoic acid; DHBA, 3,4-dihydroxybenzaldehyde; HBA, 4-hydroxy benzoic acid; CA, chlorogenic acid; HDDBA, 4-hydroxy-3,5-dimethoxybenzoic acid; VNN, vanillin; PCA + SA, p-coumaric acid plus syringaldehyde; FA, ferulic acid; and SNA, sinapic acid. (c) HPLC chromatographs of phenolic compound standards (A) and guava leaf extracts (B): (1) gallic acid (GA), (2) protocatechuic acid (PA), (3) 2,3,4-trihydroxybenzoic acid (THBA), (4) 3,4-dihydroxybenzaldehyde (DHBA), (5) 4-hydroxybenzoic acid (HBA), (6) gentisic acid, (7) chlorogenic acid (CA), (8) vanillic acid (VA) plus caffeic acid (CAA), (9) 4-hydroxy-3,5-dimethoxybenzoic acid (HDDBA), (10) vanillin (VNN), (11) p-coumaric acid (PCA) + syringaldehyde (SA), (12) ferulic acid (FA), (13) sinapic acid (SNA), and (14) salicylic acid (SAL). (d) Possible phenolic compounds identified in guava leaf extract. (1) Gallic acid (GA), (2) protocatechuic acid (PA), (3) 2,3,4-trihydroxybenzoic acid (THBA); (4) 3,4-dihydroxybenzaldehyde (DHBA), (5) 4-hydroxybenzoic acid (HBA), (6) gentisic acid, chlorogenic acid (CA), (7) 4-hydroxy-3,5-dimethoxybenzoic acid (HDDBA), (8) vanillin (VNN), (9) and (10) p-coumaric acid (PCA) + syringaldehyde (SA), (11) ferulic acid (FA), and (12) sinapic acid (SNA).
… 
(a) Total phenolic and flavonoid contents of guava leaf (dry weight basis) by using different solvent systems. (b) Quantification of phenolic compounds in guava leaf extracts by high performance liquid chromatography (HPLC). GA, gallic acid; PA, protocatechuic acid, THBA, 2,3,4-trihydroxybenzoic acid; DHBA, 3,4-dihydroxybenzaldehyde; HBA, 4-hydroxy benzoic acid; CA, chlorogenic acid; HDDBA, 4-hydroxy-3,5-dimethoxybenzoic acid; VNN, vanillin; PCA + SA, p-coumaric acid plus syringaldehyde; FA, ferulic acid; and SNA, sinapic acid. (c) HPLC chromatographs of phenolic compound standards (A) and guava leaf extracts (B): (1) gallic acid (GA), (2) protocatechuic acid (PA), (3) 2,3,4-trihydroxybenzoic acid (THBA), (4) 3,4-dihydroxybenzaldehyde (DHBA), (5) 4-hydroxybenzoic acid (HBA), (6) gentisic acid, (7) chlorogenic acid (CA), (8) vanillic acid (VA) plus caffeic acid (CAA), (9) 4-hydroxy-3,5-dimethoxybenzoic acid (HDDBA), (10) vanillin (VNN), (11) p-coumaric acid (PCA) + syringaldehyde (SA), (12) ferulic acid (FA), (13) sinapic acid (SNA), and (14) salicylic acid (SAL). (d) Possible phenolic compounds identified in guava leaf extract. (1) Gallic acid (GA), (2) protocatechuic acid (PA), (3) 2,3,4-trihydroxybenzoic acid (THBA); (4) 3,4-dihydroxybenzaldehyde (DHBA), (5) 4-hydroxybenzoic acid (HBA), (6) gentisic acid, chlorogenic acid (CA), (7) 4-hydroxy-3,5-dimethoxybenzoic acid (HDDBA), (8) vanillin (VNN), (9) and (10) p-coumaric acid (PCA) + syringaldehyde (SA), (11) ferulic acid (FA), and (12) sinapic acid (SNA).
… 
(a) Total phenolic and flavonoid contents of guava leaf (dry weight basis) by using different solvent systems. (b) Quantification of phenolic compounds in guava leaf extracts by high performance liquid chromatography (HPLC). GA, gallic acid; PA, protocatechuic acid, THBA, 2,3,4-trihydroxybenzoic acid; DHBA, 3,4-dihydroxybenzaldehyde; HBA, 4-hydroxy benzoic acid; CA, chlorogenic acid; HDDBA, 4-hydroxy-3,5-dimethoxybenzoic acid; VNN, vanillin; PCA + SA, p-coumaric acid plus syringaldehyde; FA, ferulic acid; and SNA, sinapic acid. (c) HPLC chromatographs of phenolic compound standards (A) and guava leaf extracts (B): (1) gallic acid (GA), (2) protocatechuic acid (PA), (3) 2,3,4-trihydroxybenzoic acid (THBA), (4) 3,4-dihydroxybenzaldehyde (DHBA), (5) 4-hydroxybenzoic acid (HBA), (6) gentisic acid, (7) chlorogenic acid (CA), (8) vanillic acid (VA) plus caffeic acid (CAA), (9) 4-hydroxy-3,5-dimethoxybenzoic acid (HDDBA), (10) vanillin (VNN), (11) p-coumaric acid (PCA) + syringaldehyde (SA), (12) ferulic acid (FA), (13) sinapic acid (SNA), and (14) salicylic acid (SAL). (d) Possible phenolic compounds identified in guava leaf extract. (1) Gallic acid (GA), (2) protocatechuic acid (PA), (3) 2,3,4-trihydroxybenzoic acid (THBA); (4) 3,4-dihydroxybenzaldehyde (DHBA), (5) 4-hydroxybenzoic acid (HBA), (6) gentisic acid, chlorogenic acid (CA), (7) 4-hydroxy-3,5-dimethoxybenzoic acid (HDDBA), (8) vanillin (VNN), (9) and (10) p-coumaric acid (PCA) + syringaldehyde (SA), (11) ferulic acid (FA), and (12) sinapic acid (SNA).
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Research Article
Guava Leaf Extract Diminishes Hyperglycemia and
Oxidative Stress, Prevents 𝛽-Cell Death, Inhibits Inflammation,
and Regulates NF-kB Signaling Pathway in STZ Induced
Diabetic Rats
Muthukumaran Jayachandran,1Ramachandran Vinayagam,1Ranga Rao Ambati ,1,2
Baojun Xu ,1and Stephen Sum Man Chung 1
1Food Science and Technology Program, Beijing Normal University-Hong Kong Baptist University United International College,
Zhuhai, Guangdong 519087, China
2Department of Biotechnology, Vignan’s Foundation for Science, Technology and Research (Deemed to Be University),
Vadlamudi,Guntur,AndhraPradesh522213,India
Correspondence should be addressed to Baojun Xu; baojunxu@uic.edu.hk and Stephen Sum Man Chung; smchung@uic.edu.hk
Received 17 July 2017; Revised 18 November 2017; Accepted 11 December 2017; Published 18 February 2018
Academic Editor: Stelvio M. Bandiera
Copyright ©  Muthukumaran Jayachandran et al. is is an open access article distributed under the Creative Commons
Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.
Traditional Chinese medication has been utilized by Chinese medical practitioners to treat the varied symptoms of diabetes mellitus
(DM). Notably, guava leaf has been used to treat diabetes in Asia. Our present study has been designed to analyze the action of guava
leaf extract (GLE) at the molecular level in treating DM. A low dose of streptozotocin (STZ) was used to induce experimental
diabetes in animals. Rats were treated with GLE at dierent concentrations (, , and  mg/kg b.w.). e standard drug
glibenclamide (GB) (  𝜇g/kg b.w.) was used for comparison. e diabetic rats showed a reduced level of insulin, accompanied by
exaggerated levels of blood glucose, lipid peroxidation product, and augmented expressions of inammatory cytokines, and showed
reduced levels of antioxidants compared to the control rats. Supplementation with GLE counteracted the consequences of STZ. It
suppresses the oxidative stress and inhibits the state of inammation and the results are almost similar to that of standard drug
group (GB group ). Our present research, therefore, provides useful data concerning guava leaf extract by a thorough assessment
in diabetes management. Being a natural product, additional analysis on GLE can shed light on nding eective phytochemicals
within the eld of diabetes mellitus.
1. Introduction
Diabetes mellitus (DM) is a group of metabolic diseases
resulting in an increased blood glucose level (hyperglycemia).
A prolonged hyperglycemia is the key indicator of the
metabolic illness diabetes mellitus. Oxidative stress plays a
vitalroleinthepathogenesisofDM[].Animbalancewithin
the redox status or the production and detoxication of
reactive oxygen species (ROS) end up in injury to varied
tissues and therefore the condition is termed as oxidative
stress. It is assessed by the extent of the reaction product
of oxidative harm, DNA oxidation, lipid peroxidation, and
protein oxidation []. In diabetes, oxidative stress is caused
by both an increased formation of plasma free radicals
and a diminution in antioxidant defenses. Hyperglycemia
might enhance the production of free radicals and provoke
oxidativestresswhichwillalsoaddtotheredoubledriskfor
coronary artery illness in diabetes []. erapeutic choices for
treating diabetes embrace sulfonylureas and alternative hypo-
glycemic agent secretagogues, alpha-glucosidase inhibitors,
biguanides, thiazolidinediones, and insulin.
Guava (Psidium guajava L.) is a  m height tree with
numerous nutritional values. e nutritional value of guava
fruit is known throughout the globe. Aside from this, many
Hindawi
BioMed Research International
Volume 2018, Article ID 4601649, 14 pages
https://doi.org/10.1155/2018/4601649
BioMed Research International
chemical and pharmaceutical companies use numerous parts
of guava tree []. e guava leaf has gone through phyto-
chemical analysis and found to have alkaloids, carotenoids
anthocyanins, vitamin-C, and triterpenes [–]. e anti-
inammatory and analgesic eects of P. g u a j a v a leaves (%
ethanolic extract) were established to be eective in rats using
the carrageen induced hind paw oedema model []. e
guava leaves conjointly cure numerous other diseases []. A
study states that to treat the inammation of the kidney, the
fresh guava leaves are used []. e pulped leaves are used
for treating piles in Congo [].
Utilization of folk herbal medication knowledge by
autochthonal cultures is not solely useful for preserving
their culture however conjointly useful for synthesizing new
medicine. ere’s less systematic study on the eectiveness,
let alone the mechanism of guava leaf in treating diabetes.
HencewetendtoinvestigatetheeectualityofGLEagainst
DM and its associated hyperglycemia, oxidative stress, and
inammation (NF-kB regulation). e results from our study
clearly indicate that GLE has the power to inhibit numerous
pathological conditions related to DM. e study on the
regulation of NF-kB by GLE evidences that inammation
plays a major role within the DM. In the future, studies
on the role of GLE on insulin signaling pathway and it is
interwoven with oxidative stress and inammation might
derive a conclusion on the therapeutic eects of GLE on
DM.
2. Materials and Methods
2.1. List of Chemicals. Sigma-Aldrich (Shanghai, China) is
the source for streptozotocin (STZ) and glibenclamide (GB).
Primary antibodies for interleukin- (IL-), nuclear factor-
kappa B (NF-kB), and tumor necrosis factor-𝛼(TNF-𝛼)
were purchased from Abcam (China). Secondary antibody
goat-anti-rabbit (CW) was purchased from CW Biotech,
China. All dierent analytical grade chemicals and reagents
were purchased from Shanghai Yuanye Biotechnology Co.
(Shanghai, China).
2.2. Collection and Sample Preparation of Guava Leaves.
e guava leaves from the species P. g u a j a v a were collected
from the plant gardens from southern China (Meizhou,
Guangdong, China). Guava leaves were carefully separated
and cut into small thin pieces and dried at room temperature
for  days.
2.3. Preparation of Guava Leaf Extract (GLE). e dried
leaves were ground and changed into powder form. e
desktop decoction extractor (YFT, Beijing Donghuayuan
Medical Equipment Co., Ltd., Beijing, China) was used to
prepare the extract. In this process,  g of dried guava leaves
wasboiledin.Lofwaterforabouthours.Furtherthe
extract was ltered using a Whatman No.  lter paper and
dried using a rotary evaporator at C. e dried extract
was converted into a powder form which was utilized for
the preparation of desired concentrations of the extracts. e
extracts were stored at C in sterile bottles until further use.
While performing the experiments the powdered GLE was
dissolved in water and administered to the animals.
2.4. Experimental Animals and Conditions. Healthy male
Wistar rats (–g) were procured from Southern Med-
ical University (Guangzhou, China). e experiments were
executed in accordance with the principles issued by the
National Institute of Health (NIH) Guideline for the experi-
mental animals care and use. e ethics committee (Animal)
of Zhuhai Campus of Zunyi Medical University, China, issued
the approval to hold out this experimental protocol that
additionally conforms to the rules for ethical conduct within
theanimalsuseandcare.Ratswereplacedinapolypropylene
cageandinatemperatureof±C with relative humidity
(% ±%) in  h light and  h dark condition. Before
experiments, rats were placed within the animal house for
the period of two weeks. e standard pellet diet (obtained
from Southern Medical University, Guangzhou, China) was
a balanced food composed of protein .%, fat .%, carbo-
hydrates .%, ber .%, vitamins .%, and minerals .%.
2.5. Induction of Experimental Diabetes in Animals. Afreshly
prepared . M, pH . citrate buer was used to dissolve the
STZ and maintained on ice prior to use. Animals were kept on
overnight fasting to induce diabetes (low dose STZ model) by
an intraperitoneal injection of STZ at a dose of  mg/kg b.w.
e elevated plasma glucose was determined by Accu–Chek
commercial kit (Roche diagnostics, Mannheim, Germany)
and rats having fasting glucose greater than  mg/DL were
screened and considered as diabetic rats and used for further
studies.
2.6. Experimental Design of Animal Study. e animals were
grouped into seven groups (𝑛 = 6), and a total of  rats
( diabetic and  control) underwent the study. Treatment
with GLE was started on the third day aer STZ induction.
GLE was dissolved in water and variant doses of GLE were
administered using an intragastric tube orally for -day
duration.
Experimental group I: control rats.
Experimental group II: GLE control ( mg/kg b.w.).
Experimental group III: diabetic rats.
Experimental group IV: diabetic + GLE ( mg/kg
b.w.).
Experimental group V: diabetic + GLE ( mg/kg
b.w.).
Experimental group VI: diabetic + GLE ( mg/kg
b.w.).
Experimental group VII: diabetic + GB ( 𝜇g/kg
b.w.).
When the experimental period has come to an end, the
animals were kept in the fasting condition whole night and
then animals were anesthetized by an intramuscular injection
of ketamine hydrochloride at a dose of  mg/kg b.w. and
the animals were killed. For the estimation of insulin and
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glucose, blood samples were collected into tubes containing
an anticoagulant. An ice cold saline was kept ready to wash
the dissected tissues (liver, kidney, and pancreas) and later
stored at C for further experiments.
2.7. Quantication of Phenolics in Guava Leaf. To q u a n t i fy t h e
total phenolic and total avonoids contents in guava leaves,
the dry guava leaves were extracted with dierent solvents
(ethanol/water ( : , v/v), methanol/water ( : , v/v), ethanol,
water, and methanol), respectively.
2.7.1. Determination of Total Phenolic Compounds. e meth-
od of Taga et al. [] was used to conrm the total phenolic
content in guava leaf extract (GLE) and expressed as equiva-
lents of gallic acid. e acidied ( g/L HCl) methanol/water
( :  v/v) and  𝜇L of each were added separately to
mL of % Na
2CO3to prepare the samples and standards.
Five minutes later  𝜇L of % Folin–Ciocalteu reagent
was added to the samples and kept at RT for  min and
employing a spectrophotometer absorbance was measured at
 nm (Shimadzu, A). A blank has been prepared with all
solvents, excluding samples and standards. e – 𝜇g/mL
concentration of gallic acid was prepared. e phenolic acid
concentration obtained from guava leaf extract was compared
with standards. A triplicate of analyses was carried out and
calculated.
2.7.2. Determination of Flavonoid Content Assay. e Moreno
et al. method was employed to estimate the avonoid content
[]. In the experimental procedure, the following chemicals
were added: (1) . mL of % aluminum nitrate, () . mL
of  mL/L aqueous potassium acetate, and () . mL of %
ethanol added along with  mL of dierent concentration of
extracts. Absorbance ( nm) was measured in a dark room
aer a  min incubation at RT. Quercetin was used as a
standard for estimating avonoids.
2.7.3.DeterminationofPhenolicAcidsCompositioninGuava
Leaves by High Performance Liquid Chromatography (HPLC).
e method of Luthria and Pastor–Corrales () was
used to extract the overall phenolic acids. A  mL of
methanol/water/acidic/butylated hydroxytoluene at a ratio of
 :  : . : . was used to extract the leaf sample (. g). e
content was kept in an orbital shaker for  h at RT at 
revolutions per minute, followed by a centrifugation at 𝑔
for  min. To get rid of the surplus solvents the extracts
were concentrated under vacuum at C.  mL of %
methanol was used to dissolve the dry residue. e methanol
sample aliquot was ltered into HPLC sample container using
a.mmltertogetridoftheimpuritiesandinsoluble
substances present in it and therefore the sample was stored
at CforHPLCanalysis.
HPLC phenolic acid analysis was performed in line
with the method demonstrated by Xu and Chang []. For
a water associated (Milford, MA, USA) chromatography
system alongside a  model system controller, a model
 sample injector, and a  model “A” solvent delivery
system, the values are detected at  nm employing a model
 LC uv detector. For separation at C, a . × mm,
𝜇m, Zorbax Stable bond analytical SB–C column (Agilent
Technologies, Rising Sun, MD, USA) was used. e mobile
phase comprised solvent “A” (.% TFA) and solvent “B”
(methanol), at . mL/min of ow rate and  𝜇Lofinjection
volume.egoodseparationwasfoundthroughgradient
elution and all peaks were identied, and phenolic acids were
quantied with a relative retention time of external standards.
e phenolic acids contents were expressed as 𝜇g/mg on dry
weight basis.
2.8. Biochemical Parameters
2.8.1. Insulin and Glucose Estimation. Accu–Chek commer-
cial kit was used to determine the blood glucose levels (Roche
Diagnostics, Mannheim, Germany). A Merck Millipore com-
mercial kit was used to analyze the levels of plasma insulin
(Darmstadt, Germany).
2.8.2. Estimation of Lipid Peroxidation (LPO) Markers. LPO
markers in liver, kidney, and pancreas were estimated with
UV/VIS spectrophotometer adopting the methods and pro-
cedures made by Fraga et al. [] and Jiang et al. []. A
mL volume of thiobarbituric acid (TBA)–trichloroacetic
acid (TCA)–HCl reagent (.% TBA, . M HCl, and TCA,
 :  :  ratio) was mixed with . mL of tissue homogenate and
warmed in an exceeding water bath for a period of  min and
further it underwent centrifugation at a speed of xg for
about  min at C, and later  nm was used to measure
the intensity of the supernatant. Lipid hydroperoxide values
were manifested as mM/ g of tissue. To .mL of Fox
reagent ( mg of butylated hydroxyl toluene (BHT), . mg
of ammonium iron sulfate, and . mg of xylenol orange) were
added  mL of alcohol and  mL of  mm sulfuric acid.
. mL of tissue homogenate was added and incubated at RT
forminandnmwasusedtomeasuretheabsorbance.
2.8.3. Determination of Catalase Activity (CAT). Beers and
Sizer[]wereusedtomeasuretheactivityofcatalase.
e reaction mixture is the combination of .mL of tissue
homogenate (. mL), . mL of . M phosphate buer (pH
.), and . mL of  M H2O2.ereactionwasarrested
by the addition of . mL of dichromate-acetic acid reagent
(potassium dichromate (%) and glacial acetic acid in a ratio
of:).nmwasusedtoreadtheabsorbance;𝜇MofH
2O2
consumed/min/mg protein is the unit used to denote CAT
activity.
2.8.4. Determination of Superoxide Dismutase Activity (SOD).
e Sun and Oberley [] methodology was used to verify the
activity of SOD. Using  mL of water the tissue homogenate
was diluted. To this diluted homogenate were added a cooled
. mL of chloroform and . mL of ethanol and then they
were centrifuged. e supernatant was used to estimate the
activity of the enzyme. e mixture of this assay is a com-
position of . mL of sodium pyrophosphate buer (. M,
pH .), . mL of  𝜇Mpotassiummetabisulte,.mL
of  𝜇M NADH, . mL of 𝜇M nitroblue tetrazolium
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(NBT), bettingly diluted enzyme preparation, and water.
e total volume was made in  mL. NADH was added as
an initiation of the reaction. Followed by min incubation
at C, one mL of glacial acetic acid was used to arrest
the reaction. e mixture was jolted vigorously upon mixing
with  mL of n-butanol. A  nm was used to measure
the compound concentration and the specic activity of the
enzyme was expressed as enzyme required for % inhibition
of NBT reduction/min/mg protein.
2.8.5. Determination of Glutathione Peroxidase Activity (GPx).
e Rotruck et al. [] methodology was used to measure
the activity of GPx. Briey, the reaction mixture contain-
ing . mL tissues was homogenized in . mL of . M
phosphate buer (pH .), . mL of  mM sodium azide,
phosphate buer, . mL of . mM H2O2,pH.,and.mL
glutathione. e incubation time was  min at Cand
the reaction was arrested by the addition of . mL %
TCA and subsequent centrifugation at 𝑔for  min was
done. e glutathione content of the supernatant was assayed
usingEllmansreagent(.mg,-dithiobisnitrobenzoic
acid in  mL .% sodium nitrate). e 𝜇gofGSHcon-
sumed/min/mg protein is denoted as a unit to measure the
activity of the enzyme.
2.8.6. Determination of Glutathione Reductase Activity (GR).
e Horn and Burns [] methodology was used to verify
theGRactivity.ereactionmixtureiscomposedof.mL
of GSSG,  mL of phosphate buer, . mL of NADPH, and
. mL of EDTA. e reaction mixture was made to the nal
volume by adding  mL of distilled water. A  nm was
used to measure the absorbance aer the addition of . mL
of tissue homogenate. e activity of GR was measured as
𝜇moles of NADPH oxidized/min/mg protein.
2.8.7. Estimation of Reduced Glutathione (GSH). e Beutler
andKelly[]methodwasusedtomeasuretheconcentration
of GSH. A yellow derivative has been formed aer the
reaction between the supernatant with , -dithio-bis--
nitrobenzoic acid (DTNB). e intensity was read at  nm.
2.8.8. Determination of Vitamin-C (VIT-C). e Omaye et al.
[]methodwasusedtoestimateVIT-C.Amixtureismade
of . mL of %, and TCA and . mL of tissue homogenate
were centrifuged at xg for  min. . milliliters of
dithiobis--nitrobenzoic acid (DNPH) reagent was mixed
with the above supernatant and incubated at Cforabout
hours, followed by an addition of . mL of % sulfuric acid,
and kept for incubation about  min. A  nm was used to
measure the absorbance. A standard series of ascorbic acid
was taken as – mg and compared with the samples. e
𝜇M/mg tissue is denoted as the unit to measure the levels of
ascorbic acid.
2.8.9. Determination of Vitamin-E (VIT-E). eBakeretal.
[] method was used to measure the activity of VIT-E. A
reaction mixture was made by adding  mL of lipid extract
with . mL of ethanol and mL of petroleum ether, mixed.
e resultant mixture was further centrifuged at a speed of
xg for  min. At C the supernatant was evaporated
to become dry, and then to the dry sample was added . mL
of ferric chloride solution and of --dipyridyl solution each.
e mixture was kept in the dark for min, followed by
addition of  mL of butanol.  nm was used to measure
the ultimate absorbance. A standard 𝛼-tocopherol in a range
of – mg was compared with the results of samples. e
𝜇M/mgoftissueisdenotedastheunittomeasuretheactivity
of VIT-E.
2.8.10. Estimation of Protein. e total protein was estimated
by the method of Lowry et al. []. An aliquot of cell lysate
andtissuehomogenatewasdilutedto.mLwithsaline,
then . mL % TCA was added. e resulting mixture was
centrifuged, supernatant was discarded, and using . mL
of . N NaOH the precipitate was dissolved. Aliquots were
taken for the estimation from the above said step. A . mL
alkaline copper reagent was added and the contents were
incubated at C for  min, followed by the addition of
Folin–Ciocalteu reagent. A series of standard solution in a
range of – 𝜇g and a blank were processed in the same
way. e intensity of the blue color developed was read at
 nm aer  min. e protein values are expressed as mg/g
tissue.
2.9. Expression of Inammatory Cytokines. RIPA lysis buer
was used to homogenize the pancreas. en, the homogenate
was placed on cold condition for min, followed by
centrifugation at C,thecentrifugewasprecooledbefore
beginning the experiment, and a speed of ,𝑔was
used for  min and also the supernatant obtained aer
the centrifugation was used as a sample and pellets were
thrown. Samples containing  𝜇g of total protein were
loaded on a SDS polyacrylamide gel and then separated by
electrophoresis. e SDS-PAGE gel was then transferred onto
a PVDF membrane (Millipore) followed by electrophoresis.
A block buer containing -hitter nonfat dry milk powder
or -hitter BSA was used to incubate the membrane for  h.
is was done to cut back the nonspecic binding areas and
then incubated in NF-kB-p monoclonal, TNF-𝛼,andIL-
(rabbit monoclonal;  : ) and 𝛽-actin (rabbit monoclonal;
 : ) in BSA throughout the night at C. Next day the
membranes were treated with their corresponding secondary
antibodies (anti-rabbit igg conjugated to horseradish perox-
idase) for  h at normal room conditions. TBST buer was
used to wash the blot membrane and the washing was carried
out for  min. Chemiluminescence protocol was used to
visualize the immune-reactive protein (GenScript ECL kit,
Piscataway, NJ, USA, and Image Quant LAS ) to review
the densitometric analysis of the respective protein bands
within the gel and a gel image study program was used. A
standard protein 𝛽-actin was used to compare the bands with
other proteins.
2.10. Histopathological Study. e tissues to be examined
(liver, kidney, and pancreas) are xed in % normal saline
for  h and for dehydration a dierent mixture of water and
BioMed Research International
ethyl alcohol was used and xylene was used to clean the slides
andparanwaxwasusedtoxthetissuesectionsonslides.
A - 𝜇mofliver,kidney,andpancreassectionswasmade
and then stained with hematoxylin and eosin (H & E) dye,
the stained slides were mounted with a neutral deparanated
xylene medium, and the visualization of slides was done using
a light microscope at x.
2.11. Statistical Analysis of Variance. All data were presented
as the mean ±standard deviation (SD) of experiment num-
bers (𝑛=6). One-way analysis of variance (ANOVA)
using SPSS Version  (SPSS, Cary, NC, USA) was used
to determine the statistical signicance and variance and
Duncans multiple range test (DMRT) was used to determine
the individual comparisons. When 𝑝 < 0.05,valuesare
considered statistically signicant.
3. Results
3.1. Total Phenolic and Flavonoid Contents in Guava Leaf
Extracts by Dierent Solvent. e variant of solvent extract
obtained from guava leaf was tested for its total phenolic and
avonoid content and the results were shown in Figure (a).
e yield of guava leaf extracts (GLE) was found to be .%,
.%, .%, .%, and .% in various solvent extracts such as
ethanol/water ( : , v/v), methanol/water ( : , v/v), ethanol,
water,andmethanol,respectively.eyieldofGLEswas
calculated on the dry weight basis. Maximum total phenolic
content (. mg/g on dr y weight basis) was achieved in
the ethanol/water ( : , v/v) extract, while the maximum
total avonoid content (. mg/g on dry weight basis) was
found in ethanol alone extract. Of all the solvents used, total
phenolic contents were found to be lower in methanol/water
( : ) extract followed by ethanol, water, and methanol alone
extracts. e avonoid contents were found to be lower
in water followed by methanol, methanol/water ( : ), and
ethanol/water ( : ) extracts. Previous studies report that
guava leaf contains terpenoids [, ], avonoids [, ], and
tannins [].
3.2. Identication and Quantication of Phenolic Compounds
in Guava Leaf Extract by HPLC. e Folin–Ciocalteu meth-
od gives total phenolic contents in the guava leaf extracts
since the reactivity is dierent for dierent polyphenolics
so the determination of phenolic compounds is not specic,
whereas HPLC analysis oers more exact information about
individual compounds. e phenolic acids are generally read
at  nm. Standards were used to compare the results by
comparing the retention times and UV spectra of sam-
ples. e phenolic compounds in the guava leaf extracts
areidentiedandquantiedbycomparisonwithauthentic
standards and the quantication is expressed as mg/g weight
of the extract. e quantication of phenolic compounds
in the guava leaf extract is presented in Figure (b) and
a typical HPLC chromatogram was shown in Figure (c).
Fourteen phenolic compounds such as gallic acid, ,-
dihydroxybenzoic acid, ,,-trihydroxybenzoic acid, ,-
dihydroxybenzaldehyde, caeic acid, gentisic acid, chloro-
genic acid, -hydroxybenzoic acid, vanillic and syringic acid,
vanillin, p-coumaric acid plus syringaldehyde, ferulic acid,
sinapic acid, and salicylic acid were analyzed, and thirteen
compounds were detected in guava leaf extracts. Among
 detected phenolic acids, sinapic acid (SNA,  mg/g)
was found predominant phenolic compound in guava
leaf extracts followed by -hydroxy-,-dimethoxybenzoic
acid (HDDBA, . mg/g); -hydroxy benzoic acid (HBA,
. mg/g); ferulic acid (FA, . mg/g); chlorogenic acid
(CA, . mg/g); vanillin (VNN, .mg/g); protocatechuic
acid (PA, . mg/g); gallic acid (GA, . mg/g); p-coumaric
acid plus syringaldehyde (PCA + SA, .mg/g); ,,-
trihydroxybenzoic acid (THBA, . mg/g); and DHBA,
,-dihydroxybenzaldehyde (DHBA, . mg/g). Previously,
,-dihydroxybenzoic acid, gallic acid, chlorogenic acid, -
hydroxybenzoic acid, syringic acid, caeic acid, catechin,
-hydroxybenzoic acid, ferulic acid, epicatechin, naringin,
morin, and quercetin were reported in guava leaf extracts by
various researchers [, –].
3.3. Inuence of GLE on Insulin and Glucose. Figure  eluci-
dates the hyperglycemic condition diabetic rats, whereas, on
supplementation with GLE dierent doses at , , and
 mg/kg b.w., the plasma glucose level was signicantly (p<
.) reduced. Inversely the concentration of insulin (plasma)
was declined in the diabetic rats, and upon supplementation,
with GLE the plasma insulin was signicantly (p<.)
increased near to normal. e ecient results were seen in
group  ( mg/kg b.w.) so the remaining experiments were
carried out using  mg/kg b.w. as an eective dose. e
diabetic rats treated with GB showed similar results as GLE
treatment group with not much variance. Hence the results
of GLE can be compared with that of standard drug GB.
3.4. Inuence of GLE on Lipid Peroxidation Markers. e
levels of lipid peroxidation markers were elevated in the
diabetic rats (Table ), whereas, upon GLE supplementation
(group ), the levels were reduced markedly (𝑝 < 0.05),in
comparison to the diabetic group (group ). e similar
results were obtained in the GB treated rats. e results of
GLE and GB are not statistically signicant with each other.
3.5. Inuence of GLE on Enzymatic Antioxidants. Tab l e 
shows that the concentrations of antioxidative enzymes
(SOD, CAT, GPx, and GR) were decreased in diabetic
rats, whereas, on GLE supplementation, the levels of these
enzymes were increased signicantly (𝑝 < 0.05) as compared
to the nontreated diabetic group (group ). e similar results
were obtained in the GB treated rats. e results of GLE and
GB are not statistically signicant with each other.
3.6. Inuence of GLE on Nonenzymatic Antioxidants. Tabl e 
shows that the concentrations of Vit-E, Vit-C, and GSH were
decreased in the diabetic rats, whereas, on GLE supplemen-
tation, these cellular antioxidants were increased signicantly
(𝑝 < 0.05). e similar results were obtained in the GB
treated rats. e results of GLE and GB are not statistically
signicant with each other.
BioMed Research International
0
50
100
150
200
Methanol
Wat e r
Ethanol
Total phenolic or total avonoid
contents (mg/g)
Phenolic
Flavonoid
Ethanol:water
(1:1)
(1:1)
Metha nol : water
(a)
0
2
4
6
8
10
12
14
16
18
20
Phenolic acid content (mg/g)
Phenolic compounds in guava leaf extract
GA
PA
THBA
DHBA
HBA
CA
HDDBA
VNN
PCA + SA
FA
SNA
(b)
Absorbance at 280nm
0.80
0.60
0.40
0.20
0.00
(AU)
0.00
5.00
10.00
15.00
20.00
25.00
30.00
35.00
40.00
45.00
50.00
55.00
60.00
65.00
70.00
75.00
1
2
3
4
9
10
11
12
13
14
(A)
0.070
0.060
0.050
0.040
0.030
0.020
0.010
0.000
−0.010
(AU)
1
23
4
12
Retention time (min)
Retention time (min)
0.00
5.00
10.00
15.00
20.00
25.00
30.00
35.00
40.00
45.00
50.00
55.00
60.00
65.00
70.00
75.00
(B)
5,6
7,8
5,6
(c)
OH
O
HO
HO
OH OH
OH
OH
OH
O
(1) (2) (3)
OH
O
HO
OH
(4)
OH
O
HO
OCH3
(5) (6)
(7)
OH
OH
O
(8) (9)
HO
OH
O
(10) (11) (12)
OH
OH
OH
OH
H
O
OH
COOH
OH
O
HO
OH
O
HO
H3CO
HO
OH
O
O
HO
OH
HO
OC3OC3
OC3
OC3
3CO OC3
2C
C2H
(d)
F : (a) Total phenolic and avonoid contents of guava leaf (dry weight basis) by using dierent solvent systems. (b) Quantication of
phenolic compounds in guava leaf extracts by high performance liquid chromatography (HPLC). GA, gallic acid; PA, protocatechuic acid,
THBA, ,,-trihydroxybenzoic acid; DHBA, ,-dihydroxybenzaldehyde; HBA, -hydroxy benzoic acid; CA, chlorogenic acid; HDDBA,
-hydroxy-,-dimethoxybenzoic acid; VNN, vanillin; PCA + SA, p-coumaric acid plus syringaldehyde; FA, ferulic acid; and SNA, sinapic
acid. (c) HPLC chromatographs of phenolic compound standards (A) and guava leaf extracts (B): () gallic acid (GA), () protocatechuic acid
(PA), () ,,-trihydroxybenzoic acid (THBA), () ,-dihydroxybenzaldehyde (DHBA), () -hydroxybenzoic acid (HBA), () gentisic acid,
() chlorogenic acid (CA), () vanillic acid (VA) plus caeic acid (CAA), () -hydroxy-,-dimethoxybenzoic acid (HDDBA), () vanillin
(VNN), () p-coumaric acid (PCA) + syringaldehyde (SA), () ferulic acid (FA), () sinapic acid (SNA), and () salicylic acid (SAL). (d)
Possible phenolic compounds identied in guava leaf extract. () Gallic acid (GA), () protocatechuic acid (PA), () ,,-trihydroxybenzoic
acid (THBA); () ,-dihydroxybenzaldehyde (DHBA), () -hydroxybenzoic acid (HBA), () gentisic acid, chlorogenic acid (CA), () -
hydroxy-,-dimethoxybenzoic acid (HDDBA), () vanillin (VNN), () and () p-coumaric acid (PCA) + syringaldehyde (SA), () ferulic
acid (FA), and () sinapic acid (SNA).
BioMed Research International
T : Tissue lipid peroxidation markers of the control and experimental rats.
Groups Control GLE
control Diabetic D+GLE
( mg/kg b.w.)
D+GB
( 𝜇g/kg b.w.)
TBARS (mmoles/mg tissue)
Liver 0.67 ± 0.04a0.66 ± 0.04a2.86 ± 0.26b0.99 ± 0.06c0.91 ± 0.04𝑐
Kidney 1.65 ± 0.07a1.71 ± 0.06a3.36 ± 0.34b2.01 ± 0.08c1.94 ± 0.06𝑐
Pancreas 28.29 ± 5.45a29.37 ± 4.87a41.27 ± 5.21b34.21 ± 4.19c33.29 ± 4.32𝑐
LOOH (mmoles/mg tissue)
Liver 85.38 ± 7.89a86.04 ± 7.35a110.31 ± 8.29b94.39 ± 7.39c92.76 ± 7.11c
Kidney 71.28 ± 6.75a70.37 ± 6.28a120.38 ± 8.38b85.63 ± 6.34c82.38 ± 6.29c
Pancreas 33.89 ± 7.28a32.89 ± 6.79a69.11 ± 7.16b45.39 ± 6.99c41.98 ± 6.75c
CD (mmoles/mg tissue)
Liver 65.84 ± 3.92a66.43 ± 3.47a81.29 ± 4.43b69.28 ± 3.20c64.98 ± 3.21c
Kidney 41.72 ± 2.06a42.76 ± 2.11a76.39 ± 3.86b48.37 ± 2.87c47.33 ± 3.01c
Pancreas 4.78 ± 0.28a4.81 ± 0.32a9.32 ± 0.53b5.49 ± 0.26c5.21 ± 0.22c
GLE: guava leaf extract. Values are given as means ±SD for six rats in each group. aGroup (group ) with no signicant dierence compared to control group.
bSignicantly dierent from control group at 𝑝 < 0.05.cSignicantly dierent from diabetic group at 𝑝 < 0.05. Duncan’s Multiple Range Test (DMRT).
Control
Blood glucose (mg/dL)
0
2
4
6
8
10
12
14
16
0
50
100
150
200
250
300
350
Glucose
Insulin
a
aa
b
b
bb
ccc
a
c
Diabetic
GLE control
D + GLE
(100 GA)
D + GLE
(200 GA)
D + GLE
(400 GA)
D + GB
(600 A)
Insulin (U/mL)
(kg b.w.)
F : Eect of GLE on plasma glucose and insulin levels. Each
value is mean ±SDofratsineachgroup.Ineachbar,meanswith
dierent superscript letters (a, b, and c) dier signicantly at 𝑝<
0.05 (DMRT). D: diabetic and GLE: guava leaf extract.
3.7. Inuence of GLE on the Liver, Kidney, and Pancreas
Histology. Figures (a), (b), and (a) elucidate the patho-
logical changes that occurred as a result of STZ in the liver,
kidney, and pancreas, which could exhibit the ability of
GLEinprotectingthetissuefromdamageinSTZinduced
diabetic rats. Liver of diabetic rats shows focal necrosis,
inammation of the central vein, and the congestion of
sinusoidal dilatation in the hepatocytes of diabetic rats. Aer
six weeks of treatment with GLE, the liver tissues from the D
+ GLE group show liver cells arranged neatly and no distinct
inammation of central vein was seen. A similar kind of
changes was observed in the GB group. GLE oers a vital
protection against STZ induced damage. Kidney of diabetic
rats showed necrosis, swelling of tubules, and multiple foci
of hemorrhage. Aer six weeks of treatment with GLE, the
kidney tissues from the D + GLE group show renal tubular
structures in good condition, no necrosis, and swelling of
tubules compared with the diabetic group. Glibenclamide
supplemented group showed a similar improvement. No
changesintissuearchitecturewereobservedinhepaticand
renal tissues of control rats. e histopathological study of
the pancreas shows. In Figure (a), in (A) and (B) control
group and control along with GLE show normal pancreatic
islet. In (C), STZ induced diabetic rats show inltration of
fats and damaged islet cells of pancreas as a result of which
theyweresignicantlyreducedinsizeandnumber.In(D),
diabetic + GLE ( mg/kg b.w.) treated rats show islets with
proper granules and also show the cell hyperplasty. In (E), the
similar results as group  GLE treatment rats were obtained
in the GB treated rats (Group ). is evidences that GLE
have the ability to inhibit the eects of STZ on various tissues
comparable to that of GB standard drug.
3.8. Inuence of GLE on Inammatory Markers. Figure (b)
shows the immunoblot quantifying interleukin- (IL-),
tumor necrosis factor (TNF-𝛼), and nuclear factor-kappa
B(NF-𝜅B) expression. Control group rats show normal
protein expression and diabetic rats (group ) show increased
expression of NF-𝜅B, TNF-𝛼, and IL-. Supplementation with
GLEtodiabeticrats(group)signicantlydownregulatedthe
expression of inammatory cytokines (NF-𝜅B, TNF-𝛼,and
IL-). Diabetic rats supplemented with GB showed similar
results as the GLE treatment group which could prove the
ability of GLE in inhibiting inammation compared to a
standard drug. e 𝛽-actinwasusedastheinternalstandard.
BioMed Research International
T : Tissue enzymatic antioxidant status of the control and experimental rats.
Groups Control GLE
control Diabetic D+GLE
( mg/kg b.w.)
D+GB
( 𝜇g/kg b.w.)
SOD (% NBT
reduction/min/mg protein)
Liver 7.67 ± 0.67a7.81 ± 0.27a3.86 ± 0.25b5.32 ± 0.33c5.67 ± 0.35c
Kidney 7.27 ± 0.55a7.13 ± 0.45a3.89 ± 0.65b5.49 ± 0.27c5.83 ± 0.29c
Pancreas 5.89 ± 0.48a5.95 ± 0.53a2.43 ± 0.42b4.13 ± 0.56c4.64 ± 0.58c
CAT (𝜇moles of H2O2
utilized/min/mg protein)
Liver 84.81 ± 5.29a83.29 ± 4.97a57.28 ± 5.11b71.29 ± 5.33c73.45 ± 5.47c
Kidney 41.78 ± 3.98a42.38 ± 3.26a26.39 ± 2.54b32.39 ± 3.21c34.85 ± 3.33c
Pancreas 21.87 ± 2.11a22.04 ± 2.41a7.88 ± 1.25b16.28 ± 1.78c16.89 ± 1.68c
GPx (𝜇moles of GSH
utilized/min/mg protein)
Liver 9.01 ± 0.87a8.99 ± 0.74a4.87 ± 0.45b8.11 ± 0.78c8.27 ± 0.72c
Kidney 8.12 ± 0.69a8.34 ± 0.45a4.91 ± 0.39b7.54 ± 0.81c7.84 ± 0.83c
Pancreas 8.27 ± 0.75a8.43 ± 0.78a4.38 ± 0.37b7.11 ± 0.57c7.43 ± 0.58c
GR (𝜇moles of NADPH
oxidized/min/mg protein)
Liver 0.72 ± 0.06a0.71 ± 0.06a0.42 ± 0.02b0.61 ± 0.06c0.63 ± 0.06c
Kidney 0.62 ± 0.04a0.63 ± 0.05a0.34 ± 0.03b0.50 ± 0.05c0.54 ± 0.07c
Pancreas 0.71 ± 0.05a0.70 ± 0.04a0.32 ± 0.02b0.59 ± 0.04c0.65 ± 0.04c
GLE: guava leaf extract. Values are given as means ±SD for six rats in each group. aGroup (group ) with no signicant dierence compared to control group.
bSignicantly dierent from control group at 𝑝 < 0.05.cSignicantly dierent from diabetic group at 𝑝 < 0.05. Duncan’s Multiple Range Test (DMRT).
T : Tissue nonenzymatic antioxidant status of the control and experimental rats.
Groups Control GLE control Diabetic D+GLE
( mg/kg b.w.)
D+GB
( 𝜇g/kg b.w.)
Vitamin C (mg/dL)
Liver 1.45 ± 0.13a1.43 ± 0.12a0.77 ± 0.11b1.30 ± 0.08c1.33 ± 0.07c
Kidney 1.21 ± 0.11a1.25 ± 0.13a0.68 ± 0.09b1.07 ± 0.12c1.13 ± 0.13c
Plasma 1.57 ± 0.12a1.60 ± 0.13a1.17 ± 0.11b1.38 ± 0.11c1.48 ± 0.13c
Vitamin E (mg/dL)
Liver 0.83 ± 0.08a0.87 ± 0.05a0.42 ± 0.03b0.77 ± 0.06c0.76 ± 0.07c
Kidney 0.67 ± 0.04a0.68 ± 0.02a0.33 ± 0.04b0.54 ± 0.04c0.62 ± 0.05c
Plasma 1.26 ± 0.08a1.28 ± 0.08a0.63 ± 0.05b0.93 ± 0.06c0.94 ± 0.05c
GSH (mg/dL)
Liver 4.01 ± 0.32a4.08 ± 0.27a2.28 ± 0.21b3.87 ± 0.24c3.91 ± 0.26c
Kidney 3.26 ± 0.34a3.36 ± 0.29a1.89 ± 0.18b2.93 ± 0.21c2.99 ± 0.27c
Plasma 25.28 ± 3.25a24.38 ± 3.76a13.20 ± 2.17b19.95 ± 2.81c20.01 ± 2.98c
GLE: guava leaf extract. Values are given as means ±SD for six rats in each group. aGroup (group ) with no signicant dierence compared to control group.
bSignicantly dierent from control group at p<.. cSignicantly dierent from diabetic group at p<.. Duncans Multiple Range Test (DMRT).
4. Discussion
Diabetes mellitus (DM) is a combination of heterogeneous
disarray commonly presenting with the incidence of hyper-
glycemia and glucose intolerance, as a result of lack of
insulin, defective insulin action, or both []. ere is enough
proof to demonstrate that hyperglycemia plays an important
role in initiating oxidative stress and dierent complications
associated with DM. In the current study, we have examined
theroleofbioactivecompoundsinGLEsuchasphenolic
compounds on antidiabetic activity in STZ evoked diabetic
rat model system. ere are several reasons for choosing GLE;
the important aspect we considered is that it has no side
eects and no toxicity even at higher doses. In an interesting
BioMed Research International
(A) (B)
(C) (D)
(E)
20 GG
(a)
(A) (B)
(C) (D)
(E)
20 GG
(b)
F : (a) Eect of GLE on the liver histology of the control and experimental rats (A) and (B) shows normal histology of liver with
central vein. (C) Liver histology of diabetic rats (group ) shows congestion of sinusoidal dilatation (indicated by arrow ), inammation of
the central vein (indicated by arrow [), and focal necrosis in the hepatocytes in diabetic control rats. (D) Liver histology of treatment group
(group ) shows reduced inammation, a signicant reduction in sinusoidal dilatation (indicated by arrow [), and tissue architecture near
to that of normal. (E) Liver histology of standard drug group (group ) also shows reduced inammation, a signicant reduction in sinusoidal
dilatation (indicated by arrow [), and tissue architecture near to that of normal. (b) Eect of GLE on the kidney histology of the control
and experimental rats (A) and (B) shows normal kidney histology of control (group ) and GLE control (group ). (C) Kidney histology of
diabetic rats (group ) showed multiple foci of hemorrhage, necrosis, and swelling of tubules (indicated by arrow ). (D) Kidney histology
of treatment group (group ) shows reduced necrosis and no swelling of tubules (indicated by arrow [). (E) Kidney histology of standard
drug group (group ) also shows reduced necrosis and no swelling of tubules (indicated by arrow [).
study by Kobayashi et al. [], the results suggest that the
oral administration of guava leaf extract at two dierent con-
centrations of  and  mg/kg/day caused no abnormal
toxic eects in rats which indicates that guava leaf extract has
no side eects even at very high doses. Another interesting
study has revealed that guava leaf extract did not induce
chromosomal aberrations; hence it proved that it does not
exhibit any genotoxic eects at a high dose of  mg/kg
[]. We determined the overall phenolic and total avonoid
content in the guava leaf (Figure (a)). Further, the phenolic
compounds in the guava leaf were quantied and identied
by high performance liquid chromatography (Figures (b),
(c), and (d)). e foremost phenolic compound was found
to be sinapic acid followed by dierent phenolic compounds
in the extract. Phenolic compounds in guava leaf such as
gallic acid, chlorogenic acid, ,-dihydroxybenzoic acid, -
hydroxybenzoic acid, syringic acid, caeic acid, catechin,
-hydroxybenzoic acid, ferulic acid, epicatechin, naringin,
morin, and quercetin were reported by numerous researchers
[, –]. e major phenolic compound sinapic acid
undergoes absorption and metabolism and was excreted
in the urine as -hydroxy--methoxyphenylpropionic acid,
 BioMed Research International
(A)
(D)(C)
(B)
(E)
20 GG
(a)
(B) Intensity of band scanned by densitometer
0
50
100
150
200
TNF-alpha
IL-6
NF-kB
###
Control
###
Expression
(%)
Diabetic
D + GLE
(200 GA/kg b.w.)
D + GB
(600 A/kg b.w.)
42
Lane
17
24
69
1234
(KDa)
TNF-
IL-6
NF-B p65
-Actin
(1) Control
(2) Diabetic
(3) D + GLE (200 GA/kg b.w.)
(4) D + GB (600 A/kg b.w.)
(A) Representative immunoblot showing pancreatic
, IL-6, and NF-B expression
TNF-
(b)
F : (a) Eect of GLE on the pancreas histology of the control and experimental rats. (A) and (B) show normal pancreas histology of
control (group ) and GLE control (group ), (C) pancreas histology of diabetic rats (group ) showed inltration and destroyed islet cells
of pancreas as a result of which they were signicantly reduced in size and number, (D) pancreas histology of treatment group (group )
shows well-granulated and prominent hyperplasticity of islets, and (E) pancreas histology of GB group (group ) shows well-granulated and
prominent hyperplasticity of islets. (b) Immunoblot of NF-kB, TNF-𝛼, and IL- protein samples ( 𝜇g/lane) resolved on SDS-PAGE was
probed with corresponding antibodies. Each lane was analyzed by densitometry and the expression in the control was considered as %.
e column heights are the means ±SD of six determinants. Signicantly (𝑝 < 0.05)dierent from control groups and #signicantly dierent
from STZ alone treated groups (𝑝 < 0.05).
dihydrosinapic acid, -hydroxy--methoxycinnamic acid,
and unchanged sinapic acid. Previous reports have men-
tioned that the phenolic compounds in GLE showed a possi-
blebiologicalactivityininvitroandinvivomodelsystems
[, , , ]. In the method of eliminating free radicals,
GLE plays a signicant role []. STZ, a chemical that causes
toxicity specically in the insulin synthesizing 𝛽-cell (Islet
of Langerhans) of the pancreas in mammals, is employed
oentimes to develop a typical diabetic model []. e
mechanismofactionofSTZtakesplacebyexcessproduction
BioMed Research International 
of ROS that induces cytotoxicity in beta-cells of the pancreas,
followed by reduced insulin production. Type  diabetes with
partial destruction of the pancreas was evoked by a single STZ
injection. We have chosen a low dose of STZ to induce mild
type  DM.
Free radicals play a serious role in the onset and progres-
sion of late diabetic complication. is action could also be
owing to its ability to wreck numerous components of the cell
such as proteins, lipids, and DNA []. Results of our study
found that the amount of glucose was inated considerably
within the diabetic group and reciprocally the levels of
insulin were diminished; this condition was attenuated with
treatment with GLE ( mg/kg b.w.) (Figure ). e levels
of lipid peroxidation markers, inated in diabetic rats, were
considerably prevented within the treatment group (Table ).
With this context, Soman et al. [] veried that GLE can
act as good antioxidant by reducing the blood glucose. e
standard drug group shows reduced glucose levels by its
action on calcium channels of pancreas for the release of
insulin which reverses the hyperglycemic condition.
e formation of O2and its removal were kept under
check in unstressed conditions. However, beneath the severe
oxidative stress attack can overwhelm the production of
O2. Hence, antioxidant enzymes create the foremost defense
against ROS playing a major role in eliminating the toxic
incomplete oxidations toxic intermediates. e superoxide
dismutase (SOD) is a major antioxidant enzyme involved in
direct ROS elimination and superoxide radical made within
the cells which are further converted into H2O2and later on
eliminated as H2O and singlet oxygen []. Catalase (CAT)
(EC ...) is present predominantly within the peroxi-
somes, which quickly convert toxic H2O2into H2O[].CAT
is modied along with glutathione peroxidase (GPx) and
antioxidantenzymecontainsselenium[].eleveloflipid
hydroperoxides (LOOH) was reduced by antioxidant enzyme
GPx in the presence of glutathione. NADPH-dependent
reduction of oxidized glutathione (GSSG) to reduced glu-
tathione (GSH) was catalyzed by glutathione reductase (GR)
(EC ...). GR plays a signicant role in upholding the
adequate levels of reduced GSH through GSH redox cycle.
e levels of antioxidants enzymes were seen increased in the
group supplemented with GB and the results are compared
with the GLE and found statistically not signicant.
e nonenzymatic antioxidants, like 𝛼-tocopherol (Vit-
E), ascorbic acid (Vit-C), and glutathione play a vital role
as antioxidants. ey are interconnected by utilization pro-
cesses and have very important responsibilities, against lipid
peroxidation []. e Vit-E and Vit-C participate in a vital
task in oxidative stress by defending the cells from deleterious
eects. Vit- E is a potent antioxidant, helps in detoxifying
superoxide and H2O2free radicals, and oers stability to
the membranes []. Vit-C or ascorbate is oen referred
to as a water soluble vitamin. A reactive and presumably
detrimental radical will intermingle with ascorbate. is
reaction forms an ascorbate radical with least eects to the
membrane. GSH is an intracellular antioxidant produced by
glutathione reductase helping in scavenging the free radicals.
Under severe stress condition, this system was unable to
maintain the reaction status within the cell. In our study,
theratsprovidedGLE(mg/kgb.w.)markedlyinhibitthe
decline of those antioxidants and forestall the tissues from
the deleterious eects of oxidative stress (Tables  and ).
In support of the above ndings, Suganya et al. [] found
that GLE exhibits strong free radical scavenging eects and
thisperhapsisakeyfactorincombatingoxidativestress.e
antioxidant potential of GLE should be mainly due to the
presence of phenolic acids, in particular sinapic acid. Sinapic
acid is proven to possess strong antioxidant activity that
. 𝜇M of sinapic acid can inhibit .% of DPPH radical [].
Even though the studies of varied antioxidant molecules
are established to be vital, analyzing the histologic changes
holds vital importance in explaining the disease mechanism.
Histopathological ndings of diabetic rats displayed the cen-
tral vein inammation, sinusoidal dilatation, and hepatocyte
focal necrosis (Figure (a)). Multiple foci of hemorrhage,
swelling of tubules, and necrosis were determined within
the kidney (Figure (b)). STZ induced diabetic rats show
fatty inltration and destroyed islet cells of pancreas as a
resultofwhichtheywereconsiderablyreducedinsizeand
number (Figure (a)). e damage urged that the conven-
tional detoxication process was impaired. e abovesaid
alterations were reduced signicantly in diabetic rats treated
with GLE. us, histopathological observations conjointly
support the concept that GLE reduces the burden of oxidative
stress and protects the hepatic, renal, and pancreatic tissues in
diabetic rats.
e various transcription factors like NF-𝜅B, PPAR-
𝛾, p, Nrf, AP-, 𝛽-catenin/Wnt, and HIF-𝛼can be
activated by oxidative stress []. e activation of those
transcription factors results in about -gene expression.
With the assistance of those genes, these factors control
numerous vital aspects like cell cycle regulatory molecules,
growth factors, inammatory cytokines, chemokines, and
anti-inammatory. Embryonic development, inammation,
tissue injury, and repair are controlled by an inducible
transcription factor NF-kB []. NF-kB is present in inactive
form alongside IkB in the cytoplasm beneath unstressed
conditions. When cells are aroused by cytokines such as TNF-
𝛼, bounded IkB degrades, which results in the unmasking
of NF-kB and further permits it to enter inside the nucleus
for action. e transcription of NF-kB target inammatory
genesisinitiatedonceNF-kBbindstotheDNA.Inour
study administration of STZ results in hyperglycemia and
severe oxidative stress, followed by inammation, that was
evidenced by the changes in the cytokines like IL-, NF-
kB, and TNF-𝛼relatedtoinammation.Upontreatment
with GLE, the rats showed improvement in their inam-
matory expression (Figure (b)). e anti-inammatory
action of avonoids is mainly due to its ability to inhibit
the formation of proinammatory mediators (e.g., adhesion
molecules, cytokines, eicosanoids, and C-reactive protein)
[]. Phytochemical analysis of GLE shows a high content of
avonoids alongside alternative phytoconstituents, which can
be responsible for its antihyperglycemic, antioxidative, and
anti-inammatory properties []. Glibenclamide is shown to
inhibit inammation from our results, which was in context
with the ndings of York et al., that GB can reduce the
proinammatory cytokine IL-.
 BioMed Research International
Results from our ndings suggest that GLE has the great
abilitytocutbackplasmaglucoseandoxidativestressand
conjointly ameliorates the burden of inammation in STZ
evoked diabetic rats as conrmation by reduced glucose
and restored antioxidant levels, besides reduced expression
of inammatory proteins. e benecial eects of GLE on
oxidative stress and inammation in diabetic rats were well
visualized in our histological studies. e mechanism of
action of GLE may be owing to its ability to suppress the
hyperglycemia by regulating the secretion of insulin from
the pancreatic beta-cells and in turns it may ameliorate
the oxidative stress and conrm the availability of enough
antioxidant enzymes and it is well known that presence
of oxidative stress and inammation will initiate NF-kB
activation; thus GLE alleviated the symptoms of diabetes by
a successive regulation of hyperglycemia followed by oxida-
tive stress and NF-kB pathway regulation. e regulating
mechanism of GLE is mostly comparable to that of GB
which was evidenced from the results of our study. ough
various reports claim the antidiabetic potential of GLE, our
research ndings interrelated the main symptoms of diabetes
such as hyperglycemia, oxidative stress, and inammation
scientically and it enhances the scientic importance of this
study and additional studies on its eectuality in clinical
trials can add additional strength within the eld of diabetes
management in search of novel medicine with no side eects.
Phytocompounds Analyzed in This Article
,-Dihydroxybenzaldehyde and its PubChem CID is
.
Chlorogenic acid and its PubChem CID is .
Protocatechuic acid and its PubChem CID is .
Gallic acid and its PubChem CID is .
Vanillic acid and its PubChem CID is .
Caeic acid and its PubChem CID is .
Vanillin and its PubChem CID is .
p-Coumaric acid and its PubChem CID is .
Syringaldehyde and its PubChem CID is .
FerulicacidanditsPubChemCIDis.
SinapicacidanditsPubChemCIDis.
Salicylic acid and its PubChem CID is .
Abbreviations
STZ: Streptozotocin
CAT: Catalase
CD: Conjugated dienes
DM: Diabetes mellitus
GB: Glibenclamide
SOD: Superoxide dismutase
GLE: Guava leaf extract
GSH: Reduced glutathione
IL-: Interleukin-
LOOH: Lipid hydroperoxides
NF-kB: Nuclear factor-kappa B
ROS: Reactive oxygen species
TBARS: iobarbituric acid reactive substances
TCA: Trichloroacetic acid
TBA: iobarbituric acid
TNF-𝛼: Tumor necrosis factor-𝛼.
Conflicts of Interest
erearenoconictsofinterest.
Authors’ Contributions
Muthukumaran Jayachandran (jmkbio@uic.edu.hk) de-
signed and carried out the experiments and wrote the
manuscript. Ramachandran Vinayagam (rambio@uic.edu
.hk) helped in animal feeding and dissection and car-
ried out few biochemical assays. Ranga Rao Ambati
(arangarao@gmail.com) helped in carrying out HPLC and
its interpretation. Baojun Xu (baojunxu@uic.edu.hk) and
Stephen Sum Man Chung (smchung@uic.edu.hk) designed,
wrote, and corrected the manuscript. Muthukumaran
Jayachandran and Ramachandran Vinayagam contributed
equally to the article as rst authors.
Acknowledgments
e authors sincerely thank Ms. Yinhua Liu in Zhuhai Cam-
pus of Zunyi Medical University for her technical assistance
on the use of the animal facility. e work was funded by
grants from Beijing Normal University-Hong Kong Baptist
University United Inter national College, Zhuhai, Guang-
dong, China (Grants UIC  and UIC ).
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... This study indicates that the renoprotective effects of hidrosmin in diabetic kidneys are mediated by the concerted inhibition of NF-κBp65 and STAT3 transcription factors, although further studies are needed to identify the exact mechanism of the compound. Our findings are consistent with previous evidence showing that different flavonoids relieve renal inflammation in the diabetic milieu by inhibiting NF-κB [23,53,58] and STAT signaling [59,60]. ...
... Furthermore, the antioxidant capacity of the kidneys was improved with the hidrosmin treatment, demonstrated by an increase in the expression of SOD1 and CAT, honoring the classical antioxidant capacity of flavonoids described in the literature [53,54,58]. Nevertheless, we found a downregulation of the NRF2 pathway activation after the treatment, a fact we can attribute to hidrosmin and its clear inhibition of the NF-κB pathway, which controls inflammation and ROS generation and influences NRF2 [68]. ...
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Diabetes mellitus (DM) is a high-impact disease commonly characterized by hyperglycemia, inflammation, and oxidative stress. Diabetic nephropathy (DN) is a common diabetic microvascular complication and the leading cause of chronic kidney disease worldwide. This study investigates the protective effects of the synthetic flavonoid hidrosmin (5-O-(beta-hydroxyethyl) diosmin) in experimental DN induced by streptozotocin injection in apolipoprotein E deficient mice. Oral administration of hidrosmin (300 mg/kg/day, n = 11) to diabetic mice for 7 weeks markedly reduced albuminuria (albumin-to-creatinine ratio: 47 ± 11% vs. control) and ameliorated renal pathological damage and expression of kidney injury markers. Kidneys of hidrosmin-treated mice exhibited lower content of macrophages and T cells, reduced expression of cytokines and chemokines, and attenuated inflammatory signaling pathways. Hidrosmin treatment improved the redox balance by reducing prooxidant enzymes and enhancing antioxidant genes, and also decreased senescence markers in diabetic kidneys. In vitro, hidrosmin dose-dependently reduced the expression of inflammatory and oxidative genes in tubuloepithelial cells exposed to either high-glucose or cytokines, with no evidence of cytotoxicity at effective concentrations. In conclusion, the synthetic flavonoid hidrosmin exerts a beneficial effect against DN by reducing inflammation, oxidative stress, and senescence pathways. Hidrosmin could have a potential role as a coadjutant therapy for the chronic complications of DM.
... Type 1 diabetes is characterized by autoimmune mediated destruction of pancreatic beta cells; while type 2 diabetes, the more prevalent form is defined by progressive loss of beta cells, disturbed insulin secretion and resistance to insulin [3,4]. It is a complex metabolic disorder known to be mediated by oxidative stress led hyperglycemia [5]. Several other risk factors such as, sedentary lifestyle, genetic pre-disposition, epigenetic changes, and altered gut microbiota are associated with diabetes [2,6]. ...
... The link between all these parameters in hyperglycemia is represented by the oxidative stress, which activates transcription factors including NF-κB and HIF-1α and induces DNA lesions. The activation of these factors results in the expression of genes that control vital processes and molecules in cells, such as the cell cycle regulation or inflammatory cytokines [44]. The use of compounds with an impact on the key molecules and intracellular signaling pathways involved in the DM pathogenesis with minimal side effects and increased efficiency is welcome. ...
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The present study aimed to compare two polyphenolic-enriched extracts obtained from the Thymus marschallianus Willd. (Lamiaceae) species, harvested from culture (TMCE in doses of 0.66 μg GAE/mL and 0.066 μg GAE/mL) and from spontaneous flora (TMSE in doses of 0.94 μg GAE/mL and 0.094 μg GAE/mL) by assessing their biological effects on human umbilical vein endothelial cells (HUVECs) exposed to normoglycemic (137 mmol/L glucose) and hyperglycemic conditions (200 mmol/L glucose). Extracts were obtained by solid phase extraction (SPE) and analyzed by chromatographical (HPLC-DAD) and spectrophotometrical methods. Their effects on hyperglycemia were evaluated by the quantification of oxidative stress and NF-ĸB, pNF-ĸB, HIF-1α, and γ-H2AX expressions. The HPLC-DAD analysis highlighted significant amounts of rosmarinic acid (ranging between 0.18 and 1.81 mg/g dry extract), luteolin (ranging between 2.04 and 17.71 mg/g dry extract), kaempferol (ranging between 1.85 and 7.39 mg/g dry extract), and apigenin (ranging between 4.97 and 65.67 mg/g dry extract). Exposure to hyperglycemia induced oxidative stress and the activation of NF-ĸ increased the expression of HIF-1α and produced DNA lesions. The polyphenolic-enriched extracts proved a significant reduction of oxidative stress and γ-H2AX formation and improved the expression of HIF-1α, suggesting their protective role on endothelial cells in hyperglycemia. The tested extracts reduced the total NF-ĸB expression and diminished its activation in hyperglycemic conditions. The obtained results bring evidence for the use of the polyphenolic-enriched extracts of T. marschallianus as adjuvants in hyperglycemia.
... It can also control the oxidative stress related to the NF-kB pathway activation. Different phenolic compounds present in the guava leaf extract are behind this activity [19]. Li et al reported the anti-inflammatory property of the convallatoxin, a cardiac glycoside. ...
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Certain activation of signaling pathways plays a great role in cancer pathogenesis and the growth and progression of the tumor cells. Among the several signaling pathways intensively entangled in cancer progression, NF-κB is one of the important pathways which are actively explored by researchers. This B cell-specific transcription factor NF-κB family is first discovered by David Baltimore’s group. It contains five different DNA-binding proteins which are actively involved in the formation of homodimers and heterodimers. NF-κB proteins are key regulators of innate and adaptive immune responses that can accelerate cell proliferation, inhibit apoptosis, promote cell migration and invasion, and stimulate angiogenesis and metastasis. The normal activation of the NF-κB is necessary for the survival of the cells and immunity, its deregulation can results development of cancer and multiple inflammatory diseases. Therefore NF-κB is one of the major targets of anticancer and anti-inflammatory molecule development.
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Abstract: Meal replacement (MR) is widely used in weight and diabetes management programs due to its ease of compliance and handling. However, little is known about its impact on outcomes other than glycaemic control and weight loss. Furthermore, not many studies evaluate its cost-effectiveness and sustainability. This study aimed to evaluate the efficacy of a diabetes-specific MR for the weight reduction and glycaemic controls of overweight and obese T2DM patients, as compared to routine dietary consultation. Other health outcomes, the cost effectiveness, and the sustainability of the MR will also be evaluated. Materials and Methods: This randomised controlled clinical trial will involve 156 participants who have been randomised equally into the intervention and control groups. As a baseline, both groups will receive diet consultation. Additionally, the intervention group will receive an MR to replace one meal for 5 days a week. The duration of intervention will be 12 weeks, with 36 weeks of follow-up to monitor the sustainability of the MR. The primary endpoints are weight and Hemoglobin A1c (HbA1c) reduction, while the secondary endpoints are anthropometry, biochemical measurements, satiety, hormone changes, quality of life, and cost-effectiveness. The impact of the COVID-19 pandemic on study design is also discussed in this paper. This study has obtained human ethics approval from RECUKM (JEP-2019-566) and is registered at the Thai Clinical Trials Registry (TCTR ID: TCTR20210921004).
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Background Psidium guajava is a medicinal plant with antidiabetic properties and can be found growing in tropical and sub-tropical countries around the world. Purpose The aim of the present study was to investigate the anti-diabetic potential of Psidium guajava leaf in streptozotocin-induced diabetic rats, with particular focus on key enzymes of glycogen metabolism in skeletal muscle. Study design The study design involved preparation of an aqueous Psidium guajava leaf extract; investigation of its effect on muscle glycogen synthase and phosphorylase activities and possible protective effect on pancreas in streptozotocin induced diabetic male Sprague-Dawley rats and phytochemical study of the extract to identify further compounds with anti-diabetic potential. Method Diabetes was induced in male Sprague-Dawley rats with a single intraperitoneal injection of 40 mg/kg streptozotocin. Normal and diabetic animals were treated with 400 mg/kg body weight of Psidium guajava leaves aqueous extract for a period of 14 days. Results The treatment of diabetic animals with the Psidium guajava extract ameliorated damage to the pancreatic islets and enhanced lowering of blood glucose following a glucose load. Diabetes significantly decreased (P<0.05) glycogen synthase activity by 31 % compared to normal animals this being associated with a decrease in enzyme expression. Psidium guajava treatment returned the enzyme activity to near normal levels. In diabetic animals, glycogen phosphorylase activity was elevated but not significantly (P>0.5) and treatment of both diabetic and normal animals with the extract reduced activity of the enzyme. The Psidium guajava extract also reduced glycogen phosphorylase expression in diabetic animals. Skeletal muscle glycogen content reduced by diabetes was increased as a result of treatment of diabetic animals with the Psidium guajava extract. Phytochemical analysis of the aqueous extract of Psidium guajava using Gas Chromatography-Mass Spectroscopy indicated the presence of triterpenes and phenolic compounds and we also report what we believe are three new compounds. Conclusion The results from this study suggest that the antidiabetic effects of Psidium guajava may be due to modulation of glycogen metabolism mediated by phenolic compounds and triterpenes present in the extract.
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Chronic pancreatitis (CP) is a multifactorial, inflammatory syndrome characterized by acinar atrophy and fibrosis. Activation of NOD-like receptors family pyrin domain-containing 3 (NLRP3) inflammasome is a central mediator of multiple chronic inflammatory responses and chronic fibrosis including pancreatic fibrosis in CP. The Psidium guajavaleaf is widely used in traditional medicine for the treatment of chronic inflammation, but the anti-inflammatory effect of Psidium guajavaleaf on CP has not yet been revealed. In this study, we investigated whether the extract of total flavonoids from Psidium guajava leaves (TFPGL) plays a therapeutic mechanism on CP through NLRP3 inflammasome signaling pathway in a mouse CP model. The H&E and acid-Sirius red staining indicted that TFPGL attenuated the inflammatory cell infiltration and fibrosis significantly. The results of immunohistological staining, western blot and RT-qPCR showed that the expressions of NLRP3 and caspase-1 were significantly increased in the CP model group, while TFPGL significantly decreased the NLRP3 and caspase-1 expression at both the gene and protein levels. Moreover, ELISA assay was used to examine the levels of NLRP3 inflammasome target genes, such as caspase-1, IL-1[Formula: see text] and IL-18. We found that TFPGL treatment decreased the expression of caspase-1, IL-1[Formula: see text] and IL-18, which is critical for the NLRP3 inflammasome signaling pathway and inflammation response significantly. These results demonstrated that TFPGL attenuated pancreatic inflammation and fibrosis via preventing NLRP3 inflammasome activation and TFPGL can be used as a potential therapeutic agent for CP.
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Fermentation results in the release and biotransformation of phenolic compounds in guava leaves. This study aimed to evaluate the bioaccessibility, safety and antidiabetic effect of phenolic-rich extract from fermented guava leaves (PE-fgl) using Monascus anka and Bacillus sp. BS2. The total phenolics and flavonoids were 51.18% and 16.60% bioaccessible after gastrointestinal digestion, respectively. High bioaccessibility was observed, particularly for ellagic acid, isoquercitrin, quercetin-3-O-β-D-xylopyranoside, quercetin-3-O-α-L-arabinofuranoside and avicularin, resulted in high recovery of α-glucosidase inhibition activity and antioxidant capacity. Moreover, PE-fgl was practically nontoxic with a median lethal dose (LD50) greater than 5000 mg/kg and was without evidence of toxicity following subchronic administration up to the dose limit of 2000 mg/kg. Furthermore, PE-fgl significantly improved serum glucose and lipid levels as well as antioxidant capacity in diabetic mice. Liver, kidney and pancreas damage was also significantly alleviated. These results suggested that PE-fgl could be a nontoxic candidate for diabetes treatment.
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Medicinal mushrooms are rich sources of pharmacologically active compounds. One of the mushrooms commonly used in traditional Chinese medicine is Ganoderma lucidum (Leyss. Ex Fr.) Karst. In Asian countries it is treated as a nutraceutical, whose regular consumption provides vitality and improves health. Ganoderma lucidum is an important source of biologically active compounds. The pharmacologically active fraction of polysaccharides has antioxidant, immunomodulatory, antineurodegenerative and antidiabetic activities. In this review, we summarize the activity of Ganoderma lucidum polysaccharides (GLP).
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Diabetes is a metabolic disturbance that is described by hyperglycemia linked to impairment in insulin secretion and/or action as well as aberrations in the intermediary metabolism of carbohydrates, lipids and proteins. It is the most common prevalent disease that affected both developed and developing countries’ citizens. Psidium guajava L. is known as guava, which belongs to the family Myrtaceae. This can be useful in the management of blood glucose. The aim of the present study was to investigate the protective effect of the ethanolic extract of Psidium guajava leaves (PGE200) in alloxan-induced diabetic mice. The study revealed the presence of antidiabetic activity in PGE200. The PGE200 successfully inhibited the blood glucose level in diabetic mice. The PGE200 reduced the serum level of urea, and creatinine and significantly (P < 0.05) increase the serum total protein level in diabetic mice. The efficacy was compared to the standard hypoglycemic drug Glibenclamide. From the findings obtained in the present studies, the PGE200 has a prominent antidiabetic activity.
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In this study, soluble and insoluble phenolic acids (SPA and IPA) in selected Thai fruit such as orange, banana, guava and mango using isocratic HPLC-UV method were determined. Results showed that the predominant compounds of all fruit studied were IPA (80.2-99.5%). Gallic and hydroxybenzoic acids were identified as major SPA in guava and orange, respectively. Gallic, hydroxybenzoic, vanillic, caffeic, syringic and ferulic acids were identified as major IPA in all fruit samples. Ferulic acid was the dominant IPA in orange and banana extracts (335.8 ± 13.38 and 219.5 ± 18.47 μg/g dry weights, respectively). Whilst, gallic acid was the dominant IPA in mango extract (542.5 ± 6.80 μg/g dry weight) and hydroxybenzoic acid was the dominant IPA in guava extract (50.5 ± 8.12 μg/g dry weight). The antioxidant capacity of all fruit extracts was also evaluated using a Folin-Ciocalteu's assay and DPPH free radical-scavenging assay. The phenolics in bound form of orange extract contained the highest total phenolic content (2.6 ± 0.02 μg GAE/ml). Its antioxidant capacity was 94.9%. Whereas insoluble phenolic content of guava extract had the least antioxidant capacity (22.0%).
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Investigation of antihypertensive and antioxidant properties of fruit and leaf extracts from four varieties (giant white, small white, stripped and pink) of guava (Psidium guajava L.) in vitro was the focus of this study. Consequently, methanol/1 M HCl (20:1 v/v) extraction of fruit and leaf of the guava varieties were carried out. Thereafter, the extracts were assayed for their angiotensin I converting enzyme (ACE) inhibitory effect, total phenol and flavonoid contents, reducing property, radicals (DPPH, ABTS•+, hydroxyl and nitric oxide) scavenging ability, Fe2+ chelating ability, and inhibition of Fe2+ and Sodium nitroprusside (SNP) induced lipid peroxidation reactions (in vitro). Furthermore, the phenolic constituents of the extracts were characterised with gas chromatography (GC). The results showed that all the extracts significantly (P < 0.05) inhibited ACE activity, scavenged (DPPH, ABTS•+, nitric oxide and hydroxyl) radicals, chelated Fe2+ and also inhibited Fe2+ and SNP induced lipid peroxidation in rat heart (in vitro). Nevertheless, the pink guava variety had the highest ACE inhibitory and antioxidant properties. In addition, Rosmarinic acid, eugenol, carvacrol, catechin and caffeic acid were the dominant phenolics found in the extracts. The ACE inhibitory effects and antioxidant properties of the guava extracts, which correlates significantly with their phenolic constituents, could largely contribute to their antihypertensive properties as obtained in traditional medicine.
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Human body is continuously exposed to different types of agents that results in the production of reactive species called as free radicals (ROS/RNS) which by the transfer of their free unpaired electron causes the oxidation of cellular machinery. In order to encounter the deleterious effects of such species, body has got endogenous antioxidant systems or it obtain exogenous antioxidants from diet that neutralizes such species and keep the homeostasis of body. Any imbalance between the RS and antioxidants leads to produce a condition known as “oxidative stress” that results in the development of pathological condition among which one is diabetes. Most of the studies reveal the inference of oxidative stress in diabetes pathogenesis by the alteration in enzymatic systems, lipid peroxidation, impaired Glutathione metabolism and decreased Vitamin C levels. Lipids, proteins, DNA damage, Glutathione, catalane and superoxide dismutase are various biomarkers of oxidative stress in diabetes mellitus. Oxidative stress induced complications of diabetes may include stroke, neuropathy, retinopathy and nephropathy. The basic aim of this review is to summarize the basics of oxidative stress in diabetes mellitus.
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The effects of guava leaves extracted using solvents of water, ethanol, methanol, and different concentrations of hydroethanolic solvents on phenolic compounds and flavonoids, and antioxidant properties have been investigated. The antioxidant capability was assessed based on 2,2-diphenyl-1-picrylhydrazyl radical and 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) radical-scavenging abilities, reducing power, and nitric oxide-and nitrate-scavenging activities. The results demonstrated that the antioxidant ability of guava leaf extracts has a strong relationship with phenolic compound content rather than flavonoid content. Phenolic compound content of water extracted guava leaves was higher compared to pure ethanol and methanol extracts. However, phenolic compound content extracted using hydroethanolic solvent was higher than water, whereas 50% hydroethanolic was observed to be the most effective solvent showing high antioxidant ability.
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Fruits, vegetables and medicinal herbs rich in phenolics antioxidants contribute toward reduced risk of age-related diseases and cancer. In this study, Psidium guajava leaf extract was fractionated in various organic solvents viz. petroleum ether, benzene, ethyl acetate, ethanl and methanol and tested for their antioxidant and antimutagenic properties. Methanolic fraction showed maximum antioxidant activity comparable to ascorbic acid and butylated hydroxyl toluene (BHT) as tested by DPPH free radical scavenging, phosphomolybdenum, FRAP (Fe3 + reducing power) and CUPRAC (cupric ions (Cu2+) reducing ability) assays. The fraction was analyzed for antimutagenic activities against sodium azide (NaN3), methylmethane sulfonate (MMS), 2-aminofluorene (2AF) and benzo(a)pyrene (BP) in Ames Salmonella tester strains. The methanol extracted fraction at 80 μg/ml concentration inhibited above 70% mutagenicity. Further, phytochemical analysis of methanol fraction that was found to be most active revealed the presence of nine major compounds by gas chromatography–mass spectrometry (GC–MS). This data suggests that guava contains high amount of phenolics responsible for broad-spectrum antimutagenic and antioxidant properties in vitro and could be potential candidates to be explored as modern phytomedicine.
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