Available via license: CC BY-NC-ND
Content may be subject to copyright.
Anti-inflammatory and preventive activity of white mulberry root
bark extract in an experimental model of pancreatitis
Kavitha Yuvaraj, Geetha Arumugam
*
Department of Biochemistry, Bharathi Women's College, Broadway, Chennai, 600 108, India
article info
Article history:
Received 13 May 2017
Received in revised form
18 January 2018
Accepted 19 January 2018
Available online 2 March 2018
Keywords:
Pancreatitis
NLRP3
GC-MS
HPLC
Cudraflavone B
Morus alba
abstract
Pancreatitis is characterized by highly morbid inflammation in the pancreas. Currently, there is no
specific drug available for pancreatitis except supportive medicines. The present study assessed the
pancreato-protective effect of Morus alba root bark extract by using alcohol and cerulein-induced model
of pancreatitis. The study also investigated the phytochemical profile through GC-MS and HPLC. Meth-
anolic extract of Morus alba root bark extract (MEMARB) was subjected to GC-MS and HPLC studies. Male
albino Wistar rats were administered ethanol (0%e36%) and cerulein (20
m
g/kg b.wt. i.p.) with or without
MEMARB. Serum lipase, amylase, caspase-1, lipid peroxidation products, glutathione and enzymatic
antioxidants were determined. Histological changes in the pancreas were assessed. Cudraflavone B in
MEMARB was quantified by HPLC. Significant amount of Cudraflavone B was detected by quantitative
HPLC. Marked increase in the levels of serum amylase, lipase, caspase-1, IL-18 and IL-1
b
were observed in
ethanol and cerulein administered rats than in MEMARB co-administered rats. In MEMARB co-
administered rats, the antioxidant status was restored to near normal levels. Histological examinations
showed that MEMARB significantly reduced the inflammatory and fibrotic changes. The results reveal the
potent pancreato-protective effects of Morus alba root bark. The anti-inflammatory effect of Morus alba
root bark extract might be due to the presence of various phytonutrients including Cudraflavone B.
©2018 Center for Food and Biomolecules, National Taiwan University. Production and hosting by Elsevier
Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/
licenses/by-nc-nd/4.0/).
1. Introduction
Pancreatitis is an inflammatory response initiated in the
pancreatic parenchyma due to acinar cell injury. The underlying
pathomechanism is the premature ectopic activation of the master
proteolytic zymogen trypsinogen, to active trypsin, precipitating a
cascade of zymogen activation and ‘auto digestion’of the pancreas.
1
Persistent, irreversible inflammation and fibrosis of the
pancreas are characteristics of chronic pancreatitis (CP). Symptoms
of CP include abdominal pain, frequent acute pancreatitis (AP)
episodes, exocrine and endocrine insufficiency.
2
Alcohol abuse
causes 70% of CP while smoking, gene mutations, hyperparathy-
roidism and autoimmunity are other etiologies.
3
Chronic pancrea-
titis has a worldwide prevalence of 50/100,000 persons. Tropical
calcific pancreatitis (TCP), a rare form of CP, has a prevalence of
20e125/100,000 persons in southern India. 5% of CP patients and
40e50% of hereditary pancreatitis patients develop highly lethal
pancreatic cancer.
4
Current management guidelines recommend only supportive
measures like hospitalization, intensive fluid resuscitation, bowel
rest, parenteral nutrition, enzyme supplements and pain manage-
ment through non-steroidal anti-inflammatory drugs (NSAIDs).
In both acute and chronic pancreatitis injured and dying
pancreatic acinar cells are the primary drivers of inflammation and
initiators of necroptosis and pyroptosis, key determinants of dis-
ease severity. Acinar cells elaborate immune responses by secreting
cytokines (TNF-
a
, IL-1
b
, IL-6, and IL-10), chemokines (MCP-1) and
endogenous damage associated molecular patterns (DAMPs).
NLRP3 inflammasome is a multiprotein intracellular innate
immune sensor consisting of NLRP3, apoptosis-associated speck-
like protein (ASC) and procaspase-1. It assembles in response to
Abbreviations: AP, acute pancreatitis; CP, chronic pancreatitis; TCP, tropical
calcific pancreatitis; MCP 1, monocyte chemotactic protein 1; DAMPS, damage
associated molecular patterns; PSC, pancreatic stellate cell; NF-kappa B, nuclear
factor kappa-light-chain-enhancer of activated B cells; AP1, activator protein 1;
MEMARB, methanolic extract of Morus alba root bark.
*Corresponding author.
E-mail address: geethav21@yahoo.co.in (A. Geetha).
Peer review under responsibility of The Center for Food and Biomolecules,
National Taiwan University.
Contents lists available at ScienceDirect
Journal of Traditional and Complementary Medicine
journal homepage: http://www.elsevier.com/locate/jtcme
https://doi.org/10.1016/j.jtcme.2018.01.011
2225-4110/©2018 Center for Food and Biomolecules, National Taiwan University. Production and hosting by Elsevier Taiwan LLC. This is an open access article under the CC
BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Journal of Traditional and Complementary Medicine 8 (2018) 497e505
diverse stimuli and forms the scaffold for the activation of pro-
inflammatory cytokines IL-1beta and IL-18 and induces the
release of HMGB1 expression important in pancreatic inflamma-
tion, parenchymal cell injury and disease resolution. Pro-
inflammatory cytokines IL-1 beta and IL-18 are triggers of pyrop-
tosis, a highly lytic form of cell death, which appears to be pre-
dominant in pancreatitis.
5e7
In the face of a threat from rising pancreatitis cases and the
absence of a specific licensed drug, inflammatory pathways have
been under scrutiny to find suitable targets for potential anti-
inflammatory drug molecules. Various anti-inflammatory effec-
tors that have been tested include thalidomide (targets TNF-alpha),
panhaematin (decreases leukocyte infiltration), IL-R antagonist
montelukast, MCP 1 inhibitors, COX- 2 inhibitor flavocoxid, vitamin
K3 (inhibits autophagy) and the broad anti-inflammatory effects of
agents like quercetin, resveratrol and curcumin.
1
Morus alba L. or white mulberry is native to northern China and
has been naturalized and cultivated throughout Asia and Europe. It
has long-standing ethno medicinal significance. Various parts of
the plant have been used in traditional Asian medicine. Phyto-
chemical analyses have identified alkaloids, flavonoids, flavones,
flavanones, stilbenes, benzophenones, coumarin derivatives and
terpenoids in the root bark of M. alba
8
. Morus is one of the few
genera to contain prenylated flavonoids. Prenyl flavonoids are
credited with enhanced biological effects attributed to the prenyl
side-chains.
9
But, their scientific validation should be taken care of.
The bioactive principles from Morus alba root bark are reported
to have antibacterial, antiviral, antioxidant, hypoglycemic, neuro-
protective, nephroprotective, antiulcer, analgesic and anti-
inflammatory properties.
10
The aim of the present investigation is to assess the therapeutic
efficacy of phytonutrients of white mulberry roots with special
reference to their influence on the level of cytokine production and
their impact on acinar cell damage monitored in terms of serum
and pancreatic marker enzymes and cellular antioxidants.
2. Materials and methods
2.1. Chemicals
Reference standards for HPLC - gallic acid, galangin, rutin,
quercetin and thymoquinone - were obtained from LGC Pro-
mochem India Pvt. Ltd., (Bangalore, India). HPLC grade solvents
were purchased from Merck India. ELISA kit for IL-I
b
was purchased
from Abcam and Invitrogen ELISA kit for IL-18 was purchased from
Thermo Fisher Scientific. Lipase and alpha-amylase assay kits were
procured from Coral Clinical Systems, Goa, India. All other chem-
icals used were of analytical grade.
2.2. Plant material collection, identification and extraction
Fresh Morus alba roots were collected from the Plant Sciences
department, University of Madras, Guindy Campus, Chennai,
department of Sericulture, Vitchanthangal, Kancheepuram District,
Tamil Nadu and a private mulberry farm at Purisai, Kancheepuram
District, Tamil Nadu. The plant material was authenticated by
Professor P. Jayaraman, Director, Plant Anatomy Research Centre,
West Tambaram, Chennai. The herbarium specimen (PARC/2015/
3144) was preserved for future reference in the Department of
Biochemistry, Bharathi Women's College. The roots were pooled,
cleaned under tap water and air-dried for 2e3 weeks. Completely
dry roots were lightly scraped to reveal the yellowish layer un-
derneath. The root bark or cortex was peeled off with the help of a
knife, cut into small pieces and powdered in a blender. The crude
powder was sieved to obtain a fine homogenous powder. M.alba
root bark powder was soaked in methanol and left to agitate on a
shaker for 24hrs. The extract was filtered. The residue was extrac-
ted again with fresh methanol to ensure complete extraction. The
filtrates were air dried to a powder, sieved and stored in an air-tight
container at 4
C. For animal experimentation, a homogenous sus-
pension of the bark powder was prepared with 0.1% DMSO.
2.3. Animals
Adult male albino Wistar rats (175e200g, seven-eight weeks
old) used for the study were housed under hygienic conditions [22-
24
C] in polypropylene cages under 12 h light/12 h dark cycle. The
animals were allowed free access to water and standard pelleted rat
chow during the acclimatization period. Animal maintenance and
experimentation protocols conformed to the guidelines of the
Institutional Animal Ethics Committee constituted by the Com-
mittee for the Purpose of Control and Supervision of Experiments
on Animal (CPCSEA), Government of India, [XVII/VELS/PCOL/02/
2000/CPCSEA/IAEC/06.10.15].
2.4. Experimental protocols
2.4.1. Preliminary phytochemical screening by GC-MS
The methanolic extract of the root bark of white mulberry
(M.alba) was subjected to GC-MS analysis. The analysis was carried
out on Agilent 6890N gas chromatograph with HP-5ms column
coupled to a mass spectrometer JEOL GC-MATE II in the electron
ionization (EI) mode with ionization voltage set to 70eV. The mass
spectral scan range of the mass analyser was set to 50e600 amu.
Helium was used as a carrier gas at a constant flow of 1 mL/min. The
front inlet temperature was 220
C. The GC-oven was set for the
following temperature profile: ramp rate from 50
◦
C-250
Cat
10
C/min. NIST (National Institute of Standards and Technology)
GC-MS mass spectral database was used to interpret the mass
spectrum using the retention time.
2.4.2. HPLC for identification of flavonoids and quantification of
Cudraflavone B
Accurately weighed quantities of the standards and the sample
were transferred to separate volumetric flasks and dissolved in
methanol and diluted to a specific concentration. A specific quan-
tity of Morus alba root bark powder was refluxed with extraction
solvent (methanol: water: hydrochloric acid) for 135 min to
hydrolyse the flavonoid glycosides and contents were made up
with methanol. An equal volume of the standard and sample
(20
m
L) was then used for the HPLC analysis on Shimadzu (Japan),
HPLC Class VP series with a UVevis detector. The samples were run
on a C18 column (100 Åpore size, 3.5
m
m, 4.6 mm 250 mm)
40 min run time. The mobile phase used was a mixture of meth-
anol, water and phosphoric acid mixed in the ratio (100:100:1). The
elution was isocratic with the flow rate set at 1.5 mL/min. The fla-
vonoids were monitored by the ultraviolet detector set at 270 nm.
EZChrom Data System was used for data acquisition, processing and
report generation.The resulting chromatograms were recorded
and the areas under the major peaks measured. Flavonoids were
identified by matching the retention time and their spectral char-
acteristics against those of the standards.
2.4.3. Ethanol and cerulein-induced chronic pancreatitis
After one-week of acclimatization, the animals were randomly
divided into four groups of six animals each. Group 1: received
normal diet (standard rat chow) for 5 weeks; Group 2: received the
normal diet and MEMARB (300 mg/kg body weight/day) orally for
the last 3 weeks of the experimental period; Group 3: received an
ethanol containing isocaloric diet, and 20
m
g/kg body weight of
Y. Kavitha, A. Geetha / Journal of Traditional and Complementary Medicine 8 (2018) 497e505498
cerulein intraperitoneally for the last 3 weeks Group 4: received an
ethanol containing isocaloric diet, 20
m
g/kg body weight of cerulein
intraperitoneally and MEMARB (300 mg/kg body weight) orally, for
the last 3 weeks.
At the end of the experimental period, rats were fasted over-
night and anesthetized by intramuscular injection of ketamine
hydrochloride [30 mg/kg body wt.] and killed by cervical decapi-
tation. Blood was collected and the serum separated was stored
appropriately until further analyses.
2.4.4. Tissue homogenate preparation
Pancreas was removed carefully, washed and homogenized in
0.1M Tris-HCl buffer pH 7.4 and centrifuged at low speed to remove
all the cell debris. The supernatant was used for the determination
of caspase-1, and antioxidant enzymes such as superoxide dis-
mutase (SOD), catalase (CAT) and glutathione peroxidase (GP
x
),
estimation of reduced glutathione (GSH) and estimation of lipid
peroxides.
2.4.5. Biochemical investigations
2.4.5.1. Assay of IL-1
b
.IL-1 beta rat sandwich ELISA Kit (AB100767)
was purchased and the assay was performed conforming to man-
ufacturer's instructions.
2.4.5.2. Assay of IL-18. The IL-18 Rat ELISA Kit (KRC2341) was used
to quantify serum IL-18. Instructions provided in the kit manual
were followed.
2.4.5.3. Assay of caspase-1. For the colorimetric assay of caspase-1
activity in serum and pancreatic homogenate, the method of
Thornberry
11
was adopted. The pancreas was homogenised in a
lysis buffer (25 mM HEPES pH 7.5), 1mM EDTA, 10
m
g of aprotinin/
mL, 10
m
g of leupeptin/mL, 2 mM dithiothreitol at 5mL/100 mg of
pancreas tissue. Extracts were centrifuged at 15,000 g for 30 min at
4
C. The supernatant was recentrifuged at 200,000 g for 1 hat 4
C
and the supernatant used for the measurement of capase-1 activity.
The assay is based on the spectrophotometric detection of the
chromophore p-nitroanilide (pNA), released from the labelled
substrate YVAD-pNA by caspase-1, at 400 or 405 nm. Activity was
expressed as pg/mL serum and pM/mg protein.
2.4.5.4. Assay of myeloperoxidase. The supernatants of cell free
extracts were assayed for MPO as described by Bradley et al.
12
Briefly, the rate at which a coloured product formed during the
MPO dependent reaction with o-dianisidine dihydrochloride
(0.167 mg/mL) was measured kinetically at 460 nm and the activity
was expressed as units/mg protein.
2.4.5.5. Assay of serum lipase and serum alpha-amylase.
Serum lipase and serum alpha-amylase levels were determined by
turbidimetry and colorimetry respectively, following instructions
provided in the kit manual.
2.4.5.6. Estimation of lipid peroxides. Thiobarbituric acid reactive
substances (TBARS) assay was performed to determine level of
malondialdehyde in the pancreas according to the protocol of
Draper and Hadley.
13
Briefly, the reaction mix which consisted of
0.5 mL pancreatic homogenate, 10% PTA and TBA was incubated in a
boiling water bath, cooled and centrifuged. Absorbance of the pink
chromogen formed was measured at 540 nm to determine the level
of malondialdehyde formation which was expressed as nM/100 mg
tissue protein. Estimation of 4-HNE (4-hydroxynonenal) in the
pancreas was done following the method of Kinter et al.
14
where, 4-
HNE in the sample was derivatized with dinitrophenyl hydrazine
(DNPH). The product formed was extracted 3 times with hexane,
evaporated to dryness and solubilized with methanol prior to
measurement of absorbance at 350 nm. Levels of 4-HNE in the
samples was expressed as
m
M/mg tissue protein.
2.4.5.7. Estimation of reduced glutathione and antioxidant enzymes.
GSH level in the pancreas was measured following the protocol of
Moron et al.
15
DTNB was added to the pancreatic homogenate and
the yellow complex formed was estimated spectrophotometrically
at 412nm and the level was expressed as mg/gm tissue protein.
Glutathione peroxidase (GPx) was assayed according to the method
of Floh
e and Günzler
16
where a specific volume of the enzyme
preparation was allowed to react with H
2
O
2
for a specified time
period and the remaining GSH was estimated by Ellman's reaction.
Activity expressed as nM of glutathione oxidized/min/mg protein.
Superoxide dismutase (SOD) activity was measured according to
the protocol of Kakker et al.
17
The inhibition of reduction of nitro-
blue tetrazolium (NBT) to blue coloured formazan in the presence
of phenazine methosulfate (PMS) and NADH was measured at
560 nm using n-butanol as blank. Following the method of Sinha
et al.,
18
catalase activity was measured as the amount of enzyme
required to decompose hydrogen peroxide in the presence of di-
chromate and acetic acid. Absorbance of chromic acetate formed
was measured at 620 nm. The enzyme activity was expressed as
m
M
of H
2
0
2
consumed/min/mg protein.
2.4.5.8. Estimation of protein. Bradford method
19
was used to
determine the protein concentration in the tissue homogenate. The
protein level was used to calculate the enzyme activity in tissue
homogenate.
2.4.6. Histological assessment
Freshly dissected pancreas was washed with ice-cold 0.9% saline
and fixed in 10% formo-saline for 24 h. The tissue specimens were
dehydrated with alcohol, cleaned with methyl benzoate and
embedded in paraffin wax. Sections in cryostat from snap frozen
tissues were cut into 5
m
M thickness and stained with haematox-
ylin and eosin for microscopic evaluation.
2.4.7. Statistical analyses
The statistics software package (SPSS for Windows V.10) was
used for the data analyses. The statistical significance of mean
values between different groups was determined by applying one
way ANOVA with post hoc Bonferroni test and the P value <0.05
was considered as significant.
3. Results
3.1. Phytochemical screening by GC-MS
GC-MS analysis revealed the presence of the following com-
pounds: 2H-1-Benzopyran-2-one (Coumarin), Eugenol, 5,7-
dihydroxyflavone (Chrysin), 5,7-dihydroxyisoflavone (Mefenamic
acid), 4
0
5,7-Trihydroxy isoflavone (Genistein), 4-H1-Benzopyran-4-
one, 5,7-dihydroxy-2-(4-hydroxyphenyl)- (Apigenin), 4
0
-methoxy-
5,7-dihydroxy isoflavone (Biochanin A), Psi-baptigenin, Rhein,
Morin, 5,7-dimethoxy flavone, Vitamin E, 8-glycosyl apigenin
(Vitexin). Table 1 lists out their retention times and bioactivities.
Fig. 1 shows the GC-MS chromatogram of MEMARB.
3.2. Phytochemical analysis of MEMARB by HPLC-UV analysis
Fig. 2 displays the HPLC-UV chromatograms. HPLC-UV chro-
matogram of MEMARB generated two peaks at 270 nm with
retention times 3.19 and 8.53 min (Fig. 2C). The peaks were iden-
tified to be Cudraflavone B (0.9 mg/gm; RT 3.19 min) and Quercetin
Y. Kavitha, A. Geetha / Journal of Traditional and Complementary Medicine 8 (2018) 497e505 499
(0.1 mg/gm; RT 8.53 min), by matching the retention times with
those of the reference standards run under the same conditions
(Fig. 2A and B).
3.3. Effect of MEMARB on the levels of serum lipase and serum
alpha-amylase
Table 2 shows the levels of serum lipase and serum alpha-
amylase in the experimental groups. Rats administered ethanol
and cerulein showed significant elevation in the levels of serum
lipase and serum amylase when compared to the normal control
animals (p <0.05). Serum levels of these markers of pancreatic
injury reversed towards normal values in MEMARB treated rats
(p <0.05). Rats fed normal diet and MEMARB showed normal levels
of the enzymes.
3.4. Effect of MEMARB on serum and tissue inflammatory markers
Table 3 charts out the activity levels of MPO and caspase-1 in the
pancreas and the levels of caspase-1, IL-1beta, and IL-18 in the
serum of the experimental animals. Notable increase in the levels of
these inflammatory markers was observed in the ethanol and
cerulein administered rats when compared to rats co-administered
MEMARB. A non-significant reduction of the inflammatory markers
was seen in the MEMARB control group.
Table 1
List of compounds identified in the methanolic extract of Morus alba root bark by GC-MS.
No. RT
(min)
Name of the compound Molecular
formula
Molecular Weight
(g/mol)
Bioactivity
a
,
b
1 12.1 2H-1-Benzopyran-2-one (Coumarin) C
9
H
6
O
2
146.14 Venotonic
2 14.5 Eugenol C
10
H
12
O
2
164.2 Improves cell-mediated immunity, local antiseptic
3 15.72 Chrysin C
15
H
10
O
4
254.24 Antiinflammatory, antioxidative, anti-hypercholesterolemic
4 15.72 5,7-dihydroxyisoflavone (Mefenamic acid) C
15
H
10
O
4
254.23 Anti-inflammatory, inhibitor of prostaglandin synthesis,
analgesic,anti-pyretic
5 16.75 4
0
,5,7-Trihydroxy isoflavone (Genistein) C
15
H
10
O
5
270.24 Anti-angiogenic, vasculoprotective
6 17.1 Apigenin C
15
H
10
O
5
270.4 Anti-inflammatory, anti-proliferative, anti-metastatic
7 17.57 Biochanin A C
16
H
12
O
5
284.27 Anti-inflammatory,antidiabetic, anticarcinogenic, lipid
metabolism regulator
8 17.57 Pseudobaptigenin C
16
H
10
O
5
282.24 Nutrient isoflavone
9 17.75 Rhein (Cassic acid) C
15
H
8
O
6
284.22 Anti-microbial, antibiotic, apoptotic, antiproliferative
10 18.55 Morin C
15
H
10
O
7
302.24 Anti-inflammatory, antihypertensive, anti-angiogenic,
hepatoprotective, neuroprotective
11 18.8 5,7-dimethoxy flavone C
17
H
14
O
4
282.29 Anti-proliferative, apoptotic
12 19.7 Vitamin E C
29
H
50
O
2
430.71 Anti-oxidant
13 20.63 1,6-Heptadiene-3,5 dione,1,7-bis(4-hydroxy-3-methoxy
phenyl)-(Curcumin)
C
21
H
20
O
6
368.38 Anti-inflammatory, anti-oxidant, antimicrobial, anticancer
14 20.67 Vitexin C
21
H
20
O
10
432.38 Anti-inflammatory, anti-cancer, neuroprotective
a
Pubchem.
b
Human Metabolome Database (Ref).
41
Fig. 1. Total Ion Chromatogram generated from the GC-MS analysis of the methanolic extract of Morus alba root bark: 1) 2H-1-Benzopyran-2-one (Coumarin) 2) Eugenol 3) 5,7-
dihydroxyflavone (Chrysin) 4) 5,7-dihydroxyisoflavone (Mefenamic acid) 5) 405,7-Trihydroxy isoflavone (Genistein) 6) 4-H1-Benzopyran-4-one, 5,7-dihydroxy-2-(4-
hydroxyphenyl)- (Apigenin) 7) 40-methoxy-5,7-dihydroxy isoflavone (Biochanin A) 8) Psi-baptigenin 9) Rhein 10) Morin 11) 5,7-dimethoxy flavone 12) Vitamin E 13) 8-glycosyl
apigenin (Vitexin).
Y. Kavitha, A. Geetha / Journal of Traditional and Complementary Medicine 8 (2018) 497e505500
3.5. Effect of MEMARB on the redox status elevels of TBARS, 4-HNE
and reduced glutathione (GSH)
Table 4 displays the levels of lipid peroxidation products and the
level of the antioxidant glutathione in the pancreatic tissue. In-
crease in the levels of lipid peroxidation products were found to be
highly significant in the ethanol and cerulein administered exper-
imental group while the level of the molecular antioxidant GSH was
found to be decreased, when compared to the normal rats. MEM-
ARB administration markedly reduced the levels of TBARS and 4-
HNE and increased levels of GSH. Levels of TBARS, 4-HNE and
GSH showed no significant change in MEMARB control rats.
3.6. Effect of MEMARB on the redox status eactivity levels of
antioxidant enzymes
Activity levels of the antioxidant enzymes, GPx, SOD and cata-
lase are shown in Table 5. In the ethanol and cerulein-induced
pancreatitis group, levels of GPx, SOD and catalase were signifi-
cantly depleted relative to the normal control group. No significant
changes were observed in the MEMARB control animals. In the
Fig. 2. High Performance Liquid Chromatography (HPLC) chromatograph of (A) reference standards; (B) Cudraflavone B standard; (C) MEMARB. Conditions: Mobile phase:
methanol: acetic acid: water (100:100:1); flow rate: 1.5 ml/min; Run time: 40 min. Wavelength: 270 nm.
Table 2
Effect of MEMARB on the levels of serum lipase and serum alpha-amylase.
Group Lipase (IU/L) Amylase (IU/L)
Normal Control 182 ±19 2732.21 ±360.65
MEMARB Control 156 ±16
NS
2614.02 ±316.3
NS
EtOH þCerulein 425 ±50* 4772.47 ±629.97*
EtOH þCerulein þMEMARB 236 ±31* 3198.78 ±422.24*
Data were analysed by one-way ANOVA followed by post-hoc Bonferroni test.
Values are expressed as mean ±S.D. of 6 rats in each group. Statistical significance
was calculated by comparing normal control vs. MEMARB control; Control vs.
EtOH þCerulein; EtOH þCerulein vs. EtOH þCerulein þMEMARB. *p ¼0.000; NS
non-significant.
Y. Kavitha, A. Geetha / Journal of Traditional and Complementary Medicine 8 (2018) 497e505 501
experimental group co-administered with MEMARB, the levels of
the antioxidant enzymes were restored.
3.7. Effect of MEMARB on the histology of the pancreatic and liver
tissue
Fig. 3 and Fig. 4 show photomicrographs of hematoxylin and
eosin stained sections (400X) of the pancreas and the liver
respectively. Pancreatic and liver sections from normal control and
MEMARB control rats showed normal tissue architecture (Fig. 3 (a,
c); Fig. 4 (a, c)). Section of the pancreas from the ethanol and cer-
ulein administered group showed neutrophil infiltration and
fibrotic changes (Fig. 3 (c)) and the liver section from the same
group showed tissue infiltration of neutrophils (Fig. 4c). Tissue ar-
chitecture of the pancreas (Fig. 3d) and the liver (Fig. 4d) was found
to be restored to normal in MEMARB treated experimental group.
4. Discussion
In this investigation, the anti-inflammatory and antioxidant
capacity of MEMARB was evaluated in vivo in the best-
characterised ethanol and cerulein-induced rat model of experi-
mental pancreatitis.
Supramaximal doses of cerulein, an analogue of the physiolog-
ical secretagogue cholecystokinin, stimulates protein-rich pancre-
atic secretions and disturbs the actin cytoskeleton and vesicular
transport, thereby suppressing the export of digestive enzymes.
This precipitates premature zymogen activation within the acinar
cells with subsequent tissue injury and pathology similar to human
pancreatitis. Ethanol when co-administered potentiates the effect
of cerulein.
20
Ethanol is the principal etiological factor for acute and
Table 3
Effect of MEMARB on serum and tissue inflammatory markers.
Group Caspase-1 MPO IL-1beta IL-18
Serum (pg/ml) Pancreas (pM/mg protein) Pancreas (Units/mg protein) Serum pg/ml Serum pg/ml
Normal Control 12.6 ±1.8 12.6 ±1.5 2.16 ±0.4 15.5 ±2.6 200.5 ±30.1
MEMARB Control 11.5 ±1.3
NS
10.3 ±1.2
NS
1.82 ±0.2
NS
13.6 ±2.1
NS
170 ±21.6
NS
EtOH þCerulein 21.5 ±2.8* 49.6 ±5.2* 3.98 ±0.4* 31.5 ±4.5* 360.5 ±42*
EtOH þCerulein þMEMARB 14.3 ±1.8* 20.5 ±2.8* 2.56 ±0.3* 20.5 ±3.1* 240 ±32*
Data were analysed by one-way ANOVA followed by post-hoc Bonferroni test. Values are expressed as mean ±S.D. of 6 rats in each group. Statistical significance was calculated
by comparing normal control vs. MEMARB control; Control vs. EtOH þCerulein; EtOH þCerulein vs. EtOH þCerulein þMEMARB. *p ¼0.000; NS non-significant.
Table 4
Effect of MEMARB on the redox status elevels of TBARS, 4-HNE and reduced glutathione (GSH).
Group TBARS (nM/100 mg protein) 4-HNE (
m
M/g protein) GSH (mg/g protein)
Pancreas Pancreas Pancreas
Normal Control 1776.00 ±225.55 2.000 ±0.24 96.015±11.62
MEMARB Control 1782.40 ±190.72
NS
2.13 ±0.21
NS
98.82 ±12.06
NS
EtOH þCerulein 3408.12 ±415.8* 8.100 ±0.96* 49.600 ±6.00*
EtOH þCerulein þMEMARB 1806.66 ±229.44* 5.062 ±0.60* 93.330 ±11.29*
Data were analysed by one-way ANOVA followed by post-hoc Bonferroni test. Values are expressed as mean ±S.D. of 6 rats in each group. Statistical significance was calculated
by comparing normal control vs. MEMARB control; Control vs. EtOH þCerulein; EtOH þCerulein vs. EtOH þCerulein þMEMARB. *p ¼0.000; NS non-significant.
Table 5
Effect of MEMARB on the redox status - Activity levels of antioxidant enzymes.
Group GPx (nM GSH consumed/min/mg protein) SOD (U/mg protein) Catalase (U/mg protein)
Pancreas Pancreas Pancreas
Normal Control 324.5 ±42.5 14.2 ±1.49 114.4 ±15.90
MEMARB Control 339.2 ±40.02
NS
14.5 ±1.65
NS
112.1 ±16.37
NS
EtOH þCerulein 233.6 ±29.43* 8.2 ±0.97* 72.8 ±7.79*
EtOH þCerulein þMEMARB 353.33 ±46.29* 11.7 ±1.51* 106.3 ±12.65*
Data were analysed by one-way ANOVA followed by post-hoc Bonferroni test. Values are expressed as mean ±S.D. of 6 rats in each group. Statistical significance was calculated
by comparing normal control vs. MEMARB control; Control vs. EtOH þCerulein; EtOH þCerulein vs. EtOH þCerulein þMEMARB. *p ¼0.000; NS non-significant.
Fig. 3. Histology of the pancreas (H&E stain, 40 0X): (a) &(b) Photomicrograph of
pancreas from normal control and MEMARB control rats with intact acini and islets. (c)
Photomicrograph of pancreas from ethanol and cerulein administered rats showing
neutrophil infiltration and fibrosis. (d) Photomicrograph of pancreas from MEMARB
co-administered rats showed restored tissue architecture. H&E: Haematoxylin &Eosin;
AC: acini; IS: islet; IL &F: inflammation and fibrosis.
Y. Kavitha, A. Geetha / Journal of Traditional and Complementary Medicine 8 (2018) 497e505502
chronic pancreatitis with multifarious effects on the pancreas.
Pancreas metabolizes ethanol via oxidative and non-oxidative
pathways generating the toxic by-products, acetaldehyde and
fatty acid ethyl esters (FAEE), respectively. Acetaldehyde activates
pancreatic stellate cells (PSCs) promoting fibrosis while both
ethanol and acetaldehyde regulate transcription factors NF-kappaB
and AP1. FAEEs promote accumulation of cholesteryl esters leading
to lysosomal membrane fragility, destabilization of zymogen
granule membranes and cause a sustained increase in intra acinar
calcium levels. Recent research also indicates that ethanol in-
fluences cholinergic pathways and CFTR channels in the pancreatic
ductal epithelium.
21e23
Presently, there being no specific and effective medication to
treat highly morbid pancreatic inflammation, it becomes necessary
to seek potential therapeutic agents. The diverse array of secondary
metabolites present in plants has become the focus of research on
inflammation. According to recent reports, apart from their anti-
oxidant properties, polyphenols including flavonoids have been
found to have far-reaching modulatory effects on key mitochon-
drial pathways thus protecting the cell from the toxic effects of
xenobiotics and ROS.
24
Root bark of Morus alba, a highly valued traditional Chinese
medicine, is rich in Diels-Alder type adducts, stilbenes, alkaloids
and flavonoids.
25
Prenylated flavonoids, a sub-class of flavonoids
abundant in Morus spp., have a lipophilic prenyl side-chain attached
to the flavonoid skeleton. Prenylation is said to confer flavonoids
with improved bioactivities.
9
Morus alba root bark has been shown
to have significant antioxidant, anti-inflammatory and anti-cancer
properties.
26
Cudraflavone B, a prenylated flavone from the root
bark of Morus alba is credited with potent anti-inflammatory
properties. Current literature indicates that Morus alba root bark
is a rich source of this bioactive principle which could be a potential
anti-inflammatory drug lead.
27
GC-MS is the method of choice for the screening of plant
bioactive constituents. GC-MS results showed the presence of a
variety of phytonutrients (Fig. 1;Table 1). HPLC-UV methods are
widely used to identify and quantify prenyl flavonoids in biological
samples. Cudraflavone B is reported to be a major prenyl flavonoid
bioactive in M.alba root bark, with impressive anti-inflammatory
effects.
28
HPLC-UV analysis of MEMARB (Fig. 2) confirmed the
presence of a significant quantity of this compound in the sample
and could be responsible for its anti-inflammatory properties. HPLC
analysis also revealed the presence quercetin, a potent anti-
inflammatory, anti-oxidant and immunomodulatory flavonoid.
29,30
Significant increase in the serum levels of the digestive en-
zymes, lipase and pancreatic alpha-amylase is seen in pancreatic
diseases.
31
Serum lipase and pancreatic alpha-amylase levels were
measured to assess the extent of injury to the pancreatic tissue
caused by EtOH and cerulein administration and the pancreato-
protective effect of MEMARB. Both serum lipase and pancreatic
alpha-amylase levels were found to be increased in EtOH and cer-
ulein administered animals. The amount of these enzymes was
restored to levels close to normal physiological levels in MEMARB
treated rats underlining the pancreato-protective potency of
MEMARB.
Inflammatory pathways underpin the basic pathology of
pancreatitis. Sterile inflammation in pancreatitis leads to the acti-
vation of NLRP3 inflammasome components caspase e1 and its
effectors- IL-1beta, and IL-18, the key determinants of the extent of
injury.
32,33
Serum levels of caspase-1 and the pro-inflammatory
cytokines, IL-1beta and IL-18 are determined to evaluate the
progress of inflammation. Levels of the pro-inflammatory cytokines
and caspase-1 were found to be higher than normal in pancreatitis-
induced rats. MEMARB co-administered rats showed near-normal
levels of these markers of inflammation adding further evidence
to the anti-inflammatory property of MEMARB. Oxyresveratrol,
another major bioactive in white mulberry root bark, has been
shown to modulate inflammatory responses by inhibiting MEK/ERK
pathway that leads to the activation of pro-inflammatory cyto-
kines.
34
So, along with cudraflavone B, oxyresveratrol could syn-
ergistically contribute to the anti-inflammatory nature of Morus
alba. Oxyresveratrol from Morus bombycis, a Japanese mulberry, has
been experimentally proven to attenuate NLRP3 activation which
could be linked to its anti-inflammatory potential
35
and could ac-
count for the NLRP3 attenuating effect of MEMARB.
Neutrophil infiltration is a prominent feature of inflammation in
any tissue. Acinar cell injury leads to the release of cytokines that
recruits neutrophils. Myeloperoxidase is expressed primarily by the
phagocytic neutrophil granulocytes. It generates the powerful
oxidant HOCl from H
2
O
2
and Cl
with bactericidal properties.
Myeloperoxidase activity is measured to assess the extent of
neutrophil infiltration in pancreatic tissue.
36
Our results show sig-
nificant elevation in MPO activity in the pancreas of EtOH and
cerulein administered rats which could mirror pancreatic tissue
inflammation induced by these molecules. This increase in in-
flammatory activity is countered in MEMARB co-administered an-
imals as evidenced by a fall in MPO activity in the pancreas.
MEMARB bio constituents and cudraflavone B could be implicated
in this anti-inflammatory effect.
Reactive oxygen and reactive nitrogen species (ROS/RNS) play
key roles in the pathogenesis of acute and chronic pancreatitis.
Pancreatic acinar cells contain phase I cytochrome P450 (CYP 450)
enzymes and phase II conjugation proteins for the metabolism of
xenobiotics. Oxidative stress may result from xenobiotic overload,
including alcohol, tobacco smoke, dietary toxins etc., and the
increased activity of these enzymes, leading to overwhelming in-
crease in ROS. Free radicals may deplete the endogenous antioxi-
dant molecules (mainly GSH) and the major antioxidant enzymes,
glutathione peroxidase (GP
x
), Superoxide dismutase (SOD) and
catalase and may also lead to an increase in the peroxidation of
cellular components, mainly unsaturated lipids. In this study, the
redox status of pancreatic tissue in diseased rats versus the
MEMARB-treated rats was determined by measuring the levels of
Fig. 4. Histology of the liver (H&E stain, 400X): (a) &(b) Photomicrograph of liver from
normal control and MEMARB control rats with normal tissue structure (c) Photomi-
crograph of liver from ethanol and cerulein administered rats showing neutrophil
infiltration (d) Photomicrograph of liver from MEMARB co-administered rats with
reduced inflammatory cell infiltration. H&E: Haematoxylin &Eosin; PT: portal triad;
HP: hepatocytes; CV: central vein; IL: inflammation.
Y. Kavitha, A. Geetha / Journal of Traditional and Complementary Medicine 8 (2018) 497e505 503
GSH, GP
x,
SOD, catalase, TBARS and 4-HNE. Depletion of antioxidant
enzymes GP
x,
SOD, and catalase was seen in the pancreatic tissue
and blood of the EtOH and cerulein administered rats. The levels
were restored in MEMARB co-administered rats. A significant drop
in the molecular antioxidant GSH and an elevation in the lipid
peroxidation products malondialdehye and 4-HNE were noted in
the results obtained from the pancreatitis-induced group. With
MEMARB treatment the level of GSH increased and the levels of
malondialdehye and 4-HNE decreased to relatively normal levels.
MEMARB with a large number of polyphenols like quercetin could
be responsible for this antioxidant effect.
37,38
Oxyresveratrol from
Morus alba root cortex is a potent lipid peroxidation inhibitor evi-
denced by the inhibitory effect against FeSO4/H2O2-induced
microsomal peroxidation. It has also been shown to scavenge
DPPH radical in vitro.
39
A present review indicates that the root
barks of Morus plants contain diverse polyphenols such as Diels-
Alder-type adducts, derivatives of benzofuran, stilbenes and poly-
hydroxylated alkaloids in addition to flavonoids.
40
Generally poly-
phenols are hydroxyl group containing polar compounds which can
donate the protons to neutralize the toxic free radicals. Hence, the
antioxidant potential of the test compound could be attributed to
the presence of polyphenols.
Histopathological observations of pancreas and liver sections
from the respective experimental groups reinforce findings from
the biochemical investigations. When compared to the normal
control and the drug control groups, the liver and pancreas sections
from the diseased group show inflammatory infiltrates and the
pancreas shows fibrotic changes. Sections of liver and pancreas
from the MEMARB treated group show restored tissue architecture
with a significant reduction in leukocyte infiltration and only mild
fibrosis in the pancreas proving the pancreato-protective effect of
MEMARB.
5. Conclusion
This is the pilot study which authenticates the anti-
inflammatory activity of phytonutrients present in the root bark
extract of Morus alba. The anti-inflammatory property observed in
this study might be due to the effect of flavonoids which could
intervene in the formation of cytokines. This study represents the
preventive effect of Morus alba on the development of pancreatitis.
However, as a future perspective, the same study can be applied on
animals with fully developed pancreatitis which would prove its
curative effect.
Conflicts of interest
The authors declare no competing interests.
Funding source
This research did not receive any specific grant from funding
agencies in the public, commercial or not-for-profit sectors.
Acknowledgements
We are grateful for the generous gift of Cudraflavone B from Dr.
Karel Smejkal, Associate Professor, Department of Natural Drugs,
Faculty of Pharmacy, University of Veterinary and Pharmaceutical
Sciences, Brno, Czech Republic.
The authors thank the Sophisticated Analytical Instruments
Facility, Indian Institute of Technology-Madras, Chennai, India, for
providing the infrastructure and technical support for the GC-MS
analysis.
References
1.. Sah RP, Saluja AK. Molecular mechanisms of pancreatic injury. Curr Opin
Gastroenterol. 2012;28(5):507e515.
2.. Machicado JD, Rebours V, Yadav D. Epidemiology of Chronic Pancreatitis.
American Pancreatic Association Pancreapedia. 2016;1:1e15.
3.. Ammann RW. Diagnosis and management of chronic pancreatitis: current
knowledge. Swiss Med Wkly. 2006;136:166e174.
4.. Yadav D, Lowenfels AB. The epidemiology of pancreatitis and pancreatic can-
cer. Gastroenterology. 2013;144(6):1252e1261.
5.. Gu H, Werner J, Bergmann F, Whitcomb DC, Buchler MW, Fortunato F. Necro-
inflammatory response of pancreatic acinar cells in the pathogenesis of acute
alcoholic pancreatitis. Cell Death Dis. 2013;4, e816.
6.. Sendler M, Mayerle J, Lerch MM. Necroptosis, Apoptosis, Necroptosis, Pyrop-
tosis: it matters how cells die during pancreatitis. Cell Mol Gastroenterol Hep-
atol. 2016;2:407e408.
7.. Hoque R, Mehal WZ. Inflammasomes in pancreatic physiology and disease. Am
J Physiol Gastrointest Liver Physiol. 2015;308(8):G643eG651.
8.. Wagner H, Bauer R, Melchart D, Pei-Gen X, Staudinger A. Chromatographic
Fingerprint Analysis of Herbal Medicines: Thin-layer and High Performance Liquid
Chromatography of Chinese Drugs. Springer Science &Business Media; 2011:
535e548.
9.. Yang X, Jiang Y, Yang J, et al. Prenylated flavonoids, promising nutraceuticals
with impressive biological activities. Trends Food Sci Technol. 2015;44(1):
93e104.
10.. Chan EW, Phui-Yan LY, Siu-Kuin WO. Phytochemistry, pharmacology, and
clinical trials of Morus alba.Chin J Nat Med. 2016;14(1):17e30.
11.. Thornberry NA. Interleukin e1
b
converting enzyme. Methods Enzymol.
1974;244:615e631.
12.. Bradley PP, Christensen RD, Rothstein G. Cellular and extracellular myelo-
peroxidase in pyogenic inflammation. Blood. 1982;60(3):618e622.
13.. Draper HH, Hadley M. Malondialdehyde determination as index of lipid per-
oxidation. Methods Enzymol. 1990;186:421e431.
14.. Kinter M, Grimminger LC, Gillies PJ, Shimshick EJ, Ayers C. Whole blood and
plasma concentrations of 4-Hydroxy-2-nonenal in watanabe heritable
hyperlipidemic versus New Zealand white rabbits. Biochem Biophys Res Com-
mun. 1994;199(2):671e675.
15.. Moron MS, De Pierre JW, Vik BM. Levels of glutathione, glutathione reductase
and glutathione - S - transferase activities in rat, lung and liver. Biochim Bio-
phys Acta. 1979;582:3170e3185.
16.. Floh
e L, Günzler WA. Meth Enzymol. 1984;105(1):114e121.
17.. Kakkar P, Das B, Viswanathan PN. A modified spectrophotometric assay of
superoxide dismutase (1984). Indian J Biochem Biophys. 1984;21:130e132.
18.. Sinha AK. Colorimetric assay of catalase. Anal Biochem. 1972;47(2):389e394.
19.. Bradford MM. Rapid and sensitive method for the quantitation of microgram
quantities of protein utilizing the principle of protein-dye binding. Anal Bio-
chem. 1976;72:248e254.
20.. Aghdassi AA, Mayerle J, Christochowitz S, Weiss FU, Sendler M, Lerch MM.
Animal models for investigating chronic pancreatitis. Fibrogenesis Tissue
Repair. 2011;4(1):26.
21.. Vonlaufen A, Wilson JS, Pirola RC, Apte MV. Role of alcohol metabolism in
chronic pancreatitis. Alcohol Res Health. 2007;30(1):48e54.
22.. Lugea A, Gong J, Nguyen J, Nieto J, French SW, Pandol SJ. Cholinergic mediation
of alcohol-induced experimental pancreatitis. Alcohol Clin Exp Res.
2010;34(10):1768e1781.
23.. Mal
eth J, Balazs A, Pallagi P, et al. Alcohol disrupts levels and function of the
cystic fibrosis transmembrane conductance regulator to promote development
of pancreatitis (2015). Gastroenterology. 2015;148(2):427e439.
24.. Sandoval-Acu~
na C, Ferreira J, Speiski H. Polyphenols and Mitochondria: An
Update on their increasingly emerging ROS-scavenging independent actions.
Arch Biochem Biophys. 2014;559:75e90.
25.. Wang YN, Liu MF, Hou WZ, et al. Bioactive benzofuran derivatives from cortex
mori radicis, and their neuroprotective and analgesic activities mediated by
mGluR1. Molecules. 2017;22(2):236.
26.. Eo HJ, Park JH, Park GH, et al. Anti-inflammatory and anti-cancer activity of
mulberry (Morus alba L.) root bark. BMC Compl Alternative Med. 2014;14(1):
200.
27.. Ho
sek J, Bartos M, Chudík S, et al. Natural compound cudraflavone B shows
promising anti-inflammatory properties in vitro. J Nat Prod. 2011;74(4):
614e619.
28.. Kollar P, B
arta T, Ho
sek J, et al. Prenylated flavonoids from Morus alba L. cause
inhibition of G1/S transition in THP-1 human leukemia cells and prevent the
lipopolysaccharide-induced inflammatory response. Evid base Compl Alterna-
tive Med. 2013;13.
29.. Xu F, Nie X, Shi J, et al. Quercetin inhibits LPS-induced inflammation and ox-
LDL-induced lipid deposition. J Chromatogr Sci. 2004;42:378e382.
30.. Lin X, Lin CH, Zhao T, et al. Quercetin protects against heat stroke-induced
myocardial injury in male rats: antioxidative and anti-inflammatory mecha-
nisms. Chem Biol Interact. 2017;265:47e54.
31.. Frulloni L, Patrizi F, Bernardoni L, Cavallini G. Pancreatic hyperenzymemia:
clinical significance and diagnostic approach. JOP. 2005;6(6):536e541.
32.. Ramadani M, Gansauge F, Schlosser S, Yang Y, Beger HG, Gansauge S. Over-
expression of caspase-1 in pancreatic disorders: implications for a function
besides apoptosis. J Gastrointest Surg. 2001;5(4):352e358.
Y. Kavitha, A. Geetha / Journal of Traditional and Complementary Medicine 8 (2018) 497e505504
33.. Hoque R, Malik A, Gorelick F, Mehal W. The sterile inflammatory response in
Acute Pancreatitis. Pancreas. 2012;41(3):353e357.
34.. Chen YC, Tien YJ, Chen CH, et al. Morus alba and active compound oxy-
resveratrol exert anti-inflammatory activity via inhibition of leukocyte
migration involving MEK/ERK signalling. BMC Compl Alternative Med.
2013;13(1):45.
35.. Oh NH, Han JW, Shim DW, et al. Anti-inflammatory properties of Morus
bombycis Koidzumi via inhibiting IFN-
b
signaling and NLRP3 inflammasome
activation. J Ethnopharmacol. 2015;24(176):424e428.
36.. Sandoval D, Gukovskaya A, Reavey P, et al. The role of neutrophils and platelet-
activating factor in mediating experimental pancreatitis. Gastroenterology.
1996;111:1081e1091.
37.. Shahedi K, Pandol SJ, Hu R. Oxidative stress and alcoholic pancreatitis.
J Gastroenterol Hepatol Res. 2013;2(1):335e342.
38.. Bhardwaj P, Yadav RK. Chronic pancreatitis: role of oxidative stress and an-
tioxidants. Free Radic Res. 2013;47(11):941e949.
39.. Chung KO, Kim BY, Lee MH, et al. In-vitro and in-vivo anti-inflammatory effect
of oxyresveratrol from Morus alba L. J Pharm Pharmacol. 2003;55(12):
1695e1700.
40.. Wei H, Zhu JJ, Liu XQ, Feng WH, Wang ZM, Yan LH. Review of bioactive
compounds from root barks of Morus plants (Sang-Bai-Pi) and their phar-
macological effects. Cogent Chem. 2016;2(1), 1212320.
41.. Wishart DS, Jewison T, Guo AC, et al. HMDB 3.0-the human Metabolome
database in 2013. Nucleic Acids Res. 2013;41(D1):D801eD807.
Y. Kavitha, A. Geetha / Journal of Traditional and Complementary Medicine 8 (2018) 497e505 505
