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Journal of Traditional and Complementary Medicine Vo1. 4, No. 2, pp.

108117
Copyright 2014 Committee on Chinese Medicine and Pharmacy, Taiwan This is an open access article under the CC BY-NC-ND license.


Journal of Traditional and Complementary Medicine
Journal homepage http://www.jtcm.org

Antidiabetic Activity of Polyherbal Formulation in


Streptozotocin Nicotinamide Induced Diabetic Wistar Rats
Rajendran Ramesh Petchi1, Chockalingam Vijaya1, Subramani Parasuraman2

Department of Pharmacology, Ultra College of Pharmacy, Madurai 625020, Tamil Nadu, India.
1

Unit of Pharmacology, Faculty of Pharmacy, AIMST University, Bedong 08100, Kedah, Malaysia.
2

ABSTRACT

Glycosmis pentaphylla, Tridax procumbens, and Mangifera indica are wellknown plants available throughout India and they are commonly
used for the treatment of various diseases including diabetes mellitus. The antidiabetic activity of the individual plant parts is well known,
but the synergistic or combined effects are unclear. The concept of polyherbalism has been highlighted in Sharangdhar Samhita, an
Ayurvedic literature dating back to 1300 AD. Polyherbal formulations enhance the therapeutic action and reduce the concentrations of
single herbs, thereby reducing adverse events. The aim of the present study is to formulate a polyherbal formulation and evaluate its
antidiabetic potential in animals. The polyherbal formulation was formulated using the ethanol extracts of the stem bark of G.pentaphylla,
whole plant of T.procumbens, and leaves of M.indica. The polyherbal formulation contains the ethanol extracts of G.pentaphylla,
T.procumbens, and M.indica in the ratio of 2:2:1. The quality of the finished product was evaluated as per the World Health Organizations
guidelines for the quality control of herbal materials. The quality testing parameters of the polyherbal formulation were within the limits.
Fingerprint analysis of the polyherbal formulation showed effective separation at 366nm, and it revealed that the active compound present
in the polyherbal formulation and the active compounds present in all the three extracts were the same. The acute toxicity studies of the
polyherbal formulation did not show any toxic symptoms in doses up to 2000mg/kg over14days. The oral antidiabetic activity of the
polyherbal formulation(250 and 500mg/kg) was screened against streptozotocin(50mg/kg; i.p.) + nicotinamide(120mg/kg; i.p.) induced
diabetes mellitus in rats. The investigational drug was administered for 21 consecutive days, and the effect of the polyherbal formulation
on blood glucose levels was studied at regular intervals. At the end of the study, the blood samples were collected from all the animals for
biochemical estimation, and the animals were sacrificed and the liver and pancreatic tissues were collected for histopathologic analysis.
Polyherbal formulation showed significant antidiabetic activity at 250 and 500mg/kg, respectively, and this effect was comparable with
that of glibenclamide. The antidiabetic activity of polyherbal formulation is supported by biochemical and histopathologic analysis.

Key words: Glycosmis pentaphylla, High Performance Thin Layer Chromatography fingerprint analysis, Mangifera indica,
Polyherbal formulation, Tridax procumbens

which, in one or more of its organs, contains substances that


INTRODUCTION
can be used for therapeutic purposes, or which are precursors
Plants are very useful to mankind. Many of them are used for chemopharmaceutical semisynthesis. Such plants are in
exclusively for medicinal purposes. According to the World great demand by pharmaceutical companies for their active
Health Organization(WHO), a medicinal plant is a plant ingredients.[1,2]

Correspondence to:
Mr. Ramesh Petchi R, Department of Pharmacology, Ultra College of Pharmacy, Madurai, India. Phone No. +91 81438 02251; E-mail: rameshpetchi28@gmail.com
DOI: 10.4103/2225-4110.126174

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Petchi, etal. / Journal of Traditional and Complementary Medicine 4 (2014) 108117

Diabetes mellitus is one of the most common disorders af (CPCSEA) guidelines, Ministry of Environment and Forests,
fecting almost 6% of the world population and the dynamics of Government of India.
the diabetes are changing rapidly in lowto middleincome coun
tries.[3] According to International Diabetes Federations (IDF) Preparation of ethanolic extract of G. pentaphylla,
estimates, 80% of the world diabetic population will be from T.procumbens, and M. indica plants
lowand middleincome countries in 2030. As per IDF 2011 The shadow, airdried stem bark of G.pentaphylla, whole
report, China, India, and the United States of America have a plant of T.procumbens, and leaves of M.indica were powdered
diabetic population of 90.0, 61.3, and 23.7 million, which may and extracted with 80% absolute ethanol using Soxhlet apparatus
be increased up to 129.7, 101.2, and 29.3 million, respectively, in for 6h. The extracts were evaporated to dryness(resinous mate
2030.[4] Globally, diabetes is one of the six major causes of death rial) under reduced pressure at 60C and stored at 4C until use.
and also causing various systemic complications. Diabetes mel
Phytochemical analysis
litus is treated by hormone therapy(insulin) or by administering
One gram of each of the ethanolic extracts of G. pentaphylla,
glucoselowering agents such as alphaglucosidase inhibitors,
T. procumbens and M. indica was dissolved in 100 mlof its own
sulfonylureas, biguanides, and thiazolidinediones. Development
mother solvent to obtain a stock of concentration 1%w/v and tested
of an adverse event is one of the complications in the treatment of
for the presence of carbohydrates, proteins, sterols, alkaloids, tan
any systemic disorder;hence, many of the research institutes and
nins, glycosides, flavonoids, phenolic compounds, and saponins.[7]
pharmaceutical companies are involved in drug development to
find the molecules with good therapeutic potential and less adverse Preparation of polyherbal formulation
events.[5] In the USA, 10-25% of patients experience an adverse The polyherbal formulation(capsules) contained the ethanolic
drug reaction and these adverse drug reactions are responsible for extracts of G.pentaphylla, T.procumbens, and M.indica in the
3.4-7.0% of hospital admissions.[6] ratio of 2:2:1. The quality of the polyherbal formulation was tested
In traditional systems of medicine, many plants have been as per the WHO guidelines for the quality control of herbal materi
documented to be useful for the treatment of various systemic als.[8] As per the guidelines, specific tests such as sampling, ash
disorders. Many of the traditional/indigenous systems of medi content, extractable matter, foaming index, loss on drying, tannin
cine are effective than the modern system of medicine, but they content, foreign matters, and specific powder characteristic tests
suffer from lack of complete standardization which is one of the such as angle of repose and bulk density were undertaken and
important challenges faced by the traditional system of medicine. significant results were recorded.
The concept of polyhedral formulation is well documented in the
ancient literature. Compared to the single herb, the polyherbal Preparation of formulation by wet granulation method
formulation has better and extended therapeutic potential. Hence, The formulation preparation began with trials by adding a
the present study was planned to formulate and standardize a different ratio of binders and selecting the quantity of lubricants
polyherbal formulation using a plant having known antidiabetic and preservatives, and finally the procedure was optimized.
activity and evaluate its therapeutic effects in rodents. G.pentaphylla, T.procumbens, and M.indica extracts were finely
powdered(sieve 40), and mixed in the ratio of 2:2:1 and taken for
MATERIALS AND METHODS the preparation of capsules by wet granulation technique using 20%
lactose solution as a binder. The wet mass was passed through sieve
Collection of the plant number 22 to obtain granules. The granules were dried at 45C in
Taxonomically identified stem bark of Glycosmis pentaphyl a tray dryer. The granules were lubricated with 1% magnesium
la(Rutaceae), whole plant of Tridax procumbens(Asteraceae), stearate. Diluents and preservatives were added. The optimized
and leaves of Mangifera indica(Anacardiaceae) were collected formulation showed very good flow properties. After this, the gran
from the Alagar kovil region, Madurai district. The collected ules from the optimized batch(20% lactose) were filled incapsules
plants were authenticated at the Department of Botany, American colored yellowred of size 0 in a capsule filling machine. The
college, Madurai, Tamil Nadu. The voucher specimen of the plant capsules were then deducted and transferred into poly bags, labeled,
was deposited in the Department of Pharmacology, Ultra College and the samples were evaluated as per the testing requirements.
of Pharmacy, Madurai, India for further reference. Each 750mg of herbal capsule contained the extracts of G.pen
taphylla(100mg), T.procumbens(100mg), M.indica(50mg),
Animals
and lactose and excipientsquantity sufficient(q.s.).
Adult Wistar rats(18010g) of either sex were obtained
from Sainath Enterprises, Hyderabad, India. The animals were Preformulation studies
housed in large, spacious polyacrylic cages at an ambient room Preformulation parameters such as bulk density, tap density,
temperature with 12h light/12h dark cycle. Rats had free access Carrs index, Hausners ratio, and angle of repose were determined
to water and rodent pellets diet(Hindustan Lever Ltd, Bangalore, for the laboratory granules.[9,10]
India). The study was approved by the Institute Animal Ethics
Committee of the Ultra College of Pharmacy and all the animal Standardization of formulation
experiments were carried out according to the Committee for the Physicochemical parameters of raw materials were determined
Purpose of Control and Supervision of Experiments on Animals as per the guidelines of the WHO, which includes moisture content,

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Petchi, etal. / Journal of Traditional and Complementary Medicine 4 (2014) 108117

total ash value, watersoluble ash, acidinsoluble ash, heavy solvent system. The plates were exposed to the mobile phase for
metals, watersoluble extractive, alcoholsoluble extractive, and 20min and the airdried plates were viewed in Ultraviolet light to
acidity(pH).[1113] mid-day light. The chromatograms were scanned by densitometer
pH value: pH of 1% solution was determined by using a digital at 254, 366, and 520nm. The Rf values, peak areas, and fingerprint
pH meter. data were recorded by winCATS 1.4.3 software. The Rf values
Ash value and extractive value: Total ash, water soluble ash, were calculated using the formula: distance traveled by the solute
acid soluble ash and extractive values were determined as the from the origin/distance traveled by the solvent from the origin.
procedure described elsewhere.[1112]
Limit test for heavy metals: Qualitative estimation of heavy Development of quality control standards for the polyherbal
metals was done for the detection of arsenic and lead as per the capsule
Ayurvedic pharmacopoeial procedures. The physiochemical(moisture content, limit test for heavy
Capsule evaluation: The polyherbal capsules wereevaluated metals, monographic analysis of plant material, pH, and disinte
for their description, microbial load, uniformity of dosage units, gration time) and microbial load analyses were performed for the
weight variation, disintegration time, and moisture content, and herbal capsules to ensure the content uniformity and quality.[12]
compared with Indian pharmacopoeial standards.
Acute oral toxicity
Microbial load analysis: For the safe use of the polyherbal
Acute oral toxicity of the polyherbal formulation was carried
capsules, microbial count was done and it was checked whether
out as per the guidelines set by the Organization for Economic
the total aerobic viable count, yeasts and molds were within the
Cooperation and Development(OECD), revised draft guidelines
prescribed limits and the microorganisms, Escherichia coli, Clos
423. The principle involves a stepwise procedure with the use of a
tridia, Salmonellae, Shigella, Pseudomonas, and Staphylococcus,
minimum number of animals per step to obtain sufficient information
were absent in the final formulation.
on the acute toxicity of the test substance to enable its classification.
Weight variation: Twenty capsules were individually weighed
Healthy Wistar rats (3 animals/dose) of either sex were used for the
and the average weight of the capsule was calculated. The indi
experiment. Overnight fasted rats were orally fed with the plant
vidual weights of the each capsule should be within the limits of
extracts and polyherbal formulation in increasing dose levels of 5,
90% and 110% of the average weight.
50, 300, and 2000mg/kg body weight, respectively. The animals
Moisture content: Moisture content was determined by using
were observed for their behavioral(alertness, restlessness, irritabil
automatic Karl Fischer titration apparatus.
ity, and fearfulness), neurological(spontaneous activity, reactivity,
Disintegration time: Disintegration test was performed us
touch response, pain response, and gait), and autonomic(defecation
ing the digital microprocessor based disintegration test appara and urination) profiles continuously for 24h. After a period of 24h,
tus(Electro lab, Mumbai, India). One capsule was introduced the animals were observed for 14days for mortality.[14]
into each tube and a disk was added to each tube. The assembly
was suspended in water in a 1000ml beaker. The volume of water Antidiabetic effect of herbal formulation in streptozotocin
at its highest point was at least 25mm below the surface of the and nicotimanideinduced diabetic rats
water and at its lowest point was at least 25mm above the bot The male Wistar rats were divided into five different groups
tom of the beaker. The apparatus was operated and maintained at of six animals each as follows.
a temperature of 372C. GroupI: Normal control
Highperformance thin layer chromatography(HPTLC) fin GroupII: Diabetic control
gerprint analysis: Randomly few capsules were selected. They GroupIII: Diabetic rats treated with polyherbal prepara
were opened and the contents were collected. The capsule content tion(250mg/kg)
was dissolved in a minimum volume of mobile phase and used GroupIV: Diabetic rats treated with polyherbal prepara
for the fingerprint analysis. Twenty milligram either individual tion(500mg/kg)
herbal extract or polyherbal formulation was reconstituted in 1ml GroupV: Diabetic rats treated with glibenclamide(0.25mg/kg).
of ethanol and filtered through a 0.45 m membrane filter. The Diabetes was induced in overnightfasted rats by adminis
filtrate was used for the HPTLC analysis. The butanol:glacial acetic tering single intraperitoneal(i.p.) injection of freshly prepared
acid:water in the ratio 4:1:1, and toluene:ethyl acetate:methanol streptozotocin(STZ) 50mg/kg b.w. followed by 120mg/kg of
in the ratio 7:2:1 was used as mobile phase for the development nicotimanide(NIC) in 0.1 M citrate buffer(pH4.5) in a volume
of chromatogram. The chromatogram developed using mobile of 0.5ml/kg b.wt.[15] Diabetes was confirmed in the STZ+NIC
phase consisting of toluene:ethyl acetate:methanol (7:2:1) has treated rats by measuring fasting blood glucose levels after 48h
showed good separation. of induction. After 24h of STZ+NIC injection, the rats were
Linomat IV automated TLC applicator CAMAG(Switzer given 5%w/v of glucose solution(2ml/kg b.w.) to prevent hypo
land), densitometerCAMAG TLC scanner 3, and CAMAG CATS glycemic mortality. Rats with fasting blood glucose of more than
4 software were used for fingerprint analysis. A2 l sample(8mm 200mg/dl were considered as diabetics and they were divided
band) was spotted(Track 1: G.pentaphylla, Track 2: M.indica, randomly into four different groups. The standard(glibenclamide)
Track 3: T.procumbens, and Track 4: Polyherbal formulation) and and herbal formulation were suspended in 1%w/v carboxymethyl
the chromatogram was developed in 2010cm twin trough glass cellulose(CMC) and administered once daily through oral gavage
chamber saturated with toluene: ethyl acetate: methanol(7:2:1) for 21 consecutive days. The blood samples were collected on 1st,

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7th, 14th, and 21stdays of the treatment, through the tail vein of of 0.1 N NaOH. To this, an aliquotof 4.5ml of alkaline copper
rats by pricking and were immediately used for the estimation of reagent was added and allowed to stand at room temperature for
blood glucose with a glucometer. Weekly body weight variations 10min. To this reaction mixture, 0.5ml of Folins phenol reagent
were monitored for all the experimental animals. was added and the blue color developed was read after 20min
At the end of the experiment, the blood sample was withdrawn at 640nm. Astandard curve was obtained with standard bovine
from all the experimental animals through retroorbital plexus albumin and was used to assay the tissue protein level of enzyme
puncture/posterior vena cava in plain and sodium ethylenediamine activity. Values were expressed as mg/g of tissue.[18]
tetraacetic acid(EDTA) tubes for biochemical analysis.[16] Finally Estimation of liver glycogen: The alkali extract of the tissue
the animals were sacrificed by diethyl ether anesthesia, and liver was prepared by digesting 50mg of fresh tissue with 3ml of 30%
and pancreatic tissues were excised and used for biochemical and potassium hydroxide solution in a boiling water bath for 15min.
pathological analysis. Part of the tissue sample was preserved in The tubes were cooled and a drop of 1 M ammonium acetate was
an icecold container for biochemical analysis and the remaining added to precipitate glycogen and kept in a freezer overnight for
was stored in 10% formalin solution for histopathologic analysis. complete precipitation. Glycogen was collected by centrifuging at
300rpm for 20min. Then the precipitate was dissolved by heating
Biochemical analysis and again the glycogen was reprecipitated by adding alcohol and
The whole blood sample was used for the estimation of glu 1 M ammonium acetate and centrifuged. The final precipitate was
cose(OneTouch Horizon glucometer; OrthoClinical Diagnostics, dissolved in saturated ammonium chloride solution by heating
Johnson and Johnson Company, USA), hemoglobin, and glycosyl in a boiling water bath for 5min. Aliquots of glycogen solution
ated hemoglobin(HbA1c). The plasma sample was used for the were taken up for suitable dilution and 4ml of anthrone reagent
estimation of insulin(radioimmunoassay kit; Diasorin, Italy). The was added by cooling the tubes in an ice bath. The tubes were
serum was used for the estimation of biochemical markers such as shaken well, covered with marble caps, and heated in a boiling
creatinine, urea, protein, liver glycogen, total serum cholesterol,
water bath for 20min. After cooling, the absorbance was read
serum triglyceride, high density lipoprotein(HDL)cholesterol,
at 640nm against water blank treated in a similar manner. The
serum glutamate oxaloacetate transaminase(SGOT), and serum
standard glucose solution was also treated similarly. The glyco
glutamate pyruvate transaminase(SGPT). The biochemical
gen content was calculated from the amount of glucose present
markers were measured using a Prietest EasylabBiochemistry
in the sample by multiplying with the factor 0.91, and expressed
Analyser(Robonik[India] Private Limited) and the LABKITS
as mg/100g of tissue.
enzymatic kits. The liver tissue homogenate was used for the
estimation of protein and glycogen. Histopathologic analysis
Estimation of glucose: Adrop of the whole blood sample was Part of the liver and pancreas tissue were preserved in 10%
used for measuring glucose using OneTouch Horizonglucometer, formalin for 2days. The liver and pancreas were dehydrated with
with gluco strips. alcohol(subsequently with 70, 80, 90%, and absolute alcohol)
Estimation of hemoglobin: Hemoglobin in the blood was es for 12h each. Again the tissues were cleaned by using xylene
timated by the method of Drabkin and Austin(1932). To 0.02ml for 15-20min and they were subjected to paraffin infiltration in
of blood, 5.0ml of Drabkins reagent was added, mixed well, and automatic tissue processing unit. The tissue blocks were prepared
allowed to stand for 10min. The solution was read at 540nm and the blocks were cut using microtome to get sections of thickness
together with the standard.[17] 5 m. The sections were taken on a microscopic slide on which
Estimation of HbA1c: Erythrocytes were washed with normal egg albumin(sticky substance) was applied and allowed for dry
saline, lysed with 5ml of water, and incubated at 37C for 15min. ing. Finally, the sections were stained with eosin(acidic stain) and
The contents were centrifuged, the supernatant was discarded, and hemotoxylin(basic stain).
0.5ml of saline was added. To 2ml of aliquot, 4ml of oxalate
hydrochloride solution was added and the contents were heated at Statistical analysis
100C for 4h, cooled, and precipitated with 2ml of 40% trichlo All the data were expressed as meanSEM. Statistical sig
roacetic acid(TCA). The mixture was centrifuged, and to 0.5ml nificance between the groups were tested using oneway analysis
of supernatant, 0.5ml of 80% phenol and 3ml of concentrated of variance(ANOVA) followed by Dunnetts ttest posthoc test.
sulfuric acid were added. The developed color was read at 480nm AP less than 0.5 was considered significant.
after 30min.
Working standards (10-50 g) were prepared using 1% fructose RESULTS
solution. Then, 0.5 ml of 80% phenol and 3ml of concentrated
sulfuric acid were added to the working standards and read at
480nm after 30min. The concentration of HbA1c was expressed Photochemical analysis
as mg/g of hemoglobin. The yield of ethanolic extracts of G.pentaphylla, T.procum
Estimation of protein: The liver tissue was homogenized bens, and M.indica was 25.6%, 20.6%, and 5.34%w/w(dry weight
in 20 mM TrisHCl and used for the estimation of protein. To basis), respectively. The phytochemical analysis of the ethanolic
0.5ml of tissue homogenate, 0.5ml of 10% TCA was added and extract of G.pentaphylla showed the presence of alkaloids, tannins,
centrifuged for 10min. The precipitate was dissolved in 1.0ml flavonoids, and saponins, of T.procumbens showed the presence

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of alkaloids, flavonoids, saponins, and phenolic compounds, and the moisture content, microbial load, limit test for heavy metals,
of M.indica showed the presence of alkaloids, tannins, flavonoids, monographic analysis of plant material, pH, and disintegration
and phenolic compounds. time were calculatedand these values were found to be within
normal limits.
Preformulation studies
Preformulation parameters such as bulk density, tap density, Fingerprint analysis of polyherbal formulation
Carrs index, Hausners ratio, and angle of repose were obtained Fingerprint analysis at 366nm showed good and effective
for the laboratory granules. The granules showed excellent flow compound separation than the analysis at 254 and 520nm. The
property. The results are presented in Table1. obtained Rf values and peak areas in densitometry TLC Scan
ner 3 at 366nm are shown in Table3. The fingerprint of 3D
Development of quality control standards for polyherbal display at 366nm and in densitometry is shown in Figure1,
capsule and visualization comparison spots of all the three extracts
Final batch samples of polyherbal capsules were analyzed for with polyherbalformulation at 254, 366, and 520nm are
its organoleptic characters, physico chemical parameters, limits for shown in Figure2. The fingerprint reports of all the four tracks
heavy metals and microbial load[Table2]. The study on capsules revealedthat the active compound present in the polyherbal
revealed that they were uniform in content and weight. Further, formulation and the active compounds present in all the three
extracts were the same.
Table1. Preformulation studies and results of flow properties The same Rf values and peak areas were obtained in the formu
Parameters Results lation when compared with the individual Rf values and peak areas
Bulk density 0.721 g/ml of herbal drugs. This shows that the marker compound present in
Tapped density 0.693 g/ml individual herbals extracts and the marker compounds present in
Carrs index 4.04% the formulation are the same. The visualization comparison spots
Hausners ratio 1.140.05 of all the three extracts with polyherbal formulation at 366nm
Angle of repose 19.03 are shown in Figure3.

Toxicity study of polyherbal formulation


Table2. Standardization of polyherbal capsule Acute toxicity studies did not show any mortality up to
Name of the test Observations 2000mg/kg given as single oral administration. Hence, the study
Organoleptic characters was carried out at the dose levels of 250 and 500mg/kg.
Description Brown color powder
filled in yellow cap/red Antidiabetic activity of the polyherbal formulation
body, 0 size capsule Throughout the study, the diabetic animals showed significant
Color Brown color powder reduction in body weight when compared to the control animals.
Odor Characteristic However, the polyherbal formulation and glibenclamide inhibited
Taste Bitter the diabetesinduced body weight reduction[Figure4].
Physicochemical parameters Diabetic control animals showed severe hyperglycemia com
pH 7.8
pared to normal animals. The mean blood glucose level in the
Moisture content 1.02%
diabetic control group on day 0 was 248.63 2.11 mg/dl and on
Average weight 506 mg
day 21 was 328.92 1.20 mg/dl. It was observed that the standard
Weight variation 490-520 mg
drug glibenclamide lowered the blood glucose level significantly,
Disintegration time (Mean SEM) 3min 20sec0.25
bringing it back to near normal level, whereas the polyherbal
Loss on drying 2.54%
capsule at 250mg/kg and 500mg/kg significantly(P<0.001)
Total ash 5.67%
Acidinsoluble ash 1.24%
decreased the fasting blood serum glucose level in the diabetic
Watersoluble ash 3.47%
rats on 7th,14th, and 21stdays, as compared to the diabetic control
Watersoluble extractive value 15.36% group. The results are presented in Table4.
Ethanolsoluble extractive value 13.41% Diabetic animals showed significant decrease in plasma insu
Limits for heavy metals lin, hemoglobin, and HbA1c levels when compared with control
Arsenic not more than 5 ppm Complies animals. Herbal formulation and glibenclamide reversed the
Lead not more than 10 ppm Complies insulin depletion in diabetic condition and also brought back the
Microbial load analysis hemoglobin and HbA1c levels to normal[Table5].
Total microbial count NMT 1000 cfu/g 112 cfu/g Diabetic animals showed significant reduction in liver glyco
Yeasts and molds Nil gen and total protein levels when compared to the control animals,
Presence of E.coli(should be absent) Absent whereas herbal formulation and glibenclamide treated animals
Presence of Salmonella(should be absent) Absent showed normal liver glycogen and total protein levels. The pre
Presence of Streptococcus(should be absent) Absent vention of depletion of glycogen in the liver tissue was possibly
Presence of Pseudomonas(should be absent) Absent due to stimulation of insulin release from the cells that activates
NMT: Not-more-than the glycogen synthase system. Effects of herbal formulation and

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Petchi, etal. / Journal of Traditional and Complementary Medicine 4 (2014) 108117

Figure 1. Fingerprinting of 3D display at 366 nm in densitometry detection (pink color: Glycosmis pentaphylla, blue color: Mangifera indica, green
color: Tridax procumbens, brown color: polyherbal formulation)

serum lipid profiles except HDL when compared to the control


animals, whereas the levels in the treatment group remained within
normal limits at the end of the study. Effects of herbal formula
tion and glibenclamide on the lipid profile of diabetic animals are
presented in Table7.

Histopathology results
The histopathologic analysis of pancreas revealed severe
congestion, huge decrease in the number of islets of Langerhans
and cells, and fibrosis and inflammatory cell infiltration into the
islets of Langerhans in STZand NICinduced hyperglycemic
rats. While the polyherbal formulation at the dose of 250mg/kg
and 500mg/kg showed mild congestion and mild decrease in the
number of islets of Langerhans with normal cell population,
Figure 2. Visualization comparison spots of all the three extracts with
polyherbal formulation (GP: Glycosmis pentaphylla, MI: Mangifera indicating significant amount of recovery. Glibenclamide treat
indica, TP: Tridax procumbens, PHF: polyherbal formulation) ment showed moderate congestion with moderate decrease in the
number of islets of Langerhans and cells and mild lymphocytic
infiltration[Figure5].
Table3. Rf values and peak areas at 366 nm
Histopathology of the liver[Figure6] in normal control ani
Rf value Area(AU) mals showed normal hepatic cells with wellpreserved cytoplasm,
Track Track nucleus, nucleolus, and central vein. In diabetic control animals,
1 2 3 4 1 2 3 4 the liver sections showed that the lobular architecture was main
0.07 0.12 0.04 0.05 574.6 3136.3 1112.1 419.3 tained, but there was also a severe fatty change, sinusoidal dilation
0.20 0.21 0.10 0.12 2317.9 2301.3 1993.3 2200.1 and congestion, mild portal inflammation, fibrosis, severe feathery
0.29 0.26 0.23 0.26 1896.9 2449.1 2121.9 4677.6 degeneration, and necrosis. Diabetic rats treated with herbal for
0.35 0.30 0.39 0.42 6592.0 3593.3 1094.2 9406.1 mulation showed hepatocytes with nearly normal appearance and
0.38 0.37 0.44 0.55 4356.8 10314.4 1724.0 3038.7 minimal necrosis. The sections of glibenclamide treated animals
0.42 0.51 0.69 0.70 3167.0 3088.6 1529.8 2693.0 showed normal hepatic cells and no abnormality.
0.45 0.68 0.82 0.85 1483.2 2192.6 556.0 8306.6
0.53 0.84 0.98 0.92 4302.7 1584.4 276.9 937.2 DISCUSSION
0.72 0.91 0.98 2365.4 3290.3 10342.7
0.84 0.98 11882.4 1856.5 The polyherbal formulation was formulated using the etha
0.91 602.0 nolic extracts of the stem bark of G.pentaphylla, whole plant of
0.98 6654.1 T.procumbens, and leaves of M.Indica, which are mixed properly
Track 1: Glycosmis pentaphylla; Track 2: Mangifera indica; Track 3: Tridax in 2:2:1 ratio. The antidiabetic activity of the individual plants has
procumbens; Track 4: Polyherbal formulation
been proven. The stem bark of G.pentaphylla showed significant
antidiabetic and antiarthritic activities against STZinduced diabe
glibenclamide on the liver and renal markers of diabetic animals tes and Freunds complete adjuvant induced arthritis, respectively,
are presented in Table6. in rats at the dose levels of 400 and 800mg/kg.[19] The 50% ethanol
The diabetic rats showed significant(P<0.001) increase in extract of the leaves of M.indica showed a significant hypogly

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Petchi, etal. / Journal of Traditional and Complementary Medicine 4 (2014) 108117

Figure 3. The chromatograms of all the four tracks at 366 nm (Track 1: Glycosmis pentaphylla, Track 2: Mangifera indica, Track 3: Tridax procumbens,
Track 4: polyherbal formulation)

Table4. The effect of polyherbal formulation on fasting blood glucose levels(mg/dl) in STZand NICinduced diabetic rats
Treatment Blood glucose level in mg/dl
0 day
th
7 day
th
14th day 21st day
Vehicle-normal control 90.20.60 94.181.34 92.141.27 91.311.10
Diabetic control 248.632.11 280.112.24 315.190.73 328.921.20
PHF 250 mg/kg 244.671.57*** 200.63.38*** 150.501.69*** 121.101.94***
PHF 500 mg/kg 242.242.66*** 193.22.04*** 142.211.91*** 117.202.12***
Glibenclamide 0.25 mg/kg 240.232.14*** 180.732.87*** 130.732.01*** 112.412.96***
PHF: Polyherbal formulation. Values are expressed as meanSEM(n=6). ***P<0.001 compared to diabetic control(oneway ANOVA followed by a
Dunnetts t test) STZ: Streptozotocin; NIC: Nicotimanide

cemic effect against STZinduced diabetes in rats at a dose of importance than ever before, mainly due to their efficacy and
250mg/kg.[20] In humans, the aqueous and alcoholic extracts of easy availability,[24] as well as less side effects as compared to the
the leaves of M.indica showed a significant hypoglycemic effect synthetic drugs.[25] These advantages have led the people move
at a dose level of 1g/kg/d.[21] Methanolic extract of the whole plant toward herbal preparations, for disease treatment and preven
of T. procumbens showed significant antidiabetic activity against tion, as they are claimed to displaysynergistic, potentiative, and
alloxaninduced diabetes in rats at 250 and 500mg/kg b.wt.[22] agonistic/antagonistic actions and the mixture of species in them
In some studies, the leaves of T. procumbens showed significant shows better therapeutic effect than either species on its own.[26]
antidiabetic potential in rodents.[23] The concept of polyherbalism has been highlighted in Sharangdhar
In the modern era, herbal formulations have gained greater Samhita, an Ayurvedic literature dating back to 1300 AD.[27] Poly

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Table5. Effect of polyherbal formulation on plasma insulin level in STZand NICinduced diabetic rats
Treatment Plasma insulin level(IU/ml) Hb(mg/dl) HbA1C(mg/g of Hb%)
0th day 7th day 14th day 21st day 21st day 21st day
Normal control 21.010.01 21.050.37 21.410.69 21.240.01 13.80.99 0.310.01
Diabetic control 6.800.01 5.101.98 4.840.54 4.150.42 5.320.31 1.950.60
PHF 250 mg/kg 7.001.54 12.710.40** 17.110.43*** 19.960.14*** 12.000.63** 0.530.04**
PHF 500 mg/kg 6.901.67 13.061.02** 18.691.03*** 20.680.21*** 13.400.57*** 0.440.06***
Glibenclamide 0.25 mg/kg 7.330.50 13.250.45** 19.000.36*** 20.940.68*** 13.700.89*** 0.410.05***
PHF: Polyherbal formulation; Hb: Hemoglobin; HbA1C: Glycosylated hemoglobin. Values are expressed as meanSEM(n=6). **P<0.01, ***P<0.001
compared to diabetic control animals(oneway ANOVA followed by a Dunnetts t-test) STZ: Streptozotocin; NIC: Nicotimanide

Table6. Effect of polyherbal formulation on serum creatinine, protein, urea, and liver glycogen levels in STZand NICinduced diabetic rats
Treatment Liver glycogen Total protein(mg/dl) Urea(mg/dl) Creatinine(mg/dl) SGOT(IU/l) SGPT(IU/l)
(g/100 mg wet tissue)
Normal control 5.721.12 9.650.21 220.11 0.430.11 100.332.91 81.141.00
Diabetic control 2.140.08 4.250.09 500.64 1.250.08 152.661.40 149.002.04
PHF 250 mg/kg 5.041.04*** 8.220.07* 240.61*** 0.510.71* 111.112.36*** 87.241.30***
PHF 500 mg/kg 5.310.61*** 8.410.66** 230.47*** 0.490.05** 107.322.36*** 85.431.88***
Glibenclamide 0.25 mg/kg 5.510.54*** 230.67*** 8.840.67** 0.400.09** 104.112.14*** 83.351.41***
PHF: Polyherbal formulation. Values are expressed as meanSEM(n=6). *P<0.05, **P<0.01, ***P<0.001 compared to diabetic control animals(oneway
ANOVA followed by a Dunnetts t-test) STZ: Streptozotocin; NIC: Nicotimanide

Table7. Effect of ethanolic extracts of the polyherbal formulation on serum lipids


Group Treatment Lipid profile (mg/dl)
Triglyceride Total cholesterol HDL LDL VLDL
I Vehicle-normal control 69.130.32 76.141.98 31.060.98 38.121.58 13.200.67
II Diabetic control 161.040.63 142.010.62 13.230.20 92.620.17 41.220.27
III PHF 250 mg/kg 65.100.46*** 80.110.52*** 27.640.95*** 42.100.08*** 15.120.77***
IV PHF 500 mg/kg 67.121.02*** 78.201.06*** 29.100.84*** 39.180.94*** 14.650.47***
V Standard(GC 0.25 mg/kg) 68.050.10*** 77.110.44*** 30.470.37*** 39.971.09*** 14.000.18***
PHF: Polyherbal formulation. Values are expressed as the meanSEM(n=6). *P<0.05, **P<0.01, ***P<0.001 compared to diabetic control animals
(oneway ANOVA followed by a Dunnetts t-test) HDL: High density lipoprotein; LDL: Low density lipoprotein; VLDL: Very low density lipoprotein

 lopathic system of medicine. Polyherbal formulation comprises


hundreds of unique or speciesspecific compounds and it is
 difficult to completely characterize these compounds. When
formulating a polyhedral formulation, there are chances for
 1RUPDO FRQWURO
chemical interaction and loss of a particular bioactive com
'LDEHWLF FRQWURO pound.[28] In the present study, single herbs, viz., G.pentaphylla,
 3+) PJNJ T.procumbens, M.indica, in the radio of 2:2:1 showed clear
3+) PJNJ compound separation.

*OLEHQFODPLGH PJNJ
The toxicity studies were carried out as per the OECD guide
lines. The polyherbal formulation did not show any mortality or
 adverse event up to 2000mg/kg. Hence, the study was carried out
WK GD\ WK GD\ WK GD\ VW GD\
at the dose levels of 250 and 500mg/kg.
Figure 4. Effect of polyherbal formulation on body weight in STZ- and STZ is toxic glycoside obtained from Streptomyces achromo
NIC-induced diabetic rats [values are expressed as the mean SEM genes, a grampositive bacterium. It accumulates in pancreatic
(n=6); PHF: Polyherbal formulation; ***P < 0.001 compared to control cells via the glucose transporter 2(GLUT2) and reduces their
(one-way ANOVA followed by a Dunnetts t-test)] expression. The alkylating properties of the STZ modify the bio
logical macromolecules, fragment DNA, and destroy the cells,
herbal formulations enhance the therapeutic action and reduce the causing insulindependent diabetes.[29] In the diabetic control
concentrations of single herbs, thereby reducing the adverse events. group, severe body weight loss was observed, which may be due
The HPTLC analysis of polyherbal formulation was carried to increased muscle wasting and loss of tissue proteins.[30] In the
out at the wavelengths of 254, 366, and 520nm. The quality present study, the treatment groups showed significant improve
control system for the herbal preparation differs from the al ment in body weight, which indicates that polyherbal formulation

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Figure 5. Histopathology of pancreas in rats [photomicrograph of


hematoxylin and eosin (H and E) stained paraffin section from the
pancreas (400); D: Damage, I: Islet cells, N: Nuclei, R: Regeneration,
PHF: Polyherbal formulation] Figure 6. Histopathology of liver in rats [photomicrograph of hematoxylin
and eosin (H and E) stained paraffin section from the liver (400); N:
and glibenclamide prevent the hyperglycemiainduced muscle normal, NI: Necrosis and inflammation, R: Regeneration, PHF: Polyherbal
wastage. The reduction in glucose levels may be due to increase in formulation]
plasma insulin levels or enhanced transport of blood glucose in the
peripheral tissue.[31] Our study gives evidence that the polyherbal peroxidation of unsaturated fatty acids.[34] The polyherbal formula
formulation increases the plasma insulin levels and has promising tion treated animals inhibited the hyperglycemia induced by STZ,
antidiabetic activity. which may be due to the free radical scavenging properties of the
Diabetic animals showed enhanced levels of HbA1c due to individual herbs present in it.
excessive production of glucose in blood, which further reacts Histopathology of the pancreas of STZ diabetic animals
with blood hemoglobin and produces HbA1c.[32]
showed severe decrease in the number of islets of Langerhans
The diabetic hyperglycemia induced by STZ and NIC causes
and cells, with fibrosis and inflammatory cell infiltration
elevation of plasma levels of SGPT, SGOT, urea, and creatinine,
into the islets of Langerhans, and these findings are sup
which are considered as significant markers of liver and renal
ported by the reports published earlier.[35] Histopathology of
dysfunction. The polyherbal formulation treated animals reversed
the effect of STZ and NIC on the liver and renal markers. This the liver of STZ diabetic animals showed a severe fatty change,
may be due to the hepatoprotective mechanism of the individual sinusoidal dilation and congestion, mild portal inflammation,
herbs present in the polyherbal formulation.[33] fibrosis, severe feathery degeneration, and necrosis. The he
STZ diabetic rat has increased levels of lipid peroxides and patic changes may be due to the hypertrophy of hepatocytes by
reactive oxygen species, which cause hyperglycemia. Incessant an increase in the intracytoplasmic eosinophilic granules.[36]
generation of free radicals can lead to tissue damage through Polyherbal formulation and glibenclamide treatment to the

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Petchi, etal. / Journal of Traditional and Complementary Medicine 4 (2014) 108117

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16. ParasuramanS, RaveendranR, KesavanR. Blood sample collection
CONCLUSION in small laboratory animals. JPharmacol Pharmacother 2010;1:8793.
17. JamesSA, AutaR, GojeDJ. In vitro study on inhibition of glycosylation
Thus, our study findings demonstrate the antidiabetic effect of of methanolic leaf extract of Hibiscus cannabinus. Sci World J 2011;6:79.
18. WaterborgHH. The Lowry Method for Protein Quantitation. The Protein
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Protocols Handbook. Berlin, Heidelberg: Springer; 2002. p.79.
The antidiabetic potential of the polyherbal formulation is compa 19. PetchiRR, VijayaC. Antidiabetic and antiarthritic potential of
rable with that of glibenclamide, which is evidenced by decreased Glycosmis pentaphylla stem bark in FCA induced arthritis and
levels of blood glucose, HbA1c, total cholesterol, triglyceride, low Streptozotocin Induced diabetic rats. Int J Pharm Bio Sci 2012;3:32836.
density lipoprotein(LDL)cholesterol, urea, creatinine, SGOT, 20. SharmaSR, DwivediSK, SwarupD. Hypoglycaemic potential of
Mangifera indica leaves in rats. Pharm Biol 1997;35:1303.
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Pak J Pharmacol 2006;23:138.
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