Pharmacological characterization of different fractions of Calotropisprocera (Asclepiadaceae) in streptozotocin induced experimental modelof diabetic neuropathy

Sandeep Kumar Yadav n, Badri Prakash Nagori, Prashant Kumar DesaiLachoo Memorial College of Science and Technology (Autonomous), Pharmacy Wing, Sector-A, Shastri Nagar, Jodhpur 342003, Rajasthan, India

a r t i c l e i n f o

Article history:Received 8 October 2013Received in revised form12 January 2014Accepted 18 January 2014Available online 29 January 2014

Keywords:Diabetic neuropathyCalotropis proceraAsclepiadaceaeStreptozotocinDiabetes mellitus

a b s t r a c t

Ethnopharmacological relevance: Calotropis procera (Ait.) R.Br. is one of an ancient traditional shrub,which has been used for the treatment of diabetes, pain and inflammation for thousands of years inIndia. The root extract of Calotropis procera has been widely used by the tribal's of district Udaipur,Rajasthan (India) for treatment of diabetes mellitus and its associated complications like diabeticneuropathy. The present study was performed to explore the protective effect of root, stem and leafextracts of Calotropis procera in diabetes and diabetic neuropathy against tactile allodynia, mechanicalhyperalgesia and thermal hyperalgesia in streptozotocin induced diabetic rats.Materials and methods: Diabetes and peripheral neuropathy were induced in Wistar rats by injection ofstreptozotocin (45 mg/kg/intraperitoneally). The roots, stem and leaves of Calotropis procera were sequen-tially extracted with petroleum ether, chloroform, ethyl acetate and methanol. All the extracts were assessedby oral administration at 100 and 250 mg/kg in streptozotocin diabetic rats. The following compounds wereused as positive controls: insulin NPH (1 IU/kg/day), metformin (500 mg/kg/day), glibenclamide (2.5 mg/kg/day) and a combination of acarbose (20 mg/kg/day) with methylcobalamine (500 mg/kg/day). In contrast, thestreptozotocin induced untreated diabetic rats termed as negative control. Thermal hyperalgesia, mechanicalhyperalgesia and tactile allodynia were evaluated in all groups of streptozotocin diabetic rats to assess theextent of neuropathy by Eddy's hot plate, tail immersion, Randall–Selitto and Von Frey hair tests. The basalnociceptive thresholds were assessed in week 4 of post streptozotocin injection. All groups received theirtreatment on a regular basis from 28 to 42 days following a confirmation of diabetic neuropathy. Thenociceptive thresholds were assessed in all groups in week 5 and 6. The histopathology of pancreas andbiochemical estimations of plasma insulin and glycosylated haemoglobin (HbA1C%) levels were alsoperformed in week 6 of post streptozotocin injection.Results: The negative control rats developed diabetes and diabetic neuropathy after 6 week of streptozotocinadministration distinguished by significant (po0.01) hyperalgesia and tactile allodynia with enhancedHbA1C% level compared to normoglycemic rats. Chronic administration of root methanol, stemmethanol andleaf ethyl-acetate extracts of Calotropis procera for 2 weeks at 100 and 250 mg/kg doses significantly(po0.01) attenuated the diabetes induced mechanical hyperalgesia, thermal hyperalgesia, tactile allodyniaand HbA1C% level in streptozotocin diabetic rats as compared to negative control rats. Further, the rootmethanol extract of Calotropis procera in 100 mg/kg dose showed the regeneration capability of β cells in thehistology of pancreas with significant (po0.01) improvement in plasma insulin level in streptozotocindiabetic rats compared to negative control rats.Conclusion: Root methanol extract of Calotropis procera (100 mg/kg) has shown ameliorative effect in diabeticneuropathy which may be attributed by its multiple actions including potent hypoglycemic and antioxidant.

& 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Diabetes mellitus (DM) is a complex metabolic disorder char-acterized by hyperglycemia due to defects in insulin secretion,insulin action or both (Edwin et al., 2007). It affects the metabo-lism of carbohydrates, proteins and fats in the body. Type 1 dia-betes mellitus (DM 1) or insulin dependent diabetes mellitus(IDDM) occurs due to immunological destruction of pancreatic βcells and consequent insulin deficiency. Type 2 diabetes mellitus

Contents lists available at ScienceDirect

journal homepage: www.elsevier.com/locate/jep

Journal of Ethnopharmacology

0378-8741/$ - see front matter & 2014 Elsevier Ireland Ltd. All rights reserved.http://dx.doi.org/10.1016/j.jep.2014.01.020

Abbreviations: diabetes mellitus, (DM); type 1 diabetes mellitus, (DM 1); type2 diabetes mellitus, (DM 2); insulin dependent diabetes mellitus, (IDDM); non insulindependent diabetes mellitus, (NIDDM); streptozotocin, (STZ); blood glucose level,(BGL); mean feet vibration pressure threshold, (MFVPT); total feet vibration pressurethreshold, (TFVPT); glycosylated haemoglobin, (HbA1C%); plasma insulin, (PI)

n Corresponding author. Tel.: þ919950280258.E-mail addresses: yadavsandeep1977@gmail.com,

yadavriya1@rediffmail.com (S.K. Yadav).

Journal of Ethnopharmacology 152 (2014) 349–357

(DM 2) or non-insulin dependent diabetes mellitus (NIDDM) ischaracterized by impaired insulin resistance or insulin secretion.Even some patients with type 2 diabetes may eventually need theexogenous administration of insulin after a few years of evolutiondue to complete loss of β cell mass. DM 2 is usually associated withobesity and hereditary disposition and it is the most common formof diabetes, affecting 90–95% of cases (Tamrakar et al., 2011). Thelong-term elevated glucose level in the blood is easy to cause avariety of diabetic complications such as neuropathy (Kamenovet al., 2010), nephropathy (Inukai et al., 2000), cardiopathy (Alanand Karin, 2009) and retinopathy (Romero-Aroca et al., 2009). Asthe prevalence of diabetes has risen to epidemic proportionsworldwide, diabetic vascular complications have now becomeone of the most challenging health problems. Indeed, diabeticvascular complication is a leading cause of coronary heart disease,stroke, end-stage renal failure, acquired blindness and a variety ofneuropathy, which could account for disabilities and high mortal-ity rates in patients with diabetes (Brownlee, 2001; Yamagishi andImaizumi, 2005). Diabetic neuropathy is one of the most commoncomplications of DM. Patients with this neuropathy experiencesymptoms such as spontaneous pain, allodynia, and hyperalgesiawith an incidence rate of 10–20% (Dyck et al., 1993; Harati, 1996).

Calotropis procera (Ait.) R.Br. is a wild growing tropical plant offamily Asclepiadaceae that has been widely used in the Sudanese,Unani, Arabic and Indian traditional medicinal system for thetreatment of various diseases namely leprosy, ulcers, tumors, pilesand diseases of the spleen, liver and abdomen (Kirtikar and Basu,1935; Kumar and Arya, 2006). Different parts of Calotropis procerahave been shown to possess various scientifically reported biolo-gical activities namely, proteolytic (Dubey and Jagannadham,2003), hepatoprotective (Qureshi et al., 2007), anti-inflammatory(Kumar and Basu, 1994), analgesic (Dewan et al., 2000), antiferti-lity (Kamath and Rana, 2002) and antiarthritic (Kumar and Roy,2007).

It had been documented that the tribal's (ethnic minorities) ofdistrict Udaipur, Rajasthan (India) used the roots extract ofCalotropis procera orally to cure fever, diabetes mellitus and itsassociated complications and accepted as traditionally folk med-icine (Singh and Panday, 1998). There are few research studiesregarding the application of Calotropis procera against diabetes,but there main focus was on its latex part. The literature surveyrevealed that some scientific work has already been done on thelatex part of the plant for antioxidant and hypoglycemic effect(Roy et al., 2005); however some of the activities are still withoutscientific backing. The hyperglycemia of DM could be favorablyinfluenced by the components derived from plants (Aquino et al.,1995). There are many compounds isolated from medicinal plantswith hypoglycemic activity, out of which, triterpene (α-amyrin-3O-β-(5-hydroxy) ferulic acid) (Deutschlander et al., 2011), tannins(epicatechin and catechin derivatives) (Perez et al., 1998), flavo-noids (Chen et al., 2013) and saponins (Mei et al., 2012) arescientifically reported isolated compounds. The whole part ofCalotropis procera has been investigated previously for its phyto-constituents, which revealed the presence of cardenolides, antho-cyanins, and triterpenoids (Mascolo et al., 1988). The α-amyrin,β-amyrin, (Rastogi and Mehrotra, 1993; Ansari and Ali, 2001),flavonoids, alkaloids, tannins and saponins (Hassan et al., 2006)have been reported in roots and leaves of Calotropis procera. Inaddition, the presence of flavonoids and Saponin glycosides werealso reported in stem part of the plant (Hassan et al., 2006).Further, phyto-chemical analysis also revealed that Calotropisprocera has many similar phyto-chemicals which may showhypoglycemic effects. However, no scientific work has beenreported for use of roots, stem and leaves of Calotropis proceraagainst diabetes and diabetic neuropathy. In continuation ofscientific validation of folkloric therapeutic use, we examined the

beneficial effects of different extracts of Calotropis procera, indiabetes and explored the protective effect of it against diabeticneuropathy in streptozotocin (STZ) induced type 1 diabetic rats.

2. Materials and methods

2.1. Collection and authentication of plant material

Plant materials used in this study, consisted of the roots, stemand leaves of Calotropis procera, were collected from the local areaof Jodhpur, Rajasthan, during April 2010. Plant was identified andauthenticated from Botanical Survey of India, Jodhpur, Rajasthan.A voucher specimen (12012/2010-11/tech (PL ID)-817)) has beenkept in herbarium, in the Department of Pharmacognosy, LachooMemorial College of Science and Technology, Pharmacy wing,Jodhpur, Rajasthan.

2.2. Preparation of plant extracts and drug solution

The roots, stem and leaves of Calotropis procera, were shadedried for 10–15 days and cut into small pieces. The pieces of plantparts were mechanically crushed to coarse powder with the helpof a hand-grinding mill individually, after that the powder of roots,stem and leaves were passed through sieve No. 60. Separateextraction of crushed roots, stem and leaves powder (1 kg indivi-dually) of Calotropis procera, were done in soxhlet up-to 48h, withpetroleum ether, chloroform, ethylacetate and methanol, succes-sively according to their polarity. The extracts were filtered andconcentrated in rotatory evaporator at 35–40 1C under reducedpressure to obtain a semisolid material. The extracts were dis-solved in a vehicle containing 0.2% polysorbate–80, 0.5% sodiumcarboxy methyl cellulose, 0.9% sodium chloride, 0.9% benzylalcohol and 97.2% distilled water (Lee, 2001).

2.3. Drugs and chemicals

Streptozotocin (STZ) was purchased from Hi Media LaboratoriesPvt. Ltd., Mumbai, India (CAS No. 18883-66-4, LOT No.0000114330, Batch No. CMS 1758-1G); methylcobalamin waspurchased from Intas Pharmaceutical Ltd., Maloda, Ahmedabad,India (Batch No. I-1373); Insulin N was purchased from RanbaxyLab. Ltd., Taloja, Navi-mumbai, India (Batch No. 9063543); glib-enclamide was purchased from Aventis Pharma. Ltd., Ankleshwar,India (Batch No.0212036); metformin was purchased from AbbottHealthcare Pvt. Ltd., Baddi, Solan, Himachal Pradesh, India (BatchNo. GFA 1013); acarbose was purchased from Bayer Pharmaceu-tical Pvt. Ltd., Malpur Baddi, Solan, Himachal Pradesh, India (BatchNo. P 12202). All other chemicals and reagents were obtained fromS. D. Fine chemicals, Mumbai, India.

2.4. Experimental animals and approval of the study

Healthy Wistar rats of approximately same age groups (2–3months old), having body weight 170–200 g were obtained fromthe animal house of Lachoo Memorial College of Science andTechnology, Pharmacy Wing, Jodhpur, Rajasthan. All the animalswere housed in polysulphone/polycarbonated plastic rodent cagesmaintained at an ambient room temperature of 2773 1C andrelative humidity of 5075%, with a 12 h each of dark and lightcycle. The animals were fed with standard commercial, rodentpelleted diet (Hindustan Lever Ltd., Bombay, India) and distilledwater ad libitum during 6 weeks of experimental period. Theexperimental studies were performed according to the guidelinesof Institutional Animal Ethical Committee (IAEC) of Lachoo Mem-orial College of Science and Technology, Pharmacy Wing, Jodhpur,

S.K. Yadav et al. / Journal of Ethnopharmacology 152 (2014) 349–357350

Rajasthan and care of the animals was carried out as per theguidelines of Committee for the Purpose of Control and Super-vision of Experiments on Animals (CPCSEA), Ministry of Environ-ment and Forest, Government of India (Reg. No. 541/02/c/CPCSEA).The experimental protocol (Protocol no. 6P-01/05/2010.) wasapproved by IAEC. The animals were used after an acclimatizationperiod of 7 days to the laboratory environment and the experi-ments were performed during the light cycle (0800–1600 h).

2.5. Induction and assessment of diabetes and diabetic neuropathyin rats

Diabetes was artificially induced by intraperitonial injection offreshly prepared solution of streptozotocin (STZ) in 0.1 M citratebuffer (0.1 M sodium citrate and 0.1 M citric acid, pH was adjusted to4.5 and its temperature was maintained in between 2 and 8 1C) at thedose of 45 mg/kg to overnight fasted rats. The age-matched controlrats received an equal volume of citrate buffer and used along withdiabetic animals. The blood samples of rats were collected throughsnipping the tail vein (tail cutting) (Burcelin et al., 1995) and thediabetic status of rats were confirmed post 48 h of STZ injection bymonitoring blood glucose levels (BGL) with the help of glucoseoxidase–peroxidase reactive strips and a glucometer (Optium Xceedglucometer and glucose strips, Abbott Healthcare Pvt. Ltd., Chembur,Mumbai-400071, India). The rats having BGL more than 350 mg/dl(Hajializadeh et al., 2010) were considered to have diabetes and usedfor the present study. The diabetic rats provoked peripheral neuro-pathy after one month of induction of diabetes with STZ, which wasevident from a marked hyperalgesia and allodynia associated withreduction in pain threshold and withdrawal threshold responses inhot plate, tail immersion and mechanical allodynia. Even diabeticneuropathy was also characterized by increased in vibration pressurethreshold in mechanical hyperalgesia. Only diabetic animals that hadpersistent hyperglycemia developed the clear cut sign of mechanicalhyperalgesia, thermal hyperalgesia and mechanical allodynia after4 week of STZ administration. Tail Immersion, hot plate, Von FreyHair and mechanical vibration pressure threshold sensitivity (Ran-dall–Selitto) tests were performed to assess the extent of neuropathywithin normal control, negative control (DC), positive controls andtest groups of rats. Heat hyperalgesia were evaluated by the two teststhat were tail immersion and hot plate assays. The tactile responseswere evaluated by Von Frey Hair test and paw pressure sensitivitywere measured with sensitometer by Randall–Selitto test.

2.6. Collection of blood sample

All animals in each group remained alive until the end of theexperimental period (6 weeks). At the end of the experiment,

overnight-fasted rats were anesthetized with intraperitonial chlor-alhydrate (400mg/kg,i.p.), while under anesthesia, they werepainlessly sacrificed between 9.00 and 11.00 a.m. to minimizethe diurnal variations. Blood samples were harvested via an intra-cardiac needle, from each rat into heparin and EDTA samplebottles. The heparin anti-coagulated blood samples were centri-fuged at 1000 rpm for 10 min, after which their plasma wascollected and stored at �20 1C for subsequent analysis, whilethe EDTA anti-coagulated blood samples were used for thebiochemical analysis. The samples were stored at �80 1C untilprocessed for biochemical estimations.

2.7. Experimental design (treatment schedule) and selection of dose

The rats having BGL more than 350 mg/dl (Hajializadeh et al.,2010) after 48h of STZ injection were considered to have diabetesand used for the present study. Following confirmation of diabetesand diabetic neuropathy after recording of basal nociceptivereaction at week 4 of post STZ injection, rats were randomizedin to different groups of 6 in each (n¼6) by sex and scheduled totreatment protocol from 28 to 42 days as follows:

� Test groups: The petroleum ether, chloroform, ethyl acetate andmethanol extracts of the roots, stem and leaves (total: 12extracts) of Calotropis procera were assessed by oral adminis-tration at 100 and 250 mg/kg in STZ diabetic rats. The STZdiabetic rats which received the different extracts of Calotropisprocera were termed as test groups.

� Positive controls: As positive controls, the following standardcompounds were used: insulin NPH (1 IU/kg/day), metformin(500 mg/kg/day), glibenclamide (2.5 mg/kg/day) and combinationof acarbose (20 mg/kg/day) with methylcobalamine (500 mg/kg/day).

� Negative control (DC): One group of STZ induced untreateddiabetic control rats termed as negative control (DC) group.

� Normal control (NC): One group of normoglycemic rats wasserved as normal control (NC) group in which diabetes was notinduced by STZ.

The treatment schedule of positive control and test group inSTZ diabetic rats are depicted in Table 1.

The diabetic rats provoked diabetic neuropathy after 4 weeks ofinduction of diabetes with STZ, so following confirmation ofdiabetes and diabetic neuropathy, rats were randomized in tonegative control (DC), positive controls and test groups andscheduled to treatment protocol from week 4 (Kandhare et al.,2012). Each group of rats received their treatment either orally(through intra-gastric gavages by oral feeding needles) and

Table 1Positive control and test group treatment schedule in STZ diabetic rats during 28 to 42 days.

Positive controls(total: 4 groups)

ID Test group treatments schedule in STZ diabetic rats (total: 24 groups)

Root extracts of Calotropisprocera with dose (mg/kg)

ID Stem extractsof Calotropis procerawith dose (mg/kg)

ID Leaf extractsof Calotropis procerawith dose (mg/kg)

ID

Insulin NPH SI Petroleum ether 100 RPE 100 Petroleum ether 100 SPE 100 Petroleum ether 100 LPE 100Petroleum ether 250 RPE 250 Petroleum ether 250 SPE 250 Petroleum ether 250 LPE 250

Metformin SM Chloroform 100 RCE 100 Chloroform 100 SCE 100 Chloroform 100 LCE 100Chloroform 250 RCE 250 Chloroform 250 SCE 250 Chloroform 250 LCE 250

Glibenclamide SG Ethyl acetate 100 REE 100 Ethyl acetate 100 SEE 100 Ethyl acetate 100 LEE 100Ethyl acetate 250 REE 250 Ethyl acetate 250 SEE 250 Ethyl acetate 250 LEE 250

Combination ofAcarboseþMethylcobalamine

SAþSMC Methanol 100 RME 100 Methanol 100 SME 100 Methanol 100 LME 100Methanol 250 RME 250 Methanol 250 SME 250 Methanol 250 LME 250

ID: Represents the identification code (abbreviations) allotted to the different treated groups.

S.K. Yadav et al. / Journal of Ethnopharmacology 152 (2014) 349–357 351

subcutaneously (only SI group). Dosing was performed once a daybetween 6:00 and 7:00 a.m. during week 4–6, post developmentof diabetic neuropathy. The dose of administration of STZ wasadopted according to Singh et al. (2009). The dose selection ofCalotropis procera was done on the basis of previous studydescribed by Kamath and Rana (2002), where the root extract ofCalotropis procera was used for anti-fertility study. A pilot studywith different extracts of Calotropis procera for this study was alsoperformed to select the dose.

2.8. Criteria of observations

2.8.1. Estimation of biochemical parametersThe animals were sacrificed under deep anesthesia and the blood

samples were harvested via an intra-cardiac needle from each rat.The blood samples were transferred into heparin and EDTA samplebottles and plasma were separated. The samples were stored at�80 1C until processed for biochemical estimations.

2.8.1.1. Estimation of glycosylated haemoglobin (HbA1C%). HbA1C%was determined at the end of study (week 6) by using the GlycoHbkit (Lifechem ™ GHb) purchased from Kamineni Life Sciences Pvt.,Ltd, Hyderabad, India.

2.8.1.2. Estimation of plasma insulin (PI) concentration. Plasmainsulin was determined at the end of study (week 6) by ratinsulin kit (Crystal Chem Inc., USA) in a Multiskans Spectrum,thermoscientific, USA and expressed as mIU/ml.

2.8.2. Behavioral assessmentBehavioral tests were carried out in all group of rats for thermal

hyperalgesia, mechanical hyperalgesia and mechano-tactile allo-dynia by Eddy's hot plate, tail immersion, Randall–Selitto and VonFrey hair tests, for assessing the nociceptive thresholds. Behavioralassessments for recording of basal nociceptive thresholds werecarried out in all animals in week 4 of post STZ injection. Thesetests were repeated in week 5 and 6 of post STZ injection innormal control, negative control (DC), positive controls and testgroups of rats.

2.8.2.1. Assessment of thermal hyperalgesia. After a basal recordingof nociceptive thresholds (pain thresholds) in week 4 of post STZinjection, the changes in pain thresholds values were assessed innormal control, negative control (DC), positive controls and testgroups of rats in week 5 and 6, respectively. Thermal hyperalgesiaof the hind paw was evaluated by using Eddy's hot plate (Rolax,Ambala Cantt., India) method (Eddy et al., 1950), for assessing thereactivity against noxious thermal stimuli. In addition, spinalthermal sensitivity was assessed by the tail immersion testaccording to previously described method Necker and Hellon(1978).

2.8.2.1.1. Tail-immersion (hot water/heat-allodynia) test. In peripheralnerve injury, initial burst from peripheral site and later sustainedactivation of peripheral nociceptors leads to a central sensitization ofthe dorsal horn neurons in the spinal cord. This central sensitizationis an important feature in inducing long-term thermal allodyniaand hyperalgesia, which may be assessed as tail heat-allodynia. Theterminal part of the tail (1 cm) of rat was immersed in a hot waterbath maintained at a temperature of 52.570.5 1C until the tail with-drawal latency (flicking response) or signs of struggle were observed(cut-off 12 s). Shortening of the tail withdrawal time indicateshyperalgesia (Kannan et al., 1996).

2.8.2.1.2. Hot-plate (heat-allodynia) test. The hyperalgesic response onthe hot-plate is considered to result from a combination of central andperipheral mechanisms (Kannan et al., 1996). Briefly, the rats wereplaced on the top of a controlled preheated (5571 1C) hot plate(Eddy's hot-plate) surface. The latency to the first sign of paw lickingor jump response to avoid the heat was taken as an index of the painthreshold. The cut-off time was 10 s in order to avoid damage tothe paw.

2.8.2.2. Assessment of mechanical hyperalgesia (Randall–Selitto pawpressure test). Mechanical nociceptive threshold, an index ofmechano-hyperalgesia, was assessed in each group of rat's post6 week of STZ injection via a method described by Randall andSelitto (1957). The recording of basal nociceptive threshold wasperformed in week 4 of post induction of diabetes with STZ.Briefly, nociceptive threshold (Paw pressure threshold) wasregistered in paw pressure sensitometer (Dhansai Laboratory,Mumbai, Model No. 503022), as measured by applyingincreasing pressure (in the form of vibration pressure threshold)to the hind paw of rats. The withdrawal of hind paw in response ofincreasing vibration pressure threshold was used to assess thenociceptive threshold and the values were expressed as vibrationpressure threshold. The cut-off vibration pressure threshold wastaken as 50 V/micron. The mean and total vibration pressurethresholds in week 6 of post induction of diabetes werecalculated for each group of rats.

2.8.2.3. Assessment of mechano-tactile allodynia (Von Frey hairtest). Mechano-tactile allodynia (non-noxious mechanical stimuli)responses were evaluated in rats by quantifying the withdrawalthreshold of the hind paw in response to stimulation with flexiblevon Frey filaments (Ilnytska et al., 2006). The criterion for thethreshold value, in grams (g), was equal to the filament evoking awithdrawal of the paw 2 times out of 4 trials, i.e., 50% response. Thewithdrawal threshold (tactile) responses in diabetic rats for recordingof basal nociceptive reaction were measured in week 4 of post STZinjection. Further, the tactile response was also measured in normalcontrol rats. The test was repeated in normal control, negativecontrol (DC), positive controls and test groups of rats in week5 and 6 of post STZ injection and the withdrawal threshold values(g) were compared with the negative control (DC) group.

2.9. Histopathological examination

After termination of experiment, pancreatic tissues from allgroups of rats were subjected to histopathological studies. Thewhole pancreas from each group of rats was removed aftersacrificing the animal under chloral hydrate (400 mg/kg, i.p.)anesthesia. Pancreas was collected in formaldehyde solution andimmediately histological preparations were made. A 5 mg thicksections were cut and stained with haematoxylin and eosin forhistological examination (Luna, 1990).

2.10. Phytochemical screening

Preliminary phytochemical screening was carried out to findthe presence of the active chemical constituents in differentextracts of Calotropis procera roots, stem and leaves such asalkaloids, flavonoids, tannins, phenolic compounds, triterpene,saponins, steroids, fixed oils and fats (Kokate et al., 2006). Ingeneral, tests for the presence of phytochemical compoundsinvolved the addition of appropriate chemical reagent(s) to theextract in test tubes. The mixture was then shaken and/or heatedas the case may be. The alkaloid was tested by using Dragendorff's,Mayer, Wagner's and Hager's test. The cardiac glycosides were

S.K. Yadav et al. / Journal of Ethnopharmacology 152 (2014) 349–357352

tested by Liberman's test and Keller Killiani test. The flavonoidswere tested by alkaline reagent and Shinoda test. The tannins weretested by ferric chloride and gelatin test. The total phenoliccontent in extract was determined by Bromine water and Lieber-mann tests. Saponin content of Calotropis procera was determinedby froth formation test. The steroids and triterpenoids were testedby Salkowski test. The presence of fats and fixed oils wasdetermined by using 2,4-dinitrophenylhydrazine test.

2.11. Statistical analysis

All the results were expressed as mean7standard error ofmeans (S.E.M.). The data from the biochemical and behavioralresults were statistically analyzed by one-way analysis of variance(ANOVA) followed by the Dunnett test, aided by Graph pad prismSoftware, version 3.0. The p-valueo0.01 was considered to bestatistically significant.

3. Results

3.1. Estimation of biochemical parameters

3.1.1. Effect of different extracts of Calotropis procera treatment onthe glycosylated haemoglobin (HbA1C%) levels

The levels of HbA1C% measured at the end of 6 weeks ofdifferent experimental groups are represented in Fig. 1. A signifi-cant (po0.01) increase in HbA1C% level was observed in thenegative control (DC) rats (12.7070.24), when compared withnormal control (NC) rats (5.6370.14). Chronic treatment of RME100 (8.0370.27), SME 100 (8.4070.18) and LEE 250 (8.2870.33)respectively in STZ diabetic rats fromweek 4 to week 6 amelioratedthe HbA1C% levels significantly (po0.01) as compared with DC

(12.7070.24) rats, indicating RME 100, SME 100 and LEE 250works as a better hypoglycemic agent than the other extracts ofCalotropis procera. Furthermore, treatment of STZ diabetic ratswith single daily dosing of SI (5.4170.13), SM (7.5170.22), SG(9.3670.35) or combination of SAþSMC (7.9870.24) respectivelyduring 28 to 42 days post induction of diabetes, reduced theHbA1C% level significantly (po0.01) near to the normal range ascompared to DC (12.7070.24) group rats.

3.1.2. Effect of different extracts of Calotropis procera treatment onthe plasma insulin (PI) levels

Fig. 2 shows the effect of different extracts of Calotropis proceraand standards on the level of plasma insulin measured at the endof 6 weeks in normal control, negative control (DC), positivecontrols and test groups of rats. The results of the present studyclearly indicated that the RME 100 (7.1870.27), SME 100 (6.9270.40), LEE 250 (6.9470.34), SI (13.2970.48), SM (8.8370.38),SG (9.4170.14), SAþSMC (7.6770.11) treatments, respectively,exhibited a significant (po0.01) improvement in PI level in STZinduced diabetic rats as compared to DC (3.1570.49) rats. Addi-tionally, SI (13.2970.48) treated rats showed significant (po0.01)improvement in the PI level and it returned the insulin level nearto normal range in the diabetic rats. Further, a significant (po0.01)reduction in PI level were also observed in the DC (3.1570.49)group rats compared to respective NC (16.0470.16) group rats,indicates the complete destruction of pancreatic β cells by STZ.

3.2. Behavioral assessment

The results of behavioral tests for basal nociceptive thresholds,shows that the diabetic animals developed the clear cut sign ofthermal hyperalgesia, mechanical hyperalgesia and mechano-tactile allodynia after 4 weeks of STZ injection, it indicates the

Glycosylated Hb(HbA1C%) Level After 6 Weeks of STZ (Inj.)

DC

NC

SI

SM

SG

SA +

SM

C

100

250

100

250

100

250

100

250

100

250

100

250

100

250

100

250

100

250

100

250

100

250

100

250

0.0

2.5

5.0

7.5

10.0

12.5

15.0

Gly

cosy

late

d H

b(H

bA1C

%)

P E C E E E M E P E C E EE M E P E C E E E M E

Root Stem Leaf

Different Groups Received Treatment Daily From 28 to 42 Days

Fig. 1. Effect of different extracts of Calotropis procera on HbA1C% levels in STZ diabetic rats. Data in bars are expressed as mean7S.E.M. (n¼6/group), ANOVA followed byDunnett test were used for comparison. n Represents statistical significance vs. negative control (DC) rats (po0.01).

Insulin (µIU/ml) Level After 6 Weeks of STZ (Inj.)

DC

NC SI SM SG

SA +

SM

C

100

250

100

250

100

250

100

250

100

250

100

250

100

250

100

250

100

250

100

250

100

250

100

250

0.0

2.5

5.0

7.5

10.0

12.5

15.0

17.5

Different Groups Received Treatment Daily From 28 to 42 Day

Seru

m In

sulin

(µIU

/ml)

P E C E E E M E P E C E EE M E P E C E E E M E

Root Stem Leaf

Fig. 2. Effect of different extracts of Calotropis procera on plasma insulin (PI) levels in STZ diabetic rats. Data in bars are expressed as mean7S.E.M. (n¼6/group), ANOVAfollowed by Dunnett test were used for comparison. n Represents statistical significance vs. negative control (DC) rats (po0.01).

S.K. Yadav et al. / Journal of Ethnopharmacology 152 (2014) 349–357 353

development of diabetic neuropathy followed by STZ treatment.The nociceptive thresholds evaluated as thermal hyperalgesia,mechanical hyperalgesia and mechano-tactile allodynia in week5 and 6 of post induction of diabetes in negative control (DC),positive controls and test groups of rats are depicted in Table 2 andFigs. 3 and 4.

3.2.1. Effects of different extracts of Calotropis procera treatment ondiabetes induced thermal hyperalgesia (heat-allodynia)

Data in Table 2 demonstrated the pain threshold in tail-immersion and hot-plate assays in normal control and STZ injecteddiabetic rats in week 5 and 6.

The nociceptive threshold was significantly (po0.01) lowered inDC rat as compared to NC rats in week 5 of post STZ injection in boththe tail immersion and hot plate assays. Hyperalgesia was evident inthe tail immersion and hot plate after 5 weeks of STZ injection in DCrats, respectively. The significant (po0.01) reduction in pain thresh-old were observed at 6 weeks post STZ injection in DC rats ascompared to NC rats in both the assays, respectively. Administrationof RME 100, RME 250, REE 250, SME 100, LEE 250 and all standards(SI, SM, SG alone or combination of SAþSMC) respectively during 28to 42 days (post induction of diabetes) to diabetic rats produced atime dependent increase in pain threshold significantly (po0.01) ascompared to DC rats. Further, RME 100, SME 100 and LEE 250treatments in diabetic rats improved the pain threshold significantly(po0.01) in tail-immersion and hot-plate assays respectively com-pared to DC rats (Table 2). The maximum increase in pain thresholdwith different extracts of Calotropis procera was observed with RME100 in both tail immersion and hot plate assays. In addition, diabeticrats treated with SI showed significant (po0.01) improvement in

pain threshold in week 6 in both the assays as compared to otherdiabetic rats, either treated with standard or test.

3.2.2. Effects of different extracts of Calotropis procera treatment ondiabetes induced mechanical hyperalgesia

The mean feet vibration pressure threshold (MFVPT) whichproduced paw withdrawal response after induction of diabeticneuropathy, was significantly (po0.01) increased in DC (97.7971.31) rats than that of NC (19.0870.97) rats after 6 weeks of STZinjection (Fig. 4). It indicates the uncontrolled diabetic rats showedthe reduction in paw withdrawal time in the Randall–Selitto test.The total feet vibration pressure threshold (TFVPT) was also aug-mented significantly (po0.01) in DC (391.1775.24) rats comparedwith NC (76.3373.88) rats (Fig. 3). Administration of RME 100, RME250, SME 100 and LEE 250 during 28–42 days in diabetic rats causedthe attenuation in the MFVPT and TFVPT significantly (po0.01) thanthat of DC rats. In addition, administration of RME 100 (33.5871.09,134.3374.39) and LEE 250 (39.3773.06, 157.5712.27) in diabeticrats caused the significant (po0.01) attenuation in the MFVPT andTFVPT respectively than that of DC (97.7971.31, 391.1775.24) rats.Furthermore, administration of SI, SM, SG and SAþSMC respectivelyin diabetic rats during 28 to 42 days post induction of diabetescaused the diminution in the MFVPT and TFVPT significantly(po0.01) in week 6 of the experiment compared to DC rats.

3.2.3. Effects of different extracts of Calotropis procera treatment ondiabetes induced mechano-tactile allodynia

Tactile withdrawal threshold in response to light touch withflexible Von Frey filaments was significantly (po0.01) reducedin DC rats compared with NC rats in week 5 and 6 post STZ injection.

Table 2Effect of different extracts of Calotropis procera and standards on pain threshold (heat allodynia) and tactile withdrawal threshold (mechano-tactile allodynia) in tail-immersion, hot plate and Von Frey Hair assays measured in week 5 and week 6 post injection of STZ in the diabetic rats.

Groupname

Pain threshold in tail-immersion assay Pain threshold in hot plate assay Tactile withdrawal threshold in Von Frey hair assay

Reaction time in week5 (s)

Reaction time inweek 6 (s)

Reaction time inweek 5 (s)

Reaction time inweek 6 (s)

Paw withdrawal threshold inweek 5 (g)

Paw withdrawal threshold inweek 6 (g)

SI 9.1670.30n 9.5070.22n 9.1670.30n 9.5070.22n 13.6670.33n 13.8370.30n

SM 8.8370.40n 9.0070.25n 9.0070.25n 9.1670.16n 12.8370.40n 12.3370.21n

SG 6.5070.56n 7.0070.51n 6.6670.49n 7.1670.47n 11.6670.33n 10.3370.33n

SAþSMC 9.3370.21n 9.5070.22n 9.3370.21n 9.3370.21n 13.0070.36n 13.1670.16n

LCE 250 5.6670.33n 1.8370.30 6.1670.47n 2.6670.42 10.0070.36n 6.8370.74RCE 250 4.6670.21n 1.6670.21 5.3370.21n 2.0070.25 8.6670.21 6.3370.33RME 100 8.6670.21n 8.6670.21n 8.6670.21n 8.8370.16n 12.3370.33n 12.0070.25n

SME 100 8.1670.40n 7.6670.49n 8.3370.33n 8.3370.33n 11.8370.30n 10.6670.33n

SCE 250 5.3370.21n 1.6670.21 670.51n 2.1670.16 9.5070.34 6.8370.16RPE 250 2.8370.16 1.6670.21 3.3370.21 2.0070.36 8.3370.33 6.1670.16REE 250 6.3370.49n 5.6670.21n 6.570.42n 4.1670.30n 10.3370.33n 9.1670.16n

LEE 250 8.3370.33n 8.0070.44n 8.5070.22n 8.5070.22n 12.1670.30n 11.6670.21n

SPE 250 4.0070.25n 1.6670.21 4.3370.33 2.1670.40 8.5070.22 6.1670.30SEE 250 5.0070.00n 1.8370.47 5.5070.34n 2.3370.33 9.0070.25 6.6670.0.49LME 100 4.8370.16n 1.8370.30 5.5070.34n 2.3370.21 9.007 .25 6.8370.30LPE 250 3.0070.25 1.6670.33 3.6670.21 2.0070.25 8.5070.22 6.1670.40LCE 100 5.1670.30n 1.8370.65 5.3370.21n 2.1670.16 9.6670.33 6.8370.16RCE 100 4.1670.30n 1.6670.33 4.6670.21n 2.1670.16 8.1670.40 6.5070.22RME 250 8.3370.21n 5.0070.68n 8.1670.16n 4.0070.36n 12.0070.36n 10.3370.33n

SME 250 7.6670.21n 2.0070.81 7.8370.30n 2.0070.25 11.5070.34n 6.5070.56SCE 100 4.6670.21n 1.6670.21 5.0070.25n 2.0070.25 9.1670.16 6.5070.34RPE 100 2.6670.21 1.6670.33 3.0070.25 2.0070.25 8.1670.30 6.3370.21REE 100 5.6670.33n 2.0070.63 6.1670.30n 2.0070.25 10.0070.25n 6.6670.33LEE 100 7.8370.16n 2.1670.16 8.1670.16n 2.0070.36 11.8370.30n 6.8370.30SPE 100 3.1670.30 1.6670.33 3.6670.21 2.0070.36 8.1670.40 6.5070.22SEE 100 4.1670.40n 1.8370.65 4.8370.16n 2.1670.40 8.6670.21 6.5070.22LME 250 4.1670.40n 1.6670.33 4.8370.16n 2.3370.49 8.6670.21 6.8370.30LPE 100 2.5070.22 1.6670.21 3.3370.21 2.0070.44 8.1670.30 6.3370.21DC 2.3370.21 1.6670.21 2.8370.16 2.0070.25 8.1670.40 6.1670.16NC 9.0070.25n 9.1670.16n 9.0070.25n 9.0070.25n 14.3370.21n 14.6670.21n

Data are expressed as mean7S.E.M. (n¼6/group), ANOVA followed by Dunnett test were used for comparison.n Represents statistical significance vs. negative control (DC) rats (po0.01).

S.K. Yadav et al. / Journal of Ethnopharmacology 152 (2014) 349–357354

The basal recording of nociceptive threshold measured after 4 weeksof STZ injection, showed the decrease in mean paw withdrawalthreshold (i.e. mechanical allodynia) in all diabetic rats, indicatingthe development of diabetic neuropathy. Administration of RME 100,RME 250, REE 250, SME 100 and LEE 250 in diabetic rats correcteddiabetes induced decrease in paw withdrawal threshold in the VonFrey test, by showing the significant (po0.01) improvement in pawwithdrawal threshold compared to DC rats in week 5 and 6. Further,RME 100, SME 100 and LEE 250 treatments in diabetic rats respec-tively ameliorated the paw withdrawal threshold significantly(po0.01) in week 6 compared to DC rats. Chronic treatment withSI, SM, SG and SAþSMC respectively for 2 weeks significantly(po0.01) ameliorated this decrease in mean paw withdrawal thresh-old in diabetic rats as compared to DC rats (Table 2).

3.3. Histopathological examinations of pancreas

Results showed that the RME 100 of Calotropis procera enhancedthe regeneration of islets of Langerhans (IL) and provide betterprotection from degenerative changes occurs due to STZ injuries;even RME 100 administration causes the restoration of normalcellular size of the islet structure of pancreas (Fig. 5).

3.4. Phytochemical screening

Phytochemical screening of plant extracts showed the presenceof cardenolides, anthocyanins, alkaloids, tannins, saponins andtriterpenoids in roots, stem and leaves of Calotropis procera.

4. Discussion

Peripheral diabetic neuropathy is a devastating complication ofdiabetes and a leading cause of foot amputation. Clinical indicationof peripheral diabetic neuropathy include increased vibration andthermal perception thresholds that progress to sensory loss,occurring in conjunction with degeneration of all fiber types inthe peripheral nerve. Diabetic neuropathy is characterized byclinical features like allodynia, hyperalgesia due to elevatednociceptive response, neuronal hypoxia and reduced threshold topainful stimuli (Gul et al., 2000). Similar symptoms are exhibited bySTZ induced diabetic animals (Calcutt and Chaplan, 1997). Hypergly-cemia and inflammation unleash a cascade of events that affectscellular proteins, gene expression and cell surface receptor expres-sion, ultimately resulting in progressive pathologic changes andsubsequent diabetic complications (Pop-Busui et al., 2006). The STZinduced diabetic rats is the most commonly employed animal modelof painful diabetic neuropathy (Hoybergs et al., 2008).

In the present study, STZ injected rats had significantly higherHbA1C% level, which provoke diabetic neuropathy that was asso-ciated with the neuropathic pain. In addition, RME 100, SME 100 andLEE 250 extracts of Calotropis procera attenuated STZ inducedthermal hyperalgesia, mechanical hyperalgesia and tactile allodynia.

The nociceptive threshold was significantly lowered in DC ratscompared to NC rats in tail immersion and hot plate assay. Thisindicates that diabetic animals exhibited thermal hyperalgesia,post development of diabetic neuropathy. Consequently, hyperalgesiaand neuropathic pain caused further decrease in pain threshold on

Total Feet Vibration Pressure Threshold (TFVPT) After 6 Weeks of STZ inj.

DC

NC SI SM SG

SA +

SM

C

100

250

100

250

100

250

100

250

100

250

100

250

100

250

100

250

100

250

100

250

100

250

100

250

0

100

200

300

400

Different Groups Received Treatment Daily From 28 to 42 Days

Vib

ratio

n Pr

essu

re T

hres

hold

(Vol

t/Mic

ron)

P E C E E E M E P E C E E E M E P E C E E E M ERoot Stem Leaf

Fig. 3. Effect of different extracts of Calotropis procera and standards on total feet vibration pressure threshold (TFVPT) in mechanical hyperalgesia measured in week 6 postinjection of STZ in the diabetic rats. Data in bars are expressed as mean7S.E.M. (n¼6/group), ANOVA followed by Dunnett test were used for comparison. n Representsstatistical significance vs. negative control (DC) rats (po0.01).

Mean Feet Vibration Pressure Threshold (MFVPT) After 6 Weeks of STZinj.

DC

NC

SI

SM

SG

SA +

SM

C

100

250

100

250

100

250

100

250

100

250

100

250

100

250

100

250

100

250

100

250

100

250

100

250

0

25

50

75

100

Different Groups Received Treatment Daily From 28 to 42 Days

Mea

n V

ibra

tion

Pres

sure

Thr

esho

ld (V

olt/M

icro

n)

P E C E E E M E P E C E E E M E P E C E E E M E

Root Stem Leaf

Fig. 4. Effect of different extracts of Calotropis procera and standards on mean feet vibration pressure threshold (MFVPT) in mechanical hyperalgesia measured in week 6 postinjection of STZ in the diabetic rats. Data in bars are expressed as mean7S.E.M. (n¼6/group), ANOVA followed by Dunnett test were used for comparison. n Representsstatistical significance vs. negative control (DC) rats (po0.01).

S.K. Yadav et al. / Journal of Ethnopharmacology 152 (2014) 349–357 355

exposure for thermal heat in tail immersion and hot plate assay inuncontrolled diabetic rats (DC rats). Patients with neuropathic paincomplain of ongoing pain as well as enhanced pain sensitivity tostimuli applied to their skin (Meyer et al., 2005). Contrary to that,administration of SI, SM, SG, SAþSMC RME 100, SME 100 and LEE250 respectively in diabetic rats not only reduced the HbA1C% levelbut also lead to maximum improvement in pain threshold inprogressive manner in tail immersion and hot plate tests, indicatingthe better control of BGL, reduction in neuropathic pain and thermalhyperalgesia. However, latex of Calotropis procera is scientificallyreported for its analgesic effect (Dewan et al., 2000), which indicatesits possible role in relieving pain of diabetic neuropathy.

Further, administration of RME 100, SME 100 and LEE 250 lead to areduction in TFVPT, MFVPT and reaction sensitivity to pain in lowerlimbs in the diabetic rats. However, RME 100 and LEE 250 offered thehighest reduction in TFVPT and MFVPT than the other extracts ofCalotropis procera, which produces paw withdrawal response in thediabetic rats, indicating the most potent extracts of Calotropis procera.

In diabetic rats, the decreased response latency for tactile with-drawal threshold and pain threshold of lower limbs, were recoveredsignificantly with RME 100, SME 100 and LEE 250 administration. RME100 and LEE 250 recovered the diabetes induced tactile allodyniasignificantly in neuropathic rats, even after sensory dysfunction hadcompletely developed.

The results of present study indicate that the RME 100, SME100 and LEE 250 extracts of Calotropis procera exhibited a markedanti-hyperglycemic activity in STZ induced diabetic rats by low-ering the HbA1C% and improving the PI levels. It indicates that theRME 100, SME 100 and LEE 250 extracts could be responsible forstimulation of insulin release. Further, the histopathology investi-gation of pancreas revealed that RME 100 treated rats had thelesser injuries in IL from STZ injection, indicated by the presence ofsome β cells. It suggests that RME 100 treatment caused the

regeneration in the IL. Consequently, regenerated and remainingβ cells in RME 100 treated group caused the hyper-secretion ofinsulin, which is also supported by the increased level of PI in RME100 treated group.

RME 100 and SME 100 treatment were more effective in reducinghyperglycemia and complications of diabetic neuropathy in diabeticrats rather than RME 250 and SME 250. It is likely that the higherdoses could not produce the expected effect by the presence of someother substances in the each extract, which may interfere its effect.However, in case of ethylacetate extract of leaf of Calotropis procera(LEE), the LEE 250 shown better effects compared to LEE 100 so itproduced its effects in dose dependent manner.

RME 100, SME 100 and LEE 250 extracts of Calotropis proceraameliorated diabetes and diabetic neuropathy in the STZ induceddiabetic rats. Further, RME 100 administration in the diabetic ratsnot only regenerated the β cells of the pancreas but also improvedthe PI and HbA1C% levels, which were responsible for most potenthypoglycemic effect than the other fractions of Calotropis procera.It clearly indicates that RME 100 treatment provide more pro-nounced anti-hyperglycemic effect, which causes the reversal ofsensory dysfunction associated with diabetic neuropathy. There-fore, the observed hypoglycemic effect of RME 100, SME 100 andLEE 250 may also be ascribed primary to decrease diabeticneuropathy. However, number of experimental reports indicatingantioxidant effect of Calotropis procera (Roy et al., 2005) stipulatesthe possibility of its antioxidant effects secondary to decreasediabetic neuropathy. Therefore, it may be proposed that RME 100induced decrease in HbA1C% level and oxidative stress are respon-sible for ameliorating axonal degeneration and diabetic neuro-pathy in STZ induced diabetic rats. Nevertheless, more elaborativestudies are required to identify the active components and precisesite of action of Calotropis procera in ameliorating the developmentof STZ induced diabetic neuropathy.

Fig. 5. Effect of RME 100 and standard compounds on pancreas (original magnification, 400� ). (a) NC vehicle treated group showing normal sized pancreatic islet structurewith a good number of β cells, even any lymphocytes is not present in between the islet cells. (b) SI treated group showing better architecture of IL with appearance of fewnumber of β cells. (c) SM treated group showing normal histological section of rat pancreas with improved structure of IL, without the presence of any lymphocytes.(d) SAþSMC treated group showing the good architecture of pancreas and lesser injuries with STZ. (e) SG treated group showing complete destruction of the structure of ILwith no appearance of β cells, indicating the poor protection of pancreas from STZ injuries. (f) RME 100 treated group showing better protection from degenerative changesand lesser injuries of IL from STZ injection, even the presence of some β cells indicating the regeneration. (g) Negative control (DC) rats showing marked degenerative andnecrotic changes in the IL like infiltration of lymphocytes in islet cells, dilation of extra-pancreatic acinii cells with marked destruction in the structure of IL. It indicates thatall of the β cells of IL were markedly shrunken due to the severe injury of STZ and the entire β cell mass was completely replaced by the fat cell.

S.K. Yadav et al. / Journal of Ethnopharmacology 152 (2014) 349–357356

5. Conclusion

RME 100 administration in diabetic rats not only attenuated thediabetic condition but also reversed diabetic neuropathy andneuropathic pain. It is potent anti-hyperglycemic and antioxidantattenuating actions may be responsible for the observed ameli-orative effects. In conclusion, the data of the present study suggesta potential protective role of Calotropis procera against STZ induceddiabetes and diabetic neuropathy in rats. These results furthervalidate the traditional use of Calotropis procera against diabetesand its complications.

References

Alan, C., Karin, E.B., 2009. Diabetes and atherosclerosis: is there a role forhyperglycemia? J. Lipid Res. 50, 335–339.

Ansari, S.H., Ali, M., 2001. Norditerpenic ester and pentacyclic triterpenoids fromroot bark of Calotropis procera (Ait) R.Br. Pharmazie 56, 175–177.

Aquino, R., De Simone, F., De Tommasi, N., Piacente, S., Pizza, C., 1995. Structure andbiological activity of sesquiterpene and diterpene derivatives from medicinalplants, Phytochemistry of Plants Used in Traditional Medicine. Oxford Uni-versity Press, New York, pp. 249–278

Brownlee, M., 2001. Biochemistry and molecular cell biology of diabetic complica-tions. Nature 414, 813–820.

Burcelin, R., Eddouks, M., Maury, J., Kande, J., Assan, R., Girard, J., 1995. Excessiveglucose production, rather than insulin resistance, account for hyperglycemiain recent onset streptozocin-diabetic rats. Diabetologia 385, 283–290.

Calcutt, N.A., Chaplan, S.R., 1997. Spinal pharmacology of tactile allodynia indiabetic rats. Br. J. Pharmacol. 122, 1478–1482.

Chen, F., Xiong, H., Wang, Ding, X., Shu, G., Mei, Z., 2013. Antidiabetic effect of totalflavonoids from Sanguis draxonis in type 2 diabetic rats. J. Ethnopharmacol. 149,729–736.

Deutschlander, M.S., Lall, N., Vandeventer, M., Hussein, A.A., 2011. Hypoglycemicevaluation of a new triterpene and other compounds isolated from Eucleaundulata Thunb. var. myrtina (Ebenaceae) root bark. J. Ethnopharmacol. 133,1091–1095.

Dewan, S., Sangraula, H., Kumar, V.L., 2000. Preliminary studies on analgesicactivity of latex of Calotropis procera. J. Ethnopharmacol. 73, 307–311.

Dubey, V.K., Jagannadham, M.V., 2003. Procerain, a stable cysteine protease fromthe latex of Calotropis procera. Phytochemistry 62, 1051–1057.

Dyck, P.J., Kratz, K.M., Karnes, J.L., Litchy, W.J., Klein, R., Pach, J.M., Wilson, D.M.,O'Brien, P.C., Melton , L.J., Service, F.J., 1993. The prevalence by staged severity ofvarious types of diabetic neuropathy, retinopathy, and nephropathy in apopulation-based cohort: the Rochester Diabetic Neuropathy Study. Neurology43, 817–824.

Eddy, N.B., Touchberry, C.F., Lieberman, J.E., 1950. Synthetic analgesics: I. Metha-done isomers and derivatives. J. Pharmacol. Exp. Ther. 98, 121–137.

Edwin, E., Sheeja, E., Dhanabal, S.P., Suresh, B., 2007. Antihyperglycemic activity ofPassiflora mollissima Bailey. Indian J. Pharm. Sci. 64, 570–571.

Gul, H., Yildiz, O., Dogrul, A., Yesilyurt, O., Isimer, A., 2000. The interaction betweenIL-1beta and morphine: possible mechanism of the deficiency of morphineinduced analgesia in diabetic mice. Pain 89, 39–45.

Hajializadeh, Z., Esmaeili-Mahani, S., Sheibani, V., Kaeidi, A., Atapour, M., Abbasnejad, M.,2010. Changes in the gene expression of specific G-protein subunits correlate withmorphine insensitivity in streptozotocin-induced diabetic rats. Neuropeptides 44,299–304.

Harati, Y., 1996. Diabetes and the nervous system. Endocrinol. Metab. Clin. NorthAm. 25, 325–359.

Hassan, S.W., Bilbis, F.L., Ladan, M.J., Umar, R.A., Dangoggo, S.M., Saidu, Y., Abubakar,M.K., Faruk, U.Z., 2006. Evaluation of antifungal activity and phytochemicalanalysis of leaves, roots and stem barks extracts of Calotropis procera(Asclepiadaceae). Pak. J. Biol. Sci. 9, 2624–2629.

Hoybergs, Y.M., Biermans, R.L., Meert, T.F., 2008. The impact of bodyweight andbody condition on behavioral testing for painful diabetic neuropathy in thestreptozotocin rat model. Neurosci. Lett. 436, 13–18.

Ilnytska, O., Lyzogubov, V.V., Stevens, M.J., Drel, V.R., Mashtalir, N., Pacher, P., Yorek,M.A., Obrosova, I.G., 2006. Poly(ADP-ribose) polymerase inhibition alleviatesexperimental diabetic sensory neuropathy. Diabetes 55, 1686–1694.

Inukai, T., Fujiwara, Y., Tayama, K., Aso, Y., Takemura, Y., 2000. Serum levels ofcarboxy-terminal propeptide of human type I procollagen are an indicator forthe progression of diabetic nephropathy in patients with Type 2 diabetesmellitus. Diabetes Res. Clin. Pract. 48, 23–28.

Kamath, J.V., Rana, A.C., 2002. Preliminary study on antifertility activity of Calotropisprocera roots in female rats. Fitoterapia 73, 111–115.

Kamenov, Z.A., Parapunova, R.A., Georgieva, R.T., 2010. Earlier development ofdiabetic neuropathy in men than in women with type 2 diabetes mellitus.Gender Med. 7, 600–615.

Kandhare, A.D., Raygude, K.S., Ghosh, P., Ghule, A.E., Bodhankar, S.L., 2012.Neuroprotective effect of naringin by modulation of endogenous biomarkersin streptozotocin induced painful diabetic neuropathy. Fitoterapia 83, 650–659.

Kannan, S.A., Saade, N.E., Haddad, J.J., Abdelnoor, A.M., Atweh, S.F., Jabbur, S.J.,Garabelian, B.S., 1996. Endotoxin induced local inflammation and hyperalgesiain rats mince, a new model for inflammatory pain. Pain 66, 373–379.

Kirtikar, K.R., Basu, B.D., 1935. Indian Medical Plants. vol. 3. Lalit Mohan Publishers,Allahabad, Uttar Pradesh, India p. 1606

Kokate, C.K., Purohit, A.P., Gokhale, S.B., 2006. Pharmacognosy, 35th ed. NiraliPrakashan, Pune, pp. 133–525

Kumar, V.L., Arya, S., 2006. Medicinal uses and pharmacological properties ofCalotropis procera. In: Govil, J.N. (Ed.), Recent Progress in Medicinal Plants, 11.Studium Press, Houston, Texas, USA, pp. 373–388.

Kumar, V.L., Basu, N., 1994. Anti-inflammatory activity of latex of Calotropis procera.J. Ethnopharmacol. 44, 123–125.

Kumar, V.L., Roy, S., 2007. Calotropis procera latex extract affords protection againstinflammation and oxidative stress in Freund's complete adjuvant-inducedmonoarthritis in rats. Mediators Inflamm. 2007, 47523–47529.

Lee, K.M., 2001. Overview of drug product development. Curr. Protoc. Pharmacol. 7,1–10.

Luna, L.C., 1990. Manual of Histological Screening Methods of Armed ForcesInstitute of Pathology. Mc Graw Hill Book, New York.

Mascolo, N., Sharma, R., Jain, S.C., Capassa, F., 1988. Ethnopharmacology ofCalotropis procera. J. Ethnopharmacol. 22, 211–221.

Mei, Z., Zhao, Y., Mo, S., Yang, Z., Zheng, T., Shu, G., 2012. Antidiabetic effect of totalsaponins from Entada phaseoloides (L.) Merr. in type 2 diabetic rats. J.Ethnopharmacol. 139, 814–821.

Meyer, R.A., Ringkamp, M., Campbell, J.N., Raja, S.N., 2005. Neural mechanisms ofhyperalgesia after tissue injury. Johns Hopkins Apl Tech. Digest 26, 56–66.

Necker, R., Hellon, R.F., 1978. Noxious thermal input from the rat tail: modulationby descending inhibitory influences. Pain 4, 231–242.

Perez, R.M., Zavala, G.M.A., Perez, S.G., Perez, C.G., 1998. Antidiabetic effect ofcompounds isolated from plants. Phytomedicine 5, 55–75.

Pop-Busui, R., Sima, A., Stevens, M., 2006. Diabetic neuropathy and oxidative stress.Diabetes Metab. Res. Rev 22, 257–273.

Qureshi, A.A., Prakash, T., Patil, T., Viswanath-Swamy, A.H., Gouda, A.V., Prabhu, K.,Setty, S.R., 2007. Hepatoprotective and antioxidant activities of flowers ofCalotropis procera (Ait) R.Br. in CCl4 induced hepatic damage. Indian J. Exp. Biol.45, 304–310.

Randall, L.O., Selitto, J., 1957. A method for measurement of analgesic activity ofinflamed tissue. Arch. Int. Pharmacodyn. Ther. 111, 209–219.

Rastogi, R.P., Mehrotra, B.N., 1993. Compendium of Indian Medicinal Plants. vol. 1.Central Drugs Research Institute, Lucknow and Publication & InformationDirectorate, New Delhi, pp. 71–72

Romero-Aroca, P., Fernández-Balart, J., Baget-Bernaldiz, M., Martinez-Salcedo, I.,Méndez-Marín, I., Salvat-Serra, M., Buil-Calvo, J.A., 2009. Changes in thediabetic retinopathy epidemiology after 14 years in a population of Type1 and 2 diabetic patients after the new diabetes mellitus diagnosis criteriaand a more strict control of the patients. J. Diabetes Complications 23, 229–238.

Roy, S., Sehgal, R., Padhy, B.M., Kumar, V.L., 2005. Antioxidant and protective effectof latex of Calotropis procera against alloxan-induced diabetes in rats. J.Ethnopharmacol. 102, 470–473.

Singh, R.K., Mehta, S., Jaiswal, D., Rai, P.K., Watal, G., 2009. Antidiabetic effect ofFicus bengalensis aerial roots in experimental animals. J. Ethnopharmacol. 123,110–114.

Singh, V., Panday, R.P., 1998. Ethnobotany of Rajasthan, Scientific, first ed. Publish-ers, Jodhpur, pp. 65–66

Tamrakar, A.K., Jaiswal, N., Yadav, P.P., Maurya, R., Srivastava, A.K., 2011. Pongamolfrom Pongamiapinnata stimulates glucose uptake by increasing surface GLUT4level in skeletal muscle. Mol. Cell. Endocrinol. 339, 98–104.

Yamagishi, S, Imaizumi, T., 2005. Diabetic vascular complications: pathophysiology,biochemical basis and potential therapeutic strategy. Curr. Pharm. Des. 11,2279–2299.

S.K. Yadav et al. / Journal of Ethnopharmacology 152 (2014) 349–357 357