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Prokinetic Effect of Herbomineral Unani Formulation ( Dolabi )in Diabetic RatsRahul Somani, Abu Shaikh, Dilpesh Jain and Rajkumar Shete
Energy Dispersive X-Ray Spectroscopy in Quality Control ofPowdered Herbal Formulation - Avipattikar ChurnaK. Jayaram Kumar and Mouli Nandi
Clinical Trial of Garbhpal Ras in Pregnancy OutcomeDeepa Mishra, Mukta Sinha and Vikas Kumar
Inhibitory Effect of the Root of Sida acuta Burm. F. onCalcium Oxalate Crystal GrowthT. Vimala and S. Gopalakrishnan
Spectroscopic Investigations of Palakarai (Cowrie shell) ParpamS. Joseph Vedhagiri, K. Ganesan and P.C. Jobe Parabakar
Hypoglycaemic Activity of Indian Medicinal Plants inStreptozotocin Diabetic RatsE.N. Sundaram, K.P. Singh and P. Umamaheswara Reddy
Pharmacological Screening of Cassine albens (Retz.) Kosterm (Celastraceae)for Antidepressant and Anxiolytic Activity in RodentsP.H. Patil, M.B. Gagarani, K.R. Patil and S.J. Surana
Screening of Ximenia americana L. forit’s Anti-inflammatory ActivityM. Siddaiah, K.N. Jaya Veera, P. Mallikarjuna Rao, K. Yogananda Reddy andC. Madhusudhana Chetty
In-vitro Antioxidant Capacity of Graded Doses ofMethanolic Extract from Luffa cylindrica (L) SeedsK. Nagarajan, Satyajit Dutta, Sumit Das, Surabhi Singhal,Pallavi Saxena, Avijit Mazumder and L.K. Ghosh
Antibacterial Activity of Alcoholic Extract of Aloe vera L. byDisc Diffusion MethodG.S. Niture, M.K. Patil, A.G. Karpe and A.V. Bhonsle
Volume XVIII : 1 e-Version Jan. - March, 2012
THE JOURNAL OFRESEARCH AND EDUCATION
IN INDIAN MEDICINEAn International Quarterly
ISSN 0970-7700
EDITOR - IN - CHIEFProf. Em. R.H. SINGH ABMS, Ph.D, D.Sc.Professor EmeritusBanaras Hindu UniversityFormerly Vice Chancellor,Rajasthan Ayurveda University;Dean, Faculty of Ayurveda,IMS, BHU, Varanasi - 221005 UP (India)FOUNDING EditorProf. (Dr.) Suresh Kumar
M.D.(Ay.), Ph.D. Kayachikitsa (BHU)Formerly Dean, Faculty of Ayurveda,Himachal Pradesh University, ShimlaDirector, Indian Institute of Panchakarma,CCRAS (AYUSH,MoH&FW,GoI) (Kerala)CONSULTANT SUBJECT EDITORSProf. Ranjana Patnaik Ph.D. (BHU)Professor, School of Bio Medical Engg,Institute of Technology, BHU,Varanasi - 221005 (India)Dr. Sanjeev Rastogi M.D.(Ay.) (BHU)Head, Dept. of Pancha KarmaState Ayurvedic College & HospitalLucknow - 226004 (India)E-mail: [email protected]. Manoj Kumar M.S.(Ay.) (BHU)Assistant Professor-Shalakya TantraInstitute of Medical Sciences,BHU, Varanasi - 221005 (India)Ravindra G. MaliAssistant Professor,Pharmacognosy & PhytochemistryL.B.Rao Institute of Pharmaceutical Educ. &Research, B.D.Rao College Campus,Khambhat - 388620 (Gujarat)ADVISORY-EDITORS (INTERNATIONAL)Dr. Jagat Kanwar M.Sc., Ph.D.Associate Professor,Immunology & Cell BiologyInstitute for Technology & ResearchInnovation (ITRI), Deakin University,Victoria - 3217 (Australia)E-mail: [email protected]. Marc HalpernPresident, California College of AyurvedaE-mail: [email protected] EDITORSDr. Girish Mishra M.D.(Ay) Kayachikitsa (BHU)
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The articles published in this Journal are selectively abstracted / indexed inMEDICINAL & AROMATIC PLANTS ABSTRACTS (MAPA) CSIR publication Reporting Current World Literature (Bimonthly),
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J. Res. Educ. Indian Med., Vol. XVIII (1) : 2012 Conten ISSN 0970-7700 (Linking)
CONTENTS
Prokinetic Effect of Herbomineral Unani Formulation ( Dolabi )in Diabetic Rats
Rahul Somani, Abu Shaikh, Dilpesh Jain andRajkumar Shete ... 1-6
Energy Dispersive X-Ray Spectroscopy in Quality Control ofPowdered Herbal Formulation - Avipattikar Churna
K. Jayaram Kumar and Mouli Nandi ... 7-11
Clinical Trial of Garbhpal Ras in Pregnancy OutcomeDeepa Mishra, Mukta Sinha and Vikas Kumar ... 13-19
Inhibitory Effect of the Root of Sida acuta Burm. F. onCalcium Oxalate Crystal Growth
T. Vimala and S. Gopalakrishnan ... 21-26
Spectroscopic Investigations of Palakarai (Cowrie shell) ParpamS. Joseph Vedhagiri, K. Ganesan andP.C. Jobe Parabakar ... 27-32
Hypoglycaemic Activity of Indian Medicinal Plants inStreptozotocin Diabetic Rats
E.N. Sundaram, K.P. Singh andP. Umamaheswara Reddy ... 33-43
Pharmacological Screening of Cassine albens (Retz.) Kosterm(Celastraceae) for Antidepressant and Anxiolytic Activity inRodents
P. H. Patil, M.B. Gagarani, K.R. Patil and S.J. Surana ... 45-50
Screening of Ximenia americana L. forit’s Anti-inflammatory Activity
M. Siddaiah, K.N. Jaya Veera, P. Mallikarjuna Rao,K. Yogananda Reddy and C. Madhusudhana Chetty ... 51-54
In-vitro Antioxidant Capacity of Graded Doses ofMethanolic Extract from Luffa cylindrica (L) Seeds
K. Nagarajan, Satyajit Dutta, Sumit Das,Surabhi Singhal, Pallavi Saxena,Avijit Mazumder and L.K. Ghosh ... 55-59
Antibacterial Activity of Alcoholic Extract ofAloe vera L. by Disc Diffusion Method
G.S. Niture, M. K. Patil, A.G. Karpe and A.V. Bhonsle ... 61-63
Conferences and Forthcoming Events ... 64
e-Version
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J. Res. Educ. Indian Med., Jan. - March, 2012 Vol. XVIII (1) : 1-6 ISSN 0970-7700
PROKINETIC EFFECT OF HERBOMINERAL UNANIFORMULATION (DOLABI) IN DIABETIC RATSRAHUL SOMANI,1* ABU SHAIKH,1 DILPESH JAIN1 AND RAJKUMAR SHETE2
Sinhgad College of Pharmacy,1 Pune - 411041 Maharashtra (India)RD’s College of Pharmacy,2 Bhor - 412206 Maharashtra (India)
Abstract: This study was undertaken to investigate the prokinetic activity of Unani herbomineralformulation (Dolabi) in streptozotocin induced diabetic rats and its in vitro antioxidant activity. Ratmodel of diabetes was established by intraperitoneal injection of streptozotocin (55 mg/kg, i.p).Rats were divided into three groups: Normal control, diabetic control and treatment groups. Aftertwo weeks of treatment, rats were administered with phenol red meal followed by last dose ofDolabi and they were screened for gastric emptying (GE), intestinal transit (IT) and in vitro studyof distal colonic smooth muscle. In vitro antioxidant activity of Dolabi was assessed on the basisof radical scavenging activity of the stable Diphenyl-2-picryl-hydrazyl (DPPH) free radical.Percentage of GE and IT was significantly (P < 0.05) decreased in diabetic rat as compared tonormal control groups. In streptozotocin induced diabetic rats, Dolabi significantly (P < 0.05)accelerated both GE and IT as compared to diabetic control rats. Significant (P<0.01) increase inEC50 of ACh in rat distal colon was observed in diabetic rats as compared to normal rats. Whereas diabetic rats treated with Dolabi showed significant (P<0.01) decrease in EC50 of ACh in distalcolon as compared to diabetic control group. In in vitro study, Dolabi showed potent radicalscavenging activity to stable DPPH-free radical with IC50 value of 231.09. Dolabi may exert itsprokinetic effect by reducing oxidative stress and therefore can be used as drug for treating diabeticpatients with gastrointestinal impairments.
Keywords: Dolabi, Unani medicine, Herbal medicine, Diabetes, Oxidative stress, Medicinalplants, Diabetic neuropathy.
IntroductionOxidative stress play an important role in
the pathogenesis of chronic complications ofdiabetes mellitus (Ziegler and Gries, 1997)including gastroparesis (James et al., 2008). Theconditions that lead to the over production of theprecursors of ROS and/or reduce the efficiencyof scavenging system are shown to be responsiblefor the development of oxidative stress.Hyperglycemia play an important role ingeneration of reactive oxygen species (ROS) andthat lead to chronic diabetic complicationsincluding diabetic autonomic neuropathy.Gastroparesis is the most common symptom ofdiabetic autonomic neuropathy (Kong et al.,1999) which is reported to cause considerablemorbidity in patients. Gastroparesis leads toabnormal gastric motility, characterized bydelayed gastric emptying (GE) and intestinal
transit (IT) (Shamaila et al., 2009). The abnormalgastrointestinal motility among diabetic patientsseemes to be a clinical manifestation of diabeticautonomic neuropathy (Punkkinen et al., 2008)and some reports concluded that thesegastrointestinal disturbances may be due to thedamage of peripheral cholinergic neurons as aresult of oxidative stress (Bijender et al., 2003;De Winter et al., 2005).
Herbomineral formulation (Dolabi) is usedfor treatment of diabetes and its complications inan Unani system of medicine. It containsGymnema sylvestre, Eugenia jambolana,Bambusa arundinacea, Rumex vescarricus,Acacia arabica, oxide of egg shell, oxide of ironrust, zinc oxide (Table 1). Some of theseingredients have been reported to possess bothanti-diabetic as well as antioxidant activity such
1* Corresponding Author
Somani et al.2
as E. jambolana (Sagrawat et al., 2006),A. arabica (Wadood et al., 1989; Sundaramand Mitra, 2007), zinc oxide (Robert andSilvestro, 2000) where as Gymnema sylvestreknown for its antidiabetic effect (Gholap andKar, 2003). So far there is no scientific evidenceabout efficacy of this formulation in preclinicalmodels of impaired gastrointestinal motility andin vitro antioxidant activity. The presentinvestigation was under taken to evaluate theeffect of Dolabi on impaired gastrointestinalmotility, colonic smooth muscle response toexogenous acetylcholine (ACh) in streptozotocin(STZ) induced diabetic rats and in vitroantioxidant activity.
Materials and MethodsDrug and Chemicals
Dolabi (Hamdard) a herbomineralformulation, purchased from local market.Streptozotocin, phenol red and DPPH werepurchased from Sigma (U.S.A.).
Rat model of diabetesWistar rats of either sex weighing 180-
230g were purchased from Haffkine Bio-PharmaCorporation Ltd., Mumbai (India). All theexperimental procedures and protocols used inthis study were reviewed and approved (SCOP/
IAEC/Approval/2008-09/05) by the InstitutionalAnimal Ethics Committee. Animals were housedunder standard laboratory conditions at controlledtemperature 25 ± 1ºC with 50-60% relativehumidity in a normal 12 h light and dark cyclewith free access to water and standard laboratoryfeed ad libitum.
Overnight fasted rats were injected withstreptozotocin (55 mg/kg, i.p.) dissolve in 0.1 Mcold citrate buffer (pH 4.45). The blood waswithdrawn 48 h later by retro orbital method,serum was separated and fasting serum glucoselevel was determined using the glucose oxidase-peroxidase method (Miskiewicz et al., 1973).Rats with a serum glucose level > 250 mg/dlwere considered as diabetic and used for furtherstudy. Age matched five non-diabetic rats wereused as normal control group and received 0.5mlof 0.1 M cold citrate buffer (pH 4.45).
Study design for gastrointestinal transit andin vitro study on rat distal colon
After persistent hyperglycemia for twoweeks, diabetic rats were divided into two groups.Group one was diabetic control, received distilledwater (10 ml/kg) and second group served astreatment, received suspension of Dolabi indistilled water (140 mg/kg, p.o) for next two
Table 1. Contents of Unani herbomineral formulation (Dolabi)
Name of content Sr.
No Unani Name Botanical Name
(Family)
English Name
Quantity
1 Aqaqiya Acacia arabica
(Fabaceae)
Gum Arabic Tree 166.6 mg
2 Banslochen Bambusa arundinaceae
(Bambusaceae)
Thorny bamboo 132.0 mg
3 Tukhm Hammaz Rumex vesicarius
(Polygonaceae)
Rosy Dock, Dock Sorrel,
Bladder Dock
83.3 mg
4 Gurmar Booti Gymnema sylvestre
(Asclepiadaceae)
Gymnema 27.7 mg
5 Maghz Jamun Labba Buz Syzygium cumini
(Myrtaceae)
Black Plum, Java Plum 27.7 mg
6 Kushta Baiz Murgh --
--
--
Egg gallius domestics
(oxide of egg shell)
13.8 mg
7 Kushata Khabsul Hadeed Iron (oxide of iron rust) 13.8 mg
8 Kushta Jast Zinc oxide 41.6 mg
9 Gond Safaid -- --
41.6 mg
200.0 mg 10 Labba Buz
-- --
Prokinetic Effect of Herbomineral Formulation in Diabetic Rats 3
weeks. Age matched five non-diabetic rats wereused as normal control group and received distilledwater (10 ml/kg). Gastrointestinal transit and invitro study on rat distal colon were performed.
Gastric emptying and intestinal transitAfter administration of last dose of Dolabi
to the overnight fasted rats, 1.5 ml of a phenol redmeal, consisting of phenol red (0.05%, w/w) in1.5% methylcellulose, was given through gavagefeeding. Twenty minutes later, the rats weresacrificed by cervical dislocation. Their stomachswere clamped with a string above the loweroesophageal sphincter and a string beneath thepylorus to prevent leakage of phenol red. Gastricemptying was determined spectrophotometrically.
The stomach of each rat resected just abovethe lower oesophageal sphincter and pyloricsphincter. Phenol red remained partly in the lumenof the stomach. The stomach and its contents wereput into 5 ml of 0.1 mol/l NaOH. The stomachwas minced. The samples containing the totalamount of phenol red present in the stomach werefurther diluted to 25 ml with 0.1 mol/l NaOH andleft at room temperature for 1 h. The supernatant(5 ml) was then centrifuged at 800 g for 20 min.
The absorbance was read at a wavelengthof 546 nm on a spectrophotometer (Shimadzu,Japan) and the phenol red content in the stomachwas calculated. Percentage of gastric emptyingof the phenol red was calculated as
[(infusion amount-remains) / infusionamount] × 100.
The intestinal transit (IT) of phenol redmeal was determined by modified Janseenmethod (Janseen and Jagenerous, 1957). Thesmall intestine was removed from the pyloricsphincter to the ileocecal junction and the distancetravelled by the phenol red meal was noted andexpressed as percentage of intestinal transitcalculated as
[distance traveled by phenol red meal/total length of small intestine] × 100In vitro study on rat distal colon
Immediately after cleaning and measuringthe length of large intestine, 2 cm distal colon
was cut and used for in vitro study. The distalcolon was dissected out and mounted underresting tension of 0.5 g in an organ bathcontaining continuously aerated tyrode’s solution.Dose response curves were obtained withascending doses of ACh (100μg/ml). EC50 valueswere calculated from graph plotted using percentresponses against log dose.
In vitro antioxidant activityIn vitro antioxidant activity of Dolabi was
assessed on the basis of radical scavenging effectof the stable DPPH-free radical. Free radicalscavenging activity of different concentration ofDolabi and ascorbic acid were measured usingDPPH, employing method of Blois (Blois, 1958).Solutions of different concentration (50, 100, 150,200, 250 μg/ml) of Dolabi and ascorbic acidwere added to 0.01mM, solution of DPPH inmethanol. After 30 min, absorbance was measuredat 517 nm, using spectrophotometer (Shimadzu,Japan). 0.01mM solution of DPPH in methanolwas used as control. All tests were performed intriplicate. IC50 value was calculated. The DPPHradical scavenging activity was calculatedaccording to the following equation,
DPPH radical scavenging activity (%) = A0 – A1/ A0 x 100
Where,A0 is the absorbance of DPPH,A1 is the absorbance of DPPH solution
in presence of the extract.
Statistical analysisStatistical analysis of data was conducted
using one-way ANOVA followed by Dunnett’stest. Data were expressed as mean ± SEM, P<0.05was considered statistically significant.
ResultsEffect of Dolabi on delayed gastric emptyingin diabetic rats
Gastric emptying (GE) was significantly(P < 0.01) decreased in diabetic rats as comparedto normal rats (46.24 ± 3.64 vs 57.57 ± 1.96 %,
Somani et al.4
Figure 1). In STZ induced diabetic rats, Dolabisignificantly (P<0.05) accelerated gastricemptying (GE) as compared to diabetic controlrats (59.05 ± 2.09 vs 46.24 ± 3.64 %, Figure 1).
Effect of Dolabi on delayed small intestinaltransit in diabetic rats
Intestinal transit (IT) was significantly(P < 0.05) decreased in diabetic rats compared tonormal rats (45.43 ± 1.94 vs 60.93 ± 5.00 %,Figure 2). In STZ induced diabetic rats, Dolabisignificantly (P<0.05) accelerated intestinal transit(IT) as compared to diabetic control rats (64.32 ±4.16 vs 45.43 ± 1.942 %, Figure 2).
Effect of Dolabi on EC50 of ACh in distalcolon of diabetic rats
EC50 value of Ach in rat distal colon wassignificantly (P<0.01) increased in diabetic rats
as compared to normal rats (41.52±3.94 vs20.89±3.25 μg, Figure 3).
In STZ induced diabetic rats, Dolabi showsignificant (P<0.01) decrease in EC50 value ofACh in distal colon as compared to diabeticcontrol rats (14.23±2.59 vs 41.52±3.94 μg,Figure 3).
Effect of Dolabi on in vitro antioxidantactivity
Dolabi exhibited in vitro antioxidantactivity with IC50 value of 232.11 μg/ml(Figure 4).
DiscussionPropulsive motility is termed as peristalsis
and it is subserved by a complex pattern of neuralreflexes that aim to relax intestinal muscledownstream (descending inhibitory reflex) and
Data represented as mean ± SEM. ## P< 0.01, compared to normal control group;**P< 0.05, compared to diabetic control group (ANOVA followed by Dunnett’test)
Figure 1. Effect of two weeks repeated dose treatment ofDolabi on % of gastric emptying (GE %) inSTZ induced diabetic rats
Data represented as mean ± SEM. ## P< 0.01, compared to normal control group;**P< 0.01, compared to diabetic control group (significance by one way ANOVA followed by Dunnett’ test)
Figure 3. Effect of two weeks repeated dose treatment ofDolabi on EC50 of ACh in rat distal colon in STZinduced diabetic rats
Figure 2. Effect of two weeks repeated dose treatment ofDolabi on % of intestinal transit (IT %) in STZinduced diabetic rats
Data represented as mean ± SEM # P< 0.05, compared to normal control group;* P< 0.05, compared to diabetic control group(significance by one way ANOVA followed by Dunnett’ test)
Data expressed as mean ± SEM from three observations
Figure 4. Effect of Dolabi on DPPH free radical scavengingactivity
Prokinetic Effect of Herbomineral Formulation in Diabetic Rats 5
contract the muscle upstream (ascendingexcitatory reflex) of the intestinal bolus. Intestinaltransit is controlled by both neural and myogenicmechanisms (Huizinga et al., 1998). An increaseof the contractile activity of the smooth musclelayers is in general responsible for accelerationof intestinal propulsion. Several mediators andneurotransmitters are responsible for these motorpatterns. Acetylcholine is the main excitatoryneurotransmitter in the enteric nervous system,whereas NO is the major transmitter of theinhibitory motor neurons (Waterman and Costa,1994). Disorder of autonomic functions(Punkkinen et al., 2008 ) and extrinsic nervesupply to the gut are known to be responsiblefor the disturbed gastric motility associated withdiabetes.
In the present study, STZ induced diabeticrats had mild or moderate gastroparesis which ischaracterized by slow gastric emptying andintestinal transit as compared with normalcontrols. Similar delayed gastric emptying,intestinal transit were seen in the STZ diabeticrats which is in agreement with previous studies(Bijender et al., 2003; Young et al., 2006; El-Salhy, 2002a; Anjaneyulu and Ramarao, 2002;El-Salhy, 2002b). Thus, rat with STZ induceddiabeties could be used as an animal model ofdiabetic gastroparesis. Disturbed motility ofgastrointestinal tract was also reported in thehuman with diabetes mellitus (Russo et al., 1997).
The exact cause of slow gastrointestinaltransit in diabetic patients is not known, butseveral mechanisms have been proposed. Mostimportant among them, is damage of peripheralcholinergic nerve as a result of oxidative stress(Bijender et al., 2003). A significant reductionin the contractile response of distal colonicsmooth muscle to exogenous Ach was reportedin STZ induced diabetic rats.
Treatment with antioxidant vitamin E,significantly increase contractile response ofdistal colonic smooth muscle to exogenous Achas well as accelerate small intestinal transit indiabetic rats which confirm role of oxidativestress in damage of peripheral cholinergic neuron
associated with diabetic autonomic neuropathy(Bijender et al., 2003).
Acute change in blood glucoseconcentration has also major effect ongastrointestinal motor function in healthy subjects(Russo et al., 1997). In particular, acutehyperglycemia inhibits both the gastrointestinaland ascending components of peristaltic reflex.Poor glycemic control has the potential to causedelayed gastrointestinal transit in diabetic patients(Jung et al., 2003). Therefore drug which controlboth blood glucose as well as having antioxidantactivity will be the best one, for treating diabeticassociated gastrointestinal trouble.
To the best of our knowledge, this is the firstreport on the effect of herbomineral formulation(Dolabi) on the gastrointestinal dysmotility instreptozotocin induced diabetic rats and its in vitroantioxidant activity using DPPH. Dolabisignificantly accelerate gastric emptying, intestinaltransit and increase contractile response of distalcolonic smooth muscle to exogenous ACh in STZinduced diabetic rats. It also exhibit in vitroantioxidant activity to DPPH.
Mechanism underlying the action of Dolabion impaired gastric motility may be neuronaldependent and its antioxidant activity may playan important role. Our data strongly reveals thatDolabi exert a prokinetic action on gastricemptying and intestinal transit in diabetic rats.The present study also suggests that antioxidantproperty of Dolabi may be responsible for haltingprogressive changes of chronic diabetes leadingto gastric impairment. However, further detailedstudies like estimation of endogenous antioxidantenzyme levels are needed.
AcknowledgmentsThe authors wish to thank Prof. M.N.
Navale, Founder President, STES and Dr. K.S.Jain, Principal, SCOP for providing facilities tocarryout this work.
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Address for correspondence: Prof. Rahul Somani, Sinhgad College of Pharmacy, Pune - 411041 Maharashtra(India). E-mail: [email protected]
011_2010
J. Res. Educ. Indian Med., Jan. - March, 2012 Vol. XVIII (1) : 7-11 ISSN 0970-7700
ENERGY DISPERSIVE X-RAY SPECTROSCOPY IN QUALITY CONTROLOF POWDERED HERBAL FORMULATION - AVIPATTIKAR CHURNAK.JAYARAM KUMAR* AND MOULI NANDI
Department of Pharmaceutical Sciences,Birla Institute of Technology, Mesra, Ranchi - 835215 Bihar (India)
Abstract: Energy dispersive X-ray spectroscopy (EDX) is an analytical technique used forthe elemental analysis and chemical characterization of a sample. Many of the Ayurvedic formulationscontain salt and trace elements, which help to optimize the therapeutic efficacy of the main drugs.The elements generally present are sodium, potassium, chlorine, mercury, sulphur, silver etc. EDXnot only helps to quantify the minerals or salts added as one of the ingredients but also helps toquantify the presence of the elements of a formulation as well. It can be used as an importantmeasure for quality control of Ayurvedic formulation. Thus, it can be used for their quantificationeven in minor amounts. In the present study quantification of different trace elements of twodifferent marketed formulations and in house formulation and a formulation without salt ofAvipattikar churna has been done by flame photometry and EDX. The result shows the presenceof sodium, potassium, chlorine, calcium, copper and zinc. Quantification of sodium, potassium hasbeen done by flame photometry but the results obtained by EDX showed better sensitivity,precision and accuracy.
Keywords: Energy dispersive X-ray spectroscopy, Flame photometry, Trace elements,Quality control, Avipattikar churna, Ayurvedic drugs.
IntroductionAccording to an estimate of World Health
Organization (W.H.O.) nearly 80% of populationof developing countries relies on traditionalmedicines, mostly on plant drugs for theirprimary health care needs. Most of the traditionalsystems of medicines are effective but the needis just to validate them. Validation of herbalmedicines is one of the toughest challenges forthe scientist. There is a need to develop standardsto bring this system of medicine in themain stream of health sciences (Mukherjee,2003). In India Ayurveda, Unani and Siddha havebeen used for many centuries. Central Councilfor Research in Ayurveda and Siddha (C.C.R.A.S)has given preliminary guidelines forstandardization of these formulations but for theuniformity of batches in production of Ayurvedicformulations. It is necessary to develop methodsusing phytochemical markers (Sane, 2005).However, the analysis and the structure of the
finished product, safety and side effects of theformulations, correct doses and duration of thetreatment, mode of action remains unanswered(Prakash, 1999). Energy dispersive x-rayspectroscopy (EDX) is a technique used for theelemental analysis and chemical characterizationof a sample. Different sample types (solidmaterials, liquids, powders, metals, minerals, etc.)can be analyzed with simple sample preparationover a wide range of concentrations (from tracesto main components) is possible without dilution(Juillet, 1996). It is a new technique used toidentify and quantify the trace elements presentin a sample. Many of the Ayurvedic formulationscontain salt and trace elements, which help tooptimize the therapeutic efficacy of the maindrugs. So EDX can be used as an important toolin standardization of Ayurvedic formulation.
In classical Ayurvedic books, Avipattikarchurna is mentioned to be used in digestive andirritable bowel disorders. It is composed of twelve
* Reader in Pharmacognosy
Kumar and Nandi8
different herbs and spices along with sugar andsalt (Table 4).
Herbs include Emblic myrobalan, Ginger,Belliric myrobalan, Nutgrass, Myrobalan, Longpepper, Black pepper, Vidang, Cardamom, Indiancinnamon, Clove, Trivrt (Indian jalap), sugarand Vida lavana (black salt). Though Indianjalap being the main active ingredient fortherapeutic efficacy but the presence ofsugar and salt is also important as sugar reducespitta. People with a predominantly pittaconstitution are thought to be susceptible tohypertension, heart disease, infectious diseases,and digestive conditions[http://www.amritaveda.c o m / l e a r n i n g / a r t i c l e s / Ta s t e
chart_ back_ bw_final.pdf]. accessed on date13.11.2010.
Vida lavana contains a small amount ofnatural amounts of trace elements such as ironand magnesium. It is carminative and tonic tothe digestive system. It replenishes salt lost inexercise and adding to trace elements essentialfor normal health and fitness. Potassium, sodium,chloride helps in regulation of Na/K/CA ATPase,glucose transport, transport of some amino acidsincluding alanine, proline, tryptophan and tyrosine.
Table 1. Sodium and potassium content by flame
photometer.
Sample Sodium content
(% weight)
Potassium content
(% weight)
A 0.2508 0.322
B 0.3812 0.417
In house 0.325 0.473
In house
without salt 0.1142 0.3554
Table 4. Constituents of herbal formulation Avipattikar churna.
Sl.
No.
Sanskrit / Hindi
Name
Botanical Name
1 Shunthi (Zingiber officinale)
2 Maricha (Piper nigrum)
3 Pippali (Piper longum)
4 Amalaki (Embelica officinalis)
5 Vibhitaki (Terminalia bellirica)
6 Haritaki (Terminalia chebula)
7 Vidang (Embelia ribes)
8 Nagarmotha (Cyperus rotundus)
9 Ela (Elettaria cardamomum)
10 Tejapatara (Cinnamomum zeylanicum)
11 Launga (Syzygium aromaticum)
12 Nishotha (Operculina turpethum)
13 Candy sugar
Potassium ions are involved in a numberof essential physiological processes, such as themaintenance of intracellular acid-base balance andtonicity. Copper aid enzymes, specifically inoxidation of iron. Zinc is essential in reactionsinvolving synthesis such as carbohydrates, lipids,proteins and nucleic acids. [http://web.uct.ac.za/depts/git/ibd/vits.htm# Minerals2] accessed on date13.11.2010.
Table 3. Comparison of sodium and potassium by
flame photometry and EDX (in % weight).
Flame photometry EDX Sample
Sodium Potassium Sodium Potassium
A 0.2508 0.322 0.50 0.46
B 0.3812 0.417 0.58 0.82
In
house 0.325 0.473 - 0.60
In
house
without
salt
0.1142 0.3554 - 0.34
Table 2. Trace element obtained by EDX (in % weight).
Sample Sodium Potassium Calcium Copper Zinc Chloride
A 0.50 0.46 0.55 1.39 1.24 0.72
B 0.58 0.82 0.46 3.15 2.71 1.04
In house - 0.60 1.42 2.58 2.24 0.65
In house without salt - 0.34 0.40 1.94 1.89 -
EDX in Quality Control of Herbal Formulations 9
Figure 1,3,5,7 shows the surface electron image of formulation A, B, in house and in house without salt respectivelyFigure 2,4,6,8 shows the graph showing the quantity of the present trace element of formulation A,B, in house and in house without salt respectively.
Figure 2
Figure 4
Figure 6
Figure 1
Figure 3
Figure 5
Kumar and Nandi10
Materials and MethodsInstruments and Chemicals
Systronics flame photometer 128 was usedfor flame photometry and energy dispersiveX-ray spectroscopy was done in JEOL-JSM -6390LV–SEM analyzer. Millipore water was usedfor flame photometry.Methods for Flame Photometry
1000 ppm standard solution of analyticalgrade sodium chloride and potassium chloridewas prepared. From that stock solution20,40,60,80,100 ppm solution of sodium chlorideand potassium chloride was prepared.
The sample was prepared by taking 1 g offour different formulations in 100 ml of water.It is then filtered and 10 ml of the filtrate wastaken and volume was made up to 100 ml. Theirsodium content and potassium content wasmeasured by Systronics flame photometer 128(McNaught and Wilkinson, 1997).
They were converted to percentage weightfor the convenience of comparison as the resultsobtained from EDX were in percentage weight.
Methods for Energy Dispersive X-raySpectroscopy
EDX analysis was done in JEOL- JSM -6390LV –SEM analyzer. At first the atmosphericair was poured inside the chamber and vacuumwas released. It is called Vent mode. Then thechamber was opened and the sample was placed.Again vacuum was regenerated with pumps. After
creation of vacuum, the sample was movedtowards the beam. Accelerating voltage was 20kv, working distance was 10 m.m. and spotsize was 40-70. Few milligrams of the sampleswere used in for the analysis. Comparison ofresults obtained from flame photometry and EDXwas done (Seiichi and Hiroyuki, 1986; Kazuoet al., 1996).
The results obtained from flamephotometry and EDX are given in Table 1 andTable 2. Comparison of the amount of sodiumand potassium by flame photometry and EDXare shown in Table 3.
ResultsIn house formulation without salt also
shows the presence of sodium, potassium,chlorine and other trace elements. The results offlame photometry show presence of sodiumranges from 0.1142% to 0.3812% weight andpotassium ranges from 0.322% to 0.473%. InEDX the presence of sodium ranges from0.50% to 0.58% weight and potassium rangesfrom 0.34% to 0.82% weight. In EDX presenceof other trace elements was also detected andquantified.
DiscussionThe results obtained in EDX and flame
photometry shows quite a difference. However,in EDX presence of other trace elements was
Figure 8
Figure 7
EDX in Quality Control of Herbal Formulations 11
found like chlorine, copper, zinc calcium, etc. Inhouse formulation without salt also shows thepresence of sodium, potassium, chlorine andother trace elements. It indicates the presence oftrace elements in the ingredients other than thesalt. Thus EDX gives a better comprehension oftrace elements.
AcknowledgementsFinancial help rendered by AICTE to carry
out this research work is gratefullyacknowledged. We are thankful to the Head,Department of Pharmaceutical Sciences, BIT,Mesra, Ranchi (India) for permission to carryout this experimental work.
References1. McNaught AD and Wilkinson A: IUPAC.
Compendium of Chemical Terminology. 2nd edition(the Gold Book). Blackwell Scientific Publications,Oxford (1997).
2. Ayurveda’s Six Tastes and their Effects on theDoshas http://www.amritaveda.com/learning/articles Taste_chart_back_bw_final.pdf(13.11.10)
3. Backgrounder Ayurvedic Medicine: AnIntroduction http://nccam.nih.gov/health/ayurveda/D287_BKG.pdf(13.11.10)
4. The Ayurvedic Formulary: Government of IndiaMinistry of Health and Family Planning,Department of Health, Part-1, 1st edition.
Government of India Press, Faridabad, Controllerof Publication, New Delhi, India. pp 87 (1978).
5. Juillet HM: Spectroscopy for the determinationof trace and main elements. SPECTRO AnalyticalInstruments, Kleve. Journal de physique IV.Colloque, supplkment au Journal de PhysiqueIII,C4 619-625 (1996).
6. Kazuo F, Mitsuaki O, Mitsuhiro A and TetsuyaS: Practical performance of energy dispersive x-ray spectroscopy with a high- voltage TEM upto 1,000 kv. Journal of Electron Microscopy. 45(4):285-290 (1996).
7. Lee. R and Kligler B: Interactive Medicine: Principlesfor Practice. McGraw-Hill Companies (2004).
8. Mukherjee PK: Exploring botanicals in Indiansystem of medicine-regulatory perspectives.Clinical Research and Regulatory Affairs. 20: 249–264 (2003).
9. Prakash VB: In Standardisation of Ayurvedicproducts including GMP of Ayurvedic industry,Kerala. Ayurvedic Medicine ManufacturerAssociation, Palakkad, Kerala, India (1999).
10. Sane VC: Past, present and future of Ayurvedicmedicines in India. The Pharma Review. 3: 29-33(2005).
11. Seiichi S and Hiroyuki K: Energy dispersiveX-ray spectroscopy in medium voltage electronmicroscope. Journal of Electron Microscopy. 35(4):335-342 (1986).
12. Vitamins, Minerals and Trace Elements http://w e b . u c t . a c . z a / d e p t s / g i t / i b d / v i t s . h t m #Minerals2(13.11.10)
Address for correspondence: K. Jayaram Kumar, Department of Pharmaceutical Sciences, Birla Institute ofTechnology, Mesra, Ranchi - 835215 Bihar (India). E-mail: [email protected]
036_2009
J. Res. Educ. Indian Med., Jan. - March, 2012 Vol. XVIII (1) : 13-19 ISSN 0970-7700
CLINICAL TRIAL OF GARBHPAL RAS IN PREGNANCY OUTCOMEDEEPA MISHRA,1* MUKTA SINHA2 AND VIKAS KUMAR3
Department of Prasuti Tantra,1,2 Institute of Medical Sciences,Pharmacology Laboratory, Department of Pharmaceutics,3 Institute of Technology,Banaras Hindu University, Varanasi – 221005 (India)
Abstract: Introduction: Ayurvedic sages suggest that a wholesome diet along with sheetvirya,balya, rasayan and madhur drugs during the period of pregnancy is must. This ultimately resultsin fetal growth, maternal health and post delivery lactation. Garbhpal ras, a drug mentioned in thetext of 17th century namely ‘Ras Chandanshu’ has such properties and is being used since then.It was claimed that Garbhpal ras is a panacea for feto-maternal well being. The aim of presentstudy was to assess the efficacy of Garbhpal ras in pregnancy outcome. Method: In present study,170 pregnant women were registered. Haematological and biochemical parameters were observed.Neonatal parameters like gestational age, weight, height, head circumference, chest circumference,mid arm circumference and Apgar score were assessed after delivery. Result and conclusion: Normalhaematological picture, maintained renal and liver function tests show non-toxic nature of Garbhpalras. Thus, it can be recommended safely in pregnancy at therapeutic doses.
Keywords: Garbhpal ras, Rasayan , Balya, Garbhsrava, Anupan, Antioxidant,Immunomodulatory, Pregnancy, Prevention of abortion, Ayurvedic medicine, Lactation, Meternalhealth.
IntroductionAyurveda, the oldest existing medical
system, recognized by World Health Organization,is widely practiced. Obstetric care in ayurveda isan unique feature. Ayurveda ensures a safe andnatural delivery of a healthy baby, which isachieved by a regulated diet and regimen duringpregnancy and administration of herbalpreparations needed in each month. A specialunusual glow, beauty, serenity is seen on apregnant woman’s face. This glow is theindicator of health of baby inside her body.Garbhpal ras, a drug mentioned in the text of17th century namely ‘Ras Chandanshu’ hasoccupied a respectable place in ayurveda, forpreventing miscarriage and ensures betternourishment to fetus (Rasyog sagar, 1907-10;Chhangadi, 1999; Goyal and Mahajan, 1988;Indian Medical Science Series, 1994).
Garbhpal ras contains minerals like Hingula(Cinnabar/Hgs), Nag (lead/Pb), Vang (Tin/Sn)
and Loh bhasma (Iron/Fe). Herbal contents ofGarbhpal ras include Dalchini (Cinnamomumzeylanicum), Ela (Elettaria cardamomum),Tejpatra (Cinnamomum tamala), Shunthi(Zingiber officinale), Marich (Piper nigrum),Dhanyak (Coriandrum sativum), Chavya (Piperretrofractum), Krishna jeerak (Carumbulbocastanum), Draksha (Vitis vinifera) andDevdaru (Cedrus deodara). All ingredients insame quantity (1 karsh each) except Loh bhasma(1/2 karsh) were triturated in extract ofVishnukranta (Clitoria ternatea) for seven days.(1 karsh = 12 grams)
Prior to this clinical study of Garbhpal rasin pregnant women, this drug has been evaluatedfor acute, sub-chronic and chronic toxicity. Itwas also evaluated for teratogenic effects, if any,in dams and was found safe at therapeutic doses(Mishra et al., 2008; Mishra et al., 2009a;Mishra et al., 2009b; Mishra et al., 2009c).Garbhpal ras was also found helpful to reducesymptoms like nausea, vomiting, leg cramps, heart
*1. Incharge and Lecturer, Department of Prasuti Tantra, A & U Tibbia College and Hospital, New Delhi (India). 2. Professor and Head of Department, Prasuti Tantra, IMS, BHU, Varanasi (India). 3. Associate Professor, Department of Pharmaceutics, IT, BHU, Varanasi (India).
Mishra, Sinha and Kumar14
burn, low back pain, abdominal pain, oedema,constipation and flatulence during pregnancy(Mishra et al., 2009d; Mishra et al., 2009e).
The aim of this study was to assess the efficacyof Garbhpal ras in pregnancy outcome, if any.
Materials and MethodsInclusion criteria
Women belonging to age group, 18 to 38years, having amenorrhoea due to pregnancy (asearly as pregnancy was detected and up to fivemonth of pregnancy), history of abortion, historyof intra uterine death/still birth, history of pretermdelivery or any other obstetric problem duringprevious pregnancies.
Exclusion criteriaWomen having essential hypertension,
severe aneamia, diabetes mellitus, hypo/hyperthyroidism and severe degree of pregnancyinduced hypertension. Patients havingtuberculosis (except women already on ATT forsuspected genital tuberculosis), liver or renaldisorder were also excluded.
Initially 178 pregnant women wererandomly selected, out of which 170 casesregistered from out patient department (duringperiod of Aug. 2006 to Sept. 2007), PrasutiTantra, Sir Sunderlal Hospital, BHU, Varanasi(India). At the time of registration written consentfor becoming a volunteer of this study was takenfrom each pregnant woman.
All the registered cases have beencategorized in 4 groups as follows:
Group 1. Patients registered from firsttrimester having normal course of pregnancywith no specific obstetric or medical history.
Group 2. Patients registered from firsttrimester with high risk factor like history ofabortion, pregnancy induced hypertension inprevious pregnancy, preterm delivery andprevious caesarean section within two years,elderly primigravida, TORCH infection, onanti tubercular treatment.
Group 3. Patients registered from secondtrimester having normal course of pregnancywith no specific obstetric or medical history.
Group 4. Patients registered from secondtrimester with high risk factor like history ofabortion, pregnancy induced hypertension inprevious pregnancy, preterm delivery andprevious caesarean section within two years,elderly primigravida, TORCH infection, onanti-tubercular treatment.
Each group was further subdivided intocontrol and trial:
Control group (C1,C2,C3,C4): Patientsreceived iron (Livogen), calcium (Calcimax forte)after the first trimester, and folic acid from thevery beginning till last month. Natural progesterone200 μg once or twice a day advised in caseshaving history of spontaneous abortion.
Trial group (T1,T2,T3,T4): Patientsadditionally received Garbhpal ras (120 mg bidwith milk and munakka or draksha). As the womenregistered for the study, they were advisedGarbhpal ras to be taken with anupan after meals.Maximum duration for drug administration wassix months.
In all the groups folic acid was advised assoon as pregnancy was detected till delivery andiron (Livogen), calcium (Calcimax forte) wereadvised after the first trimester. Blood sampleswere taken before initiation of treatment and inthe last month of gestation for analysis. Pregnantwomen were reviewed for routine antenatal check-up ( Fortnightly up to 12 weeks, monthly till 32weeks, fortnightly up to 36 weeks and weeklyafterwards).
Parameters studied1. Neonatal parameters (Anthropometry like
weight, height, head circumference, chestcircumference, mid arm circumference,
gestational age and Apgar score at the timeof delivery).
2. Outcome in cases of previous history ofspontaneous abortion(s) (Table 1).
3. Haematological and bio-chemical bloodparameters (Table 2 and 3).
Statistical analysisAll data were expressed as Mean±SD.
Treated and control groups were compared with
Clinical Trial of Garbhpal ras in Pregnancy Outcome 15
independent sample ‘t’ test, intragroup comparisonwas done with paired ‘t’ test. A 95% confidencelevel was used to determine statistically significantdifferences between Garbhpal ras treated andcontrol groups.
Results and DiscussionResults were assessed on the basis of
differences in neonatal parameters (like gestationalage at the time of delivery, weight, height, headcircumference, chest circumference, mid armcircumference and Apgar score), successfuloutcome in cases of history of spontaneousabortion(s) and untoward effect on haematologicaland bio-chemical blood parameters, if any.
Effect of Garbhpal ras on heamatologicalparameters
Haemoglobin was improved overall, butstatistically significant difference was observedin pregnant women registered in first trimestertreated with Garbhpal ras in comparison tocontrol group. It shows that Garbhpal ras causesno interference/ impairment in absorption of ironrather it might have synergistic effect.
It was observed that average mean valueof total leukocytes count was increasedsignificantly in last month, though it was withinnormal limits. It shows that Garbhpal ras doesnot produce any haemopoitic toxicity (Table 2).
Effect of Garbhpal ras on biochemicalparameters of blood
Level of blood sugar was found withinnormal range in all groups. No incidence of hypo/hyperglycemia was observed in any of the case.Liver function (serum bilirubin, SGOT, SGPTand alkaline phosphatase) and renal function(blood urea, serum creatinine) are maintainedduring antenatal period in trial group as well as
Table 1. History of spontaneous abortion(s).
Cases having 1, 2 or 3 no. of abortion(s)
Group Number
of cases
Percent-
age (%)
1 2 3
C2(n=19) 11 57.89 10 1 0
T2(n=25) 15 60.00 9 5 1
C4(n=14) 08 57.14 6 2 0
T4(n=20) 13 65.00 9 3 1
Table 2. Effect of Garbhpal ras on haematological and biochemical parameters.
Haemoglobin
(gm%)
TLC
(mm3)
FBS
(mg/dl)
Bl Urea
(mgdl)
Serum Creat
(mg/dl) Group
BT AT BT AT BT AT BT AT BT AT
T1
(n=30)
10.25
±1.22
12.14
±1.10††
8347.22
±1103.37
11244.44
±1162.87††
72.04
±12.98
73.25
±10.51*
16.29
±3.08
16.76
±2.12
0.65
±0.08
0.70
±0.08
C1
(n=20)
10.23
±1.23
11.58
±1.10††
8652.15
±1192.18
11808.71
±1240.20††
78.76
±11.98
79.42
±10.58
17.20
±3.20
17.9
±2.32†
0.66
±0.13
0.67
±0.09
T2
(n=25)
10.32
±1.63
12.20
±0.92††*
9373.33
±1038.03
11295.65
±1157.38††
76.06
±11.97
76.29
±9.38
16.36
±2.25
16.33
±1.90
0.68
±0.12
0.72
±0.09
C2
(n=19)
10.50
±1.39
11.81
±0.88†
9121.08
±1003.10
11989.47
±1191.12††
75.05
±11.29
77.93
±10.64
17.98
±3.32
16.77
±3.50
0.69
±0.13
0.71
±0.10
T3
(n=25)
9.77
±1.04
11.85
±0.73††
9733.33
±829.52
12013.33
±1046.00††
71.64
±10.71
73.74
±10.82
17.20
±4.99
16.95
±3.82
0.65
±0.05
0.66
±0.06
C3
(n=17)
10.32
±1.44
11.88
±1.00††
9494.12
±872.12
12276.47
±1056.84††
77.54
±10.62
76.65
±10.66
17.34
±4.19
17.91
±3.69
0.65
±0.09
0.68
±0.09
T4
(n=20)
10.16
±1.57
11.85
±1.22††
9552.00
±1037.87
12468.75
±1189.8††
73.43
±13.06
71.57
±6.32*
15.08
±0.93*
15.04
±0.83
0.68
±0.07
0.70
±0.08
C4
(n=14)
10.24
±1.26
11.78
±1.15††
9186.67
±1010.56
12320
±1166.96††
75.09
±11.01
80.36
±12.44†
17.49
±4.10
15.85
±2.62†
0.67
±0.09
0.69
±0.08
BT= Before treatment ; AT= At last month of pregnancy ; * p<0.01, compared to control ; † p<0.01, compared to BT ; †† p<0.001, compared to BT.
Mishra, Sinha and Kumar16
Table 3. Effect of Garbhpal ras on biochemical parameters.
Serum Bilirubin
(mg/dl) SGOT
(U/ml)
SGPT
(U/ml)
Alk Phosp
(U)
Serum Protien
(gm/dl)
Serum Alb
(gm/dl) Group
BT AT BT AT BT AT BT AT BT AT AT BT
T1
(N=30)
0.64
±0.12
0.60
±0.11
28.03
±4.67
27.56
±4.40
35.12
±4.78
35.32
±4.65
142.77
±24.34
149.80
±23.50†
6.48
±0.58
6.44
±0.50
3.59
±0.40
3.56
±0.41
C1
(N=20)
0.68
±0.08
0.56
±0.11
29.01
±5.88
29.10
±4.94
36.05
±6.39
33.90
±5.06
154.45
±25.03
158.90
±25.68
6.67
±0.60
6.62
±0.58
3.51
±0.43
3.60
±0.40
T2
(N=25)
0.62
±0.13
0.57
±0.12
27.78
±6.28
27.53
±4.19
35.40
±4.37
35.23
±4.16
142.44
±37.12
147.22
±32.28
6.47
±0.49
6.58
±0.43
3.36
±0.41
3.40
±0.40
C2
(N=19)
0.63
±0.12
0.60
±0.13
27.32
±6.57
27.21
±4.21†
34.69
±6.65
35.79
±5.80
144.21
±31.16
140.63
±34.16
6.38
±0.49
6.60±
0.36
3.44
±0.37
3.55
±0.36
T3
(N=25)
0.62
±0.11
0.57
±0.10
24.91
±5.55
26.02
±4.17
32.26
±6.15
31.7
±6.01
147.64
±29.14
157.21
±30.41†
6.66
±0.64
6.70
±0.50
3.47
±0.56
3.66
±0.41
C3
(N=17)
0.63
±0.11
0.61
±0.11
27.52
±6.89
28.28
±4.63
35.61
±4.55
36.38
±5.50
143.29
±27.85
156.29
±27.43†
6.25
±0.52
6.50
±0.41
3.45
±0.46
3.56
±0.41
T4
(N=20)
0.59
±0.12
0.55
±0.11
26.66
±6.28
27.95
±6.39
36.16
±4.06
37.29
±4.30
137.21
±42.33
140.50
±46.09
6.24 ±0.31*
6.41
±0.26*
3.28 ±0.32*
3.37
±0.36
C4
(N=14)
0.59
±0.10
0.58 ±0.09†
25.14
±7.35
26.07
±4.92
32.21
±8.28
32.64
±8.12
163.29
±24.36
167.86
±41.65
6.99
±0.67
6.98
±0.45
3.74
±0.40
3.76
±0.40
* p<0.01, compared to control ; † p<0.01, compared to BT
Table 4. Effect of Garbhpal ras on neonatal parameter.
Group
Gestational
age
(weeks)
Weight
(gm)
Height
(cm)
CC
(cm)
HC
(cm)
MAC
(cm)
APGAR
score
(1stmin)
APGAR
score
(5thmin)
APGAR
score
(10thmin)
C1
(n=20)
38.40
±1.60
2.66
±0.31
48.13
±1.70
31.89
±1.25
33.50
±1.14
9.23
±0.75
6.42
±0.61
7.52
±0.51
8.63
±0.50
T1
(n=29)
39.17
±0.93
2.99
±0.33**
49.74
±1.27**
32.24
±1.24
34.10
±0.91
9.61
±0.51*
6.69
±0.89
7.79
±0.68
8.79
±0.68
C2
(n=15)
38.40
±1.12
2.63
±0.32
48.00
±1.80
32.50
±1.10
33.68
±1.12
9.04
±0.70
6.50
±0.76
7.82
±0.50
8.78
±0.43
T2
(n=22)
38.91
±1.15
2.94
±0.29**
49.45
±1.41*
32.75
±1.32
33.81
±0.72
9.69
±0.81*
6.68
±0.65
7.64
±0.63
8.82
±0.40
C3
(n=14)
38.43
±1.02
2.65
±0.31
48.23
±1.79
32.92
±1.71
33.42
±1.32
9.19
±0.50
6.61
±0.65
7.75
±0.45
8.77
±0.44
T3
(n=16)
39
±1.21
2.92
±0.40
49.10
±1.12
32.34
±1.29
33.97
±0.85
9.78
±1.10
6.81
±0.54
7.88
±0.50
8.88
±0.50
C4
(n=13)
38.23
±1.24
2.54
±0.46
47.75
±1.91
31.71
±1.71
32.96
±1.57
9.25
±0.69
6.42
±1.00
7.41
±1.00
8.42
±1.00
T4
(n=12)
38.67
±0.94
2.76
±0.36
48.83
±1.47
31.46
±0.87
33.50
±0.80
9.33
±0.89
6.62
±0.52
7.61
±0.51
8.69
±0.48
* p<0.05, compared to control ; ** p<0.01, compared to control.
Clinical Trial of Garbhpal ras in Pregnancy Outcome 17
control group (Table 2 and 3). Normal levels ofbio-chemical parameters till the end of treatment,show non-toxic nature of Garbhpal ras.
Effect of Garbhpal ras on outcome ofpregnancy
We are able to ascertain here the outcomeof 141 pregnancies. There were 143 live birthsincluding 2 twins in Garbhpal ras group. Nostillbirth, spontaneous abortion or majormalformation was observed in any group duringstudy. However there were 3 cases of intrauterinegrowth restriction, one case of intrauterine death(due to pregnancy induced hypertension) and 2preterm deliveries (of gestational age 34-35weeks) came across during study in control group(Table 6). Two patients of preterm delivery (ofgestational age 35-36 weeks) were also observedin Garbhpal ras group (T2), though they werepositive for TB IgM and VDRL respectively. Total62.5% and 57.52% women out of total trial andtotal control group respectively had history ofspontaneous abortion(s) in their previouspregnancies (Table 1). Women belong to controlgroup, having previous history of spontaneousabortion advised progesterone therapy duringpresent study, while women of trial group didnot received such therapy. It showed thatGarbhpal ras might have some role in successfuloutcome of pregnancy. But its efficacy and mode
of action yet to be determined on largerpopulation. Ayurvedic sages have suggestedseveral precautions to minimize the risk ofabortions. If the uterus has vitiated doshas, thenit expels the fetus, termed garbhsrava / garbhpata(S.S.Ni.8/10; M.Ni.64/2; B.P.Chi.70/72; Y.R.StriGarbh Roga Ni.). Such uterus does not causeproper upbringing of fetus and sometimes babymay die soon after birth. Amongst all doshas,vata is predominant. By its dynamic andrandomness nature, it expels the fetus. Garbhpalras contains balya and vata-shamak herbs. They
Table 5. Incidence of spontaneous vaginal delivery/
caesarean section.
Group SVD LSCS
C1 (n=20) 15 5
T1 (n=29) 18 (1LPV) 11
C2 (n=15) 7 8
T2 (n=22) 12 (1-Br, 1-LPV, 2-PT) 10
C3 (n=14) 9 (1-IUGR) 5
T3 (n=16) 11 5
C4 (n=13) 3 (1-IUD, 2PT) 10
T4 (n=12) 5 7
Control (n=62) 34 (54.84%) 28 (45.16%)
Trial (n=79) 46 (58.23%) 33 (41.77%)
Table 6. Indication for caesarean section.
Group FD IUGR PIH Post
Dated
FPOL
+ LPV
Abn.
Presen.
CPD Twins Previous
CS
C1 (n=20) 1 - - - - 1 2 - 1
T1 (n=29) 1 - - - 1 1 4 - 4
C2 (n=15) 2 - 1 - 2 - - - 3
T2 (n=22) 1 - - - - 1 3 1 4
C3 (n=14) 1 1 1 - - - - - 2
T3 (n=16) 1 - - 1 - - 1 - 2
C4 (n=13) 3 - - - 1 1 2 - 3
T4 (n=12) 1 - - 1 - 1 - 1 3
(SVD= Spontaneous vaginal delivery; LSCS=Lower segment caesarean section; FD=Fetal distress; IUGR=Intra uterine growth restriction; PIH=Pregnancy induced hypertension; FPOL=Failed progress of labour; LPV= Leaking per vaginum; CPD=Cephalo-pelvic disproportion;
Br=Breech presentation; PT=preterm delivery ( 36 weeks gestational age); IUD=Intrauterine death of fetus)
Mishra, Sinha and Kumar18
may probably help in strengthening the ligamentswhich hold the uterus in place, supporting theweight of the fetus and prevent garbhsrava orgarbhpata.
The incidence of spontaneous vaginaldelivery was observed little more in Garbhpalras treated group (58.23%) in comparison tocontrol (54.84%) (Table 5).
Effect of Garbhpal ras on neonatalparameters
Average mean of anthropometry of neonate(weight, height, head circumference, chestcircumference, mid arm circumference) werefound improved in Garbhpal ras group incomparison to control group. It was also observedthat pregnant women registered during firsttrimester of pregnancy, treated with Garbhpalras have statistically significant changes in weightof baby at birth (2.99 kg in T1 and 2.94 kg inT2) with respective control (2.66 kg in C1 and2.63 kg in C2). Length (crown heel length) andmid arm circumference in T1 and T2 groups hadalso shown statistically significant changes withrespective control (Table 4). Apgar score andgestational age at the time of delivery had notshown significant difference in comparison tocontrol (Table 4). It means Garbhpal ras hasnot any effect on these parameters.
Health status of women along with goodnutrition is prime key to the programming of theproper fetal growth in the uterus (C.S.Sh.2/6).Good maternal body composition due to goodnutrition ensures good immune adaptations andit also provides plenty of antioxidants andmicronutrients for benefit of fetal growth. Ginger(Asnani and Verma, 2007), Cinnamon(Jayaprakash et al., 2006), Black pepper(Gulcin, 2005), Long pepper (Singh et al., 2007)and Munakka (Koga et al., 1999) haveantioxidant activity. In a state of oxidative stressi.e. pregnancy, antioxidants help to protect thebody from the formation of free radicals whichimpair the immune system (Palep, 2007).Piperine has immunomodulatory andcytoprotective activity, thus pregnant women
taking Garbhpal ras have better immunity toovercome several diseases (Pathak andKhandelwal, 2007; Choi et al., 2007). Herbslike ginger, black pepper, long pepper andcoriander helps in digestion, thus nourish motheras well as fetus.
Most of the contents of the Garbhpal ras(Cinnamon, long pepper, cardamom coriander,ginger, Devdaru*) have madhur vipak, hencebeing anabolic, it improves weight of mother aswell as fetus. Small doses of mercury to humanbeings cause an increase in the number of redblood corpuscles, while body weight and generalnutrition are also improved (Nadkarni, 1982).In such condition, fetus gets better nutrition.Ginger also plays a role in increased growth offetus (Wilkinson, 2000). Anupan of Garbhpalras i.e. milk and draksha are madhur in ras, addglucose in diet and consequently improve generalnutrition of fetus. These may be the possiblereasons of increase in weight of fetus of trialgroup in comparison to control group. No grosscongenital anomaly was observed in any of theneonates in all the groups.
ConclusionNormal haematological picture, maintained
renal and liver function tests show non-toxicnature of drug. Improvement in hemoglobinpercentage and normal bio-chemical parametersverify safe use of Garbhpal ras. Successfuloutcome in cases of previous spontaneousabortion with improved neonatal parametersjustify the efficacy of Garbhpal ras. It gave acrystal clear impression that Garbhpal ras canbe recommended safely at therapeutic dose inpregnancy.
AcknowledgementsAuthors are thankful to Dabur India Ltd.,
Sahibabad, U.P. (India) for providing gift sampleof Garbhpal ras.
References1. Asnani, V. and Verma, R.J: Antioxidative effect
of rhizome of Zinziber officinale on parabeninduced lipid peroxidation: an in vitro study. ActaPol Pharm. 64(1): 35-7 (2007).
* Devdaru (Devdar) = Cedrus deodara
Clinical Trial of Garbhpal ras in Pregnancy Outcome 19
2. Bhav Prakash: Vidyotini Hindi commentary andnotes by Pt. Brahm Shankar Mishra. ChaukhambhaSanskrit Sansthan, Varanasi. Ed. VIII. Vikramsamvat (2050).
3. Charak Samhita: Hindi Commentry by KashinathShastri and Dr. Gorakh Nath. Chaukhambha SanskritSansthan, Varanasi. Ed. XVII (1991).
4. Chhangadi, G.S: In Ras Tantra Sar Va SiddhiPrayog Sangrah. Krishna Gopal Ayurveda Bhavan,Ajmer. Ed. IX. Vol. I. pp 554-55 (1999).
5. Choi, B.M., Kim, S.M., Park, T.K., Li, G., Hong,S.J., Park, R., Chung, H.T. and Kim, B.R.:Piperine protects cisplatin-induced apoptosis viaheme oxygenase-1 induction in auditory cells. JNutr Biochem. 18(9): 615-22 (2007).
6. Goyal, R.K. and Mahajan, R: In Adhyatan RasShastra. Chaukhambha Surbharati Prakashan,Varanasi. Ed. I. pp 309 (1988).
7. Gulcin, I: The antioxidant and radical scavengingactivities of black pepper (Piper nigrum) seeds. IntJ Food Sci Nutr. 56(7): 491-9 (2005).
8. Indian Medical Science Series: No. 3. ClinicalApplication of Ayurvedic Research and a list ofAyurvedic preparations by a panel of Vaidyas. SriSatguru Publication, Delhi. Ed. III. (ISBN 81-7030-101-07) pp160-61 (1994).
9. Jayaprakasha, G.K., Ohnishi-Kameyama, M.,Ono, H., Yoshida, M. and Jaganmohan Rao, L:Phenolic constituents in the fruits of Cinnamomumzeylanicum and their antioxidant activity. J AgricFood Chem. 8; 54(5): 1672-9 (2006).
10. Koga, T., Moro, K., Nakamori, K., Yamakoshi,J., Hosoyama, H., Kataoka, S. and Arigo, T:Increase of antioxidative potential of rat plasmaby oral administration of proanthocyanidin-richextract from grape seeds. J Agric Food Chem.47(5): 1892-1897 (1999).
11. Madhav Nidan: Vidyotini Hindi commentary andnotes by Sri Sudarshan Shastri. ChaukhambhaSanskrit Sansthan, Varanasi. Ed. XVIII (1989).
12. Mishra, D., Sinha, M., Singh, P.N. and Kumar,V: Acute and sub-chronic toxicity study ofGarbhpal ras. Electronic J Pharmacol Therapy.1: 31-34 (2008).
13. Mishra, D., Sinha, M., Kumar, M. and Kumar,V: Chronic toxicity study of ‘Garbhpal ras’, AnAyurvedic medicine. J Herb Med Toxicol.3(1): 13-17 (2009a).
14. Mishra, D. , Sinha, M. and Kumar, V:Teratological study of ‘Garbhpal ras’, AnAyurvedic formulation. J Herb Med Toxicol.3(1): 37-40 (2009b).
15. Mishra, D. , Sinha, M. and Kumar, V: Safetyevaluation of Garbhpal ras, an Ayurvedicformulation. In National Scientific Seminar onReproductive Health of Women through Ayurveda.Rashtriya Ayurveda Vidyapeeth, Research papers,New Delhi. pp 251-261 (2009c).
16. Mishra, D., Sinha, M. and Kumar,V: Effect of‘Garbhpal ras’ on pregnancy induced nausea andvomiting (NVP). Ayurvedic Rennaince. (2009d).Accepted for publication.
17. Mishra, D., Sinha, M. and Kumar, V: Clinicalevaluation of ‘Garbhpal ras’ in pregnancy relatedgastrointestinal disorders. Ayurvedic Rennaince.7(2): 13-16 (2009e).
18. Nadkarni, A.K: Indian Materia Medica. Vol 2.Bombay Popular Prakashan. pp 72-73 (1982).
19. Palep, H.S . Beyond safe motherhood toprogramming for a super baby II (2007). availableon www. Pharmabiz.com/article/detnews.asp?Arch=&articleid=11331§ioned=46.
20. Pathak, N. and Khandelwal, S: Cytoprotectiveand immunomodulating properties of piperine onmurine splenocytes: an in-vitro study. Eur JPharmacol (2007). In press.
21. Rasyog Sagar: By Hari Prapanna Sharma. KrishnaDas Academy, Varanasi. Garbhpal ras II 1907-10/Ras Chandanshu. Vol. I. pp 374-75 (1983).
22. Singh, S.K., Shanmugavel, M., Kampasi, H.,Singh, R., Mondhe, D.M., Rao, J.M., Adwankar,M.K., Saxena, A.K. and Qazi, G.N: Chemicallystandardized isolates from Cedrus deodara stemwood having anticancer activity. Planta Med.73(6): 519-26 (2007).
23. Sushrut Samhita: Translation of Ayurveda TatvaSandeepika by Kaviraj Ambika Dutta Shastri.Chaukhambha Sanskrit Series, Varanasi. Ed. VII(1990).
24. Wilkinson, J. M: Effect of Ginger tea on thefetal development of Sprague- Dawley rats. ReprodToxicol. 507-512 (2000).
25. Yog Ratnakar: Vidyotini Hindi commentary byVaidya Sri Lakshmi Pati Shastri edited by SriBrahma Shankar Shastri. Chaukhambha SanskritSansthan, Varanasi. Ed. II (1973).
Address for correspondence: Dr. Deepa Mishra, Incharge and Lecturer, Department of Prasuti Tantra, A & UTibbia College and Hospital, New Delhi - 110005 (India). E-mail: [email protected]
009_2009
J. Res. Educ. Indian Med., Jan. - March, 2012 Vol. XVIII (1) : 21-26 ISSN 0970-7700
INHIBITORY EFFECT OF THE ROOT OF SIDA ACUTA BURM. F. ONCALCIUM OXALATE CRYSTAL GROWTHT. VIMALA AND S. GOPALAKRISHNAN*
Department of Chemistry, Manonmaniam Sundaranar University, Abishekappatti,Tirunelveli - 627012 Tamil Nadu (India)
Abstract: The root of Sida acuta Burm. F. is useful in nervous and urinary diseases, disordersof blood and bile and in chronic bowel complaints. Calcium oxalate is the most importantconstituent of urinary stone crystals and several research workers have been attracted to carry outin-vitro studies on urinary stone crystals. However, the inhibitory effect of the root of S. acutaon calcium oxalate crystal growth in-vitro has not been carried out. In the present work an attempthas been made to grow crystals of calcium oxalate and to study the inhibitory effect of themethanolic and aqueous extracts of the root of S. aucta on calcium oxalate crystal growth in-vitro. Calcium oxalate crystals were grown in silica gel media in Hane’s tubes by single diffusionmethod. The crystal growth after the addition of the methanolic and aqueous extracts in 20 mg/5 ml and 10 mg/5 ml doses was studied. In both the extracts there were reductions in sizes ofcalcium oxalate crystal columns and also the size of the individual calcium oxalate crystals whencompared to that of the controls. The results of in-vitro experimental models, IR and SEM togetherattribute an inhibitory capacity with respect to calcium oxalate crystals for the methanolic andaqueous extracts of Sida acuta.
Keywords: Urinary disorders, Calcium oxalate, Crystal growth, Sida acuta.
IntroductionSida acuta Burm.F. (syn. S. carpinifolia
Mast; family – Malvaceae) is an erect, perennialundershrub or shrub, distributed throughout thehotter parts of India and Nepal. It is commonlyknown as Vattatiruppi in Tamil and Bala inSanskrit. This species is not only important as amedicine, but also yields a good fibre. The rootis commonly used for nervous and urinarydisorders and also for curing various ailments(Anonymous, 1952). The major alkaloid of S.acuta was cryptolepine and this exhibitedantimicrobial activity against Proteus vulgaris(Leslie et al., 1980). The aqueous extract ofthe whole plant showed significant hepatoprotective activity against carbontetrachloride,paracetamol and rifampicin induced hepatotoxicities in experimental albino rats (Rao, 1998).Chemical analysis of the whole plant has led tothe isolation of many compounds (Saraswathyet al., 1998; Prakash et al., 1981). Ethanolic
extract of the whole plant of S. acuta showedpartial neutralization effect against venom lethaleffect (Otero et al., 2000).
Deposition of calcium oxalate microcrystalin human body can be a significant problem andit is recognised that 70 - 80 % of kidney stonescontain calcium oxalate (Seftel and Resnick,1990). Barring D- penicillamine and allopurinolfor cystine and uric acid stones, no effectivedrug therapy is available in Allopathy for thetreatment or prevention of other types of stones.However, a large number of indigenous drugshave been used for this purpose in our countrysince ancient times (Chopra et al., 1956).Cystone is claimed to have properties of crushingstones in-situ and expelling them without surgery(Dandia et al., 1976). Saxifraga liqulata andTribulus terrestris are two common herbs of thisherbo - mineral formulation (Handa and Kapoor,2000). The chances of another stone formationcan be minimised and dissolution of existing
* Professor and Head, Pharmaceutical Chemistry
Vimala and Gopalakrishnan22
stones enhanced if the composition of urine ismodified. The rationale behind the use of herbalremedies is not well established except for a fewplants. Studies on the growth of urinary crystalsin the presence of some substances which actas inhibitors or promoters of crystal growth mayhelp to pick out potential substances for use byrecurrent stone formers (Natarajan et al., 1997).In the present work an attempt has been madeto study the inhibitory effect of the methanolicand aqueous extracts of the root of Sida acutaon calcium oxalate crystal growth in-vitro.
Materials and MethodsThe roots of S. acuta were collected from
Tirunelveli, Tirunelveli District, Tamil Nadu, Indiaduring the month of September. The botanicalidentity was confirmed by comparing the samplewith the Herbarium specimens preserved in theDepartment of Botany, by Dr. V. Chelladurai,Govt. Siddha Medical College, Palayamkottai,Tirunelveli District, Tamil Nadu, India. A voucherspecimen of the plant had been deposited at theHerbarium, Department of Chemistry,Manonmaniam Sundaranar University, Tirunelveli,Tamil Nadu, India [Voucher specimen numberMSU 054]. The plant material was thoroughlywashed with water, cut into small pieces, driedunder shade and powdered.
The powdered root was successivelyextracted with petroleum ether (40º- 60ºC),benzene, chloroform, methanol and water. Themethanol and aqueous extracts were condensedand evaporated to dryness under vacuum.
There are previous reports on the growthof calcium oxalate crystals from aqueous solutionand gel methods (Sindhu et al., 1992;Srinivasan and Natarajan, 1996;Aravindakshan and Jayanthi Bai, 1996). Inthe present study calcium oxalate crystals weregrown in -vitro in silica gel media in Hane’stubes by single diffusion method. The gel wasprepared by treating sodium metasilicate solutionof density 1.03 g/ml with 3M acetic acid and thepH was adjusted to 6.3. One of the reactants,
calcium chloride (1M) was incorporated insidethe gel. After gellation, 1M oxalic acid was slowlyadded over the gel along with ethanol/water asthe supernatant solution for control group anddried methanolic extract dissolved in ethanol /aqueous extract of the test plant for other groups.The crystals appeared as cloudy precipitate andin due course, the depth of the column wasfound to increase. The thickness (length) ofcrystal column was measured in centimeter ondays 1, 3, 7, 10, 15, 20, 25 and 30. The sizesof the crystals formed were noted in microns(μm) on days 1, 3, 7, 14, 21 and 28 using anoptical microscope and the values thus obtainedwere compared with each other. The wholeexperiment was carried out at room temperature(27o + 3oC). The inhibitory effect of methanolicand aqueous extracts were studied at 20 mg/5ml and 10 mg/5 ml doses. The experimentswere repeated six times and the average valuewas taken. All the results were expressed asmean + SE. The test of significance wasstatistically analyzed using Student’s t-test (Fisher,1950). After the crystal growth, the crystals werecleared off the gel by repeated washing withdistilled water, filtered and air dried. The purityof the crystals thus obtained was assessed byinfra-red spectroscopy using JASCO FT-IR-410by KBr pellet method. For SEM studies a JEOLJSM 35 - C scanning electron microscope wasused.
Results and DiscussionThe inhibitory effect of the methanolic and
aqueous extracts of the root of S. acuta oncalcium oxalate crystal growth have been studiedin-vitro at 20 mg/5 ml and 10 mg/5 ml dosesand the results are presented in Tables 1 and 2.Concentration dependent inhibitory effect wasobserved in methanolic extract of S. acuta wherethe degree of inhibition was more with 20 mg/5 ml concentration. In the case of aqueousextract, the size of the crystal columns werealmost equal for higher and lower concentrationsbut slightly less than the size of crystal columnsin the control set up. Reductions in the lengths
Effect of Sida acuta on Calcium Oxalate Crystal Growth 23
of crystal columns were observed even on thefirst day of the experiment for methanolic extractand the reduction in growth rate was moresignificant from fifth day onwards. Aqueousextract of S. acuta did not produce significantreductions in the lengths of crystal columns.The mean lengths of the crystal columns in thecontrol set up were 3.48 cm and 3.97 cmrespectively on the 30th day when ethanol anddistilled water were used. The mean lengths ofcrystal columns in the extract set up were 2.88cm (20 mg/5 ml) and 2.93 cm (10 mg/5 ml) formethanolic extract and 3.83 cm (20 mg/5 ml)and 3.83 cm (10 mg/5 ml) for aqueous extract.Calcium oxalate crystals from the extract treatedtubes when viewed under microscope showedconsiderable reduction in size when compared tothat of the control. The mean lengths of thegrown crystals on 28th day of the experiment inthe control set up were 180 μm and 294 μm
respectively when ethanol and distilled water wereused. The mean lengths of the crystals in theextract set up were 96 μm (20 mg/5 ml) and162 μm (10 mg/5 ml) for methanolic extractsand 246 μm (20 mg/5 ml) and 262 μm (10 mg/5 ml) for aqueous extracts on 28th day. Theresults are presented in Fig. 1 and Fig. 2.
The drug treated crystals are found toexhibit IR spectra similar to that of the controlsuggesting no possibility of any type of chemicalbonding between the drug and the oxalatecrystals. These IR spectra are shown inFig. 3-6.
The calcium oxalate crystals were alsosubjected to SEM analysis for understanding themorphological changes if any. SEM of in-vitrogrown calcium oxalate crystals by single diffusionmethod in silica gel media are presented in
Table 1. Effect of methanolic extract of Sida acuta root on the lengths of calcium oxalate crystal columns in cm.
Days Control Sida acuta
20 mg/ 5 ml 10 mg/ 5ml
1 1.783 + 0.08 1.700 + 0.00 1.633 + 0.03
3 2.200 + 0.05 2.150 + 0.00 2.133 + 0.03
7 2.716 + 0.01 2.383 + 0.01 ** 2.383 + 0.03 *
10 3.016 + 0.03 2.533 + 0.03 ** 2.550 + 0.05 *
15 3.266 + 0.03 2.783 + 0.01 ** 2.783 + 0.08 *
20 3.483 + 0.04 2.883 + 0.01 ** 2.933 + 0.08 *
25 3.533 + 0.04 2.883 + 0.01 ** 2.933 + 0.08 *
30 3.483 + 0.04 2.883 + 0.01 ** 2.933 + 0.08 *
All values are mean + SEM of six experiments in each set * P< 0.05; ** P< 0.005
Table 2. Effect of aqueous extract of Sida acuta root on the lengths of calcium oxalate crystal columns in cm.
Days Control Sida acuta
20 mg/5 ml 10 mg/5 ml
1 1.733 + 0.02 1.700 + 0.02 1.750 +0.03
3 2.566 + 0.02 2.533 + 0.03 2.583 +0.03
7 3.133 + 0.02 3.083 + 0.04 3.166 +0.03
10 3.616 + 0.02 3.566 + 0.03 3.650 +0.03
15 4.016 + 0.02 3.950 + 0.03 4.050 +0.04
20 4.683 + 0.04 4.566 + 0.02 4.566 +0.04
25 3.966 + 0.07 3.833 + 0.02 3.766 +0.03
30 3.966 + 0.07 3.833 + 0.03 3.833 +0.04
All values are mean + SEM of six experiments in each set * P< 0.05; ** P< 0.005
Vimala and Gopalakrishnan24
Fig. 7-10. The crystals from control experimentscontained individual prismatic crystals, twinnedcrystals and some aggregates.
Crystals from methanolic extract set upcontained some deformed and smaller crystalaggregates in addition to single and twinnedcrystals. Addition of the extract resulted in partialdissolution of the crystals with cracks andcrevices and a tendency for chipping away ofthe parts as compared to control. Similar resultswere reported by Aravindakshan and JayanthiBai (1996). Aqueous extract showed a markedreduction in the tendency to form crystalaggregates as is evident from Fig. 10. Aqueousextract when included in the crystal growth setup also resulted in corrosion, crevices in thecrystals which could indicate a dissolution effectfor drug. Agglomeration of calcium oxalatecrystals plays a major role in urinary stoneformation and has the potential to produce verylarge crystals in a short period of time with no
Fig. 3. IR spectra of calcium oxalate crystals treated with ethanol.
Fig. 4. IR spectra of calcium oxalate crystals treated with water
Fig. 5. IR spectra of calcium oxalate crystals treated with methanolic extract of S. acuta
Fig. 6. IR spectra of calcium oxalate crystals treated with aqueous extract of S. acutaFig. 2. Effect of aqueous extract of S. acuta on the size of the crystals.
Fig. 1. Effect of methanolic extract of S. acuta on the size of the crystals.
Effect of Sida acuta on Calcium Oxalate Crystal Growth 25
reduction of supersaturation (Hounslow et al.,1988). The crystals also showed less definededges with dots of corrosion which could facilitate
dissolution of the crystals. Natarajan et al.(1997) have reported that no nucleation, reductionin the number and size of the crystals, reductionin total mass of the crystals formed, the changein the morphology of crystals and change in thecrystalline quality may be considered as inhibitoryeffects in crystal growth experiments.
In the present investigation the morphologyof the crystals have changed with uneven surfacesand less defined edges. Platy crystals wereobtained (Fig. 8b) when methanolic extract wasused. Change in the lengths of crystal columns,shape, size, transparency were also observedindicating the inhibitory effect of the two extractsof Sida acuta.
AcknowledgementsOne of the authors (T. Vimala) wishes to
thank the University Grants Commission, NewDelhi and the Principal and Management of St.Jude’s College, Thoothoor, Kanyakumari District(India) for selecting her under FIP Programme.The authors also thank Dr. Y.M. Fazil Marickar,Govt. Medical College, Trivandrum for technicalassistance and Dr. Peter Koshy, R. R. L.Trivandrum (India) for SEM analysis.
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Fig. 10. Scanning electron micrographs of calcium oxalate crystals(a) On adding aqueous extract of S. acuta showing minimal aggregation.(b) On adding aqueous extract of S. acuta showing less defined edges.
Fig. 8. Scanning electron micrographs of calcium oxalate crystals(a) On adding methanolic extract of S. acuta(b) On adding methanolic extract of S. acuta showing corrosion.
Fig. 9. Scanning electron micrographs of calcium oxalate crystals(a) On adding water showing twinning(b) On adding water showing aggregation.
Fig. 7. Scanning electron micrographs of calcium oxalate crystals(a) On adding ethanol(b) On adding ethanol showing sharp edges.
7a 7b
8a 8b
9a 9b
10a 10b
Vimala and Gopalakrishnan26
8. Leslie Gunatilaka, A.A., Sotheeswaran, S.,Balasubramaniam, S., Chandrasekara, A.I. andBadra Sriyani, H.T: Studies on medicinal plants ofSri Lanka III : Pharmacologically important alkaloidsof some Sida species. Planta Med.39: 66-72 (1980).
9. Natarajan, S., Ramachandran, E. and Blisin Suja,D: Growth of some urinary crystals and studies oninhibitors and promoters. II. X–ray studies andinhibitory or promotery role of some substances.Cryst. Res. Technol. 32: 553-559 (1997).
10. Otero, R., Nunez, V., Jimenez, S.L., Fonnegra,R., Osorio, R.G., Garcia, M.E. and Diaz, A:Neutralization of lethal and enzymatic effects ofBothropsatrox venom. J. Ethnopharmacol.71: 505-511 (2000).
11. Prakash Anand, Varma, R.K. and Ghosal, S:Chemical constituents of Malvaceae Part III.Alkaloidal constituents of Sida acuta, S. humilis, S.rhombifolia and S. spinosa. Planta Med.43: 384-388 (1981).
12. Rao, K.S. and Mishra, S.H: Anti–inflammatoryand hepato protective activities of Sida acuta.Indian Drugs. 35: 92-97 (1998).
13. Saraswathy, A., Susan, T., Gnana Ravi, R.,Govindarajan, S. and Kundu, A.B: Chemicalinvestigation of Sida acuta Burm. F.Bulletin of Medico – Ethnobotanical Research.19: 176-180 (1998).
14. Seftel, A. and Resnick, M.I: Metabolicevaluation of urolithiasis. Urol. Clin. North Am.17: 159-69 (1990).
15. Sindhu, S., Krishnamoorthy, H., Thomas,N.E., Roshni, S.V., Vathsala, K.R.,Aravindakshan, C. and Marickar, Y.M.F:Diffusion controlled growth of calcium oxalate.Indian J. Pure Appl. Phys. 30: 82-83 (1992).
16. Srinivasan, N. and Natarajan, S: Growth ofsome urinary crystals and studies on inhibitorsand promoters. I standardisation of parametersfor crystal growth and characterization of crystals.Indian J. Phys. 70A: 563-568 (1996).
Address for correspondence: S. Gopalakrishnan, Department of Chemistry, Manonmaniam SundaranarUniversity, Abishekappatti, Tirunelveli - 627012 Tamil Nadu (India). E-mail: [email protected]
020_2009
J. Res. Educ. Indian Med., Jan. - March, 2012 Vol. XVIII (1) : 27-31 ISSN 0970-7700
SPECTROSCOPIC INVESTIGATIONS OF PALAKARAI(COWRIE SHELL) PARPAMS. JOSEPH VEDHAGIRI,* K.GANESAN AND P.C. JOBE PRABAKAR
Research and PG Department of Physics, T.B.M.L. College, Porayar - 609307Nagai District, Tamil Nadu (India)
Abstract: Parpam (Bhasma) is one form of internal medicine widely and effectively used inSiddha system of medicine. Shells of Cypraea moneta Linn. is commonly known as cowrie orcowry. In Siddha, it is known as Palakarai and in Ayurveda it is used as Varatika bhasma. Theingredients that are being used for the preparation of a parpam have to be subjected to several stagesof pudam, known as calcination process. The three intermediate stages during the preparation ofPalakarai parpam were taken for the study. All the three stages were subjected to particle sizeanalysis, atomic and weight percentage of elemental variations using Scanning Electron Microscopyand Energy Dispersive Spectroscopy (SEM/EDS). Also the vibrational frequencies and their relatedfunctional groups were assigned using Fourier Transform Infrared Spectroscopy (FTIR) and finallythe crystallographic phase changes by X-ray diffraction (XRD) were analyzed. The results of SEM/EDS, FTIR Spectra and XRD are reported and discussed.
Keywords: Cypraea moneta shells, Palakarai parpam, SEM – EDS analysis, FTIR and XRDanalysis, Siddha medicine, Varatika bhasma.
IntroductionShells of Cypraea moneta Linn. commonly
known as cowries are called chozhi (or) palakaraiin Tamil. It belongs to the phylum mollusca andthe class gastropoda. Similar to the shells ofoyster, palakarai also is the shell for protectingthe sea mollusc Cypraea moneta. The shell,palakarai is obtained abundantly along the Indiancoast. In ancient days palakarai were valued ascurrency equal to gold, and used as ornamentalmaterials. In traditional system of medicine theshells of Cypraea moneta, have been used asmedicine to cure various ailments mainly relatedwith stomach. Palakarai parpam was found tobe effective in anti-pyretic and anti-inflammatory,in experimentally induced albino rats(Devanathan et al., 2002). Also used in treatingstomach poisoning and all toxaemic states, colic,retention of urine, gonorrhoea and inflammationof urino-genital tract (Anonymous, 1993).
Materials and MethodsThe samples of initial, intermediate and final
stages during the preparation of palakarai parpam
were collected and labeled as JVP1, JVP2, andJVP3. Scanning electron microscope (SEM-Model:JEOL JSM 5610 Series) was used to examine themorphology and the particle size of the three stagesand SEM images of four hundred magnificationsof three stages are considered. Probable elementspresent in atomic and weight percentage at eachstage are analyzed using EDS. The fundamentalvibration band frequencies are also assigned for allthe three stages by recording FTIR spectra overthe region 400-4000 cm-1 at 4 cm-1 resolutionusing KBr wafer on a Nicolet Avatar-360 Series.
The XRD technique is employed to assessthe phase purity and crystallographic changesduring calcinations process of parpam. The XRDpatterns of initial and final stages of parpam wererecorded with high-resolution powder X-raydiffractometer, PANalytical–Philip–Netherlands /Model XPert Pro, with Cu-Kα1 radiation(λ = 1.54056 A°) at 40 kev and 30 mA at thescanning rate of 2° per minute and 2θ was variedfrom 5 to 60°.
* Associate Professor (Physics)
Vedhagiri, Ganesan and Prabakar28
Results and DiscussionsSEM Particle size Analysis
Figure 1a is representing SEM images of400 magnifications of the three stages. It isobserved that the morphology of the particles isof granular amorphous nature, varies from aminimum to maximum size. In comparing all thethree stages most significant changes of particlesize have been observed due to calcinationsprocess. The range of particle size is varied fromstage one to stage two from 41.67-16.66 μm to33.67-8.34 μm due to first time calcinations.The maximum size of the particle at the secondstage is further reduced to 12.82 μm from 33.97μm and the minimum size has been retained to8.34 μm at the final stage which clearly indicatesthe particle size reducing due to the repeatedcalcinations. The size of the particles isdecreasing in the order of JVP1 > JVP2 > JVP3.
SEM-EDS AnalysisThe Electron Density Spectrum of 400
magnifications of the three stages is given inFigure 1b. The elemental compositions ofpalakarai parpam found in the three stages aregiven in Table 1.The weight percentage ofcalcium for all the stages are found to beprominent. First stage JVP1 is found to contain75.07 wt% of calcium and rose to 93.56 wt%at the second stage after calcinations. Finally theweight percentage of calcium is maintained to80.02% at the last stage. Magnesium, zinc,strontium and barium were the other elementspresent as listed in Table 1. After three repeatedcalcinations, palakarai parpam is found to containcalcium as the major constituent to the value of80.02 wt%. It is concluded that palakarai parpamis consisting mainly of calcium and magnesium,barium and a trace amount of strontium asreported.
FTIR AnalysesThe recorded FTIR spectrum of the three
stages is given in Figure 2. Each compound hasits own unique absorption pattern in the fingerprint region ~1500 to 650 cm-1. (Kalsi, 1995).
The FTIR spectra of the samples could reveal thesignificant vibrational bands (Petsch et al., 1989).The presence of hydroxyl O-H stretching andbending bands are observed around ~3660 to 3800cm-1 and ~1650cm-1 (Ana et al., 1996) in all thestages and are pronounced less in the final stagewhich could be attributed to the reduction in thehydrogen bonded structure due to repeated hightemperature treatment of the sample.
The presence of calcium carbonate isconfirmed in all the three stages due to strongvibrational band frequencies prevailed around~1420 to 1500 cm-1 and 860 to 875 cm-1
(Prabakaran et al., 2005).The vibrational band observation around
~1080 cm-1 is attributed to the presence ofcalcium carbonate in the form of aragonite. Atthe final stage due to calcinations this band getsdiminished, which clearly indicates that the formof calcium carbonate is not completely gettingchanged, from aragonite to calcite. It is noticeablethat calcite has no vibrational band assignmentat ~1080 cm-1 (Narasimhulu and Lakshmanan,2000).
The presence of calcium carbonate in all thethree stages is further confirmed by the existenceof vibrational frequencies at ~875 cm-1 and~712 cm-1 (Konwar et al., 2004). In the finalstage, calcium carbonate has been furtherconfirmed by the strong vibrational bandfrequencies exhibited at ~1420, ~874 and ~712cm-1 (Gosh, 1978). In comparing the FTIRspectra of JVP1 and JVP2, it is observed thatthe vibrational band frequencies due to methylamide and hydroxyl compounds related C-H, N-H and O-H stretching have been shifted. At thefinal stage all these bands are found weakindicating that due to calcinations, the organicsubstances have been progressively reduced.
XRD AnalysesThe XRD patterns are analyzed and the peaks
are indexed. XRD patterns of JVP1 and JVP3 arepresented in Figure 3. Powdered XRD patterns ofinitial stage showed the characteristic peaks of
Spectroscopic Investigations of Palakarai (Cowrie Shell) Parpam 29
Figure 1a: Three stages of SEM images of Palakarai Parpam (400 magnification)
Figure 1b: Three stages of EDS of Palakarai Parpam (400 magnification)
Figure 1a: Stage I Figure 1b: Stage I
Figure 1a: Stage II Figure 1b: Stage II
Figure 1a: Stage IIIFigure 1b: Stage III
Vedhagiri, Ganesan and Prabakar30
poorly crystalline calcium carbonate. All the peaksare indexed from their observedd-values. The structure is found to beorthorhombic with lattice parameters a =3.8218A°, b = 4.9827 A°, c = 24.2633 A° and α = β= γ = 90° with unit cell volume 462.04 (A°). 3
The lattice parameters of JVP3 (a =3.8502 A°,b = 10.8675 A°, c = 23.3284 A° and α = β =γ = 90° with unit cell volume 976.19 A°)3 areslightly changed and crystal system prevails thesame as orthorhombic. It is a known fact thatcalcite form of calcium carbonate is always ofhexagonal structure. If it changes from aragoniteto calcite, the crystal system would be of hexagonal.
Wave numbers (cm-1)
Wave numbers (cm-1)
% T
Wave numbers (cm-1)
% T
% T
Wave numbers (cm-1)
Figure 3. XRD Palakarai Parpam
Figure 2. FTIR – Spectrum of Palakarai Parpam
Spectroscopic Investigations of Palakarai (Cowrie Shell) Parpam 31
No such change of crystal structure from initial tofinal stage is observed, except the variations in sidesand volume of unit cell. Moreover the peak around2θ of 29° at the initial stage has not been shiftedand remains almost the same in the final stage,confirming no phase transition of crystal system.The increase in the intensity of the peaks isindicating the improvement of crystallinity due tocalcinations process. The increase in unit cellvolume is attributed to the increase of surface areawhich is a vital factor in drug interaction. The peaksaround 2θ of 29.2° to 29.6° are attributed to thepresence of calcium carbonate (Davis et al.,1997). The sharp peaks observed at JVP3 clearlyimply that the repeated calcinations promotecrystallinity which is accountable for bioavailabilityand dissolution rate.
ConclusionThe particle size of parpam is reduced due
to repeated heat treatment. SEM-EDS analysisreveals that palakarai parpam is having calciumas prominent element and magnesium, bariumand strontium as trace elements. The organicsubstances are progressively reduced from initial tothe final stage. The presence of calcium carbonatein aragonite form is observed. The XRD studyreveals that the crystallinty has been improved dueto calcinations. The orthorhombic crystal structurehas not been changed and remains as orthogonal upto the final stage.
AcknowledgementsAuthors are grateful to Kaviraj
Pharmaceuticals, Erode, Tamil Nadu (India) forproviding the samples. Research grant receivedunder a minor research project of UGC by one ofthe authors (SJV) is gratefully acknowledged.
References1. Ana H Delgado, Ralph M, Paroli and James J
Beaudock: Applied Spectroscopy. 50: 970 (1996).2. Anonymous: Formulary of Siddha Medicines.
IVth ed. IMPCOPS, Chennai. pp 228 (1993).3. Davis EE, Fisher AT and Firth JV Procee: Ocean
Drilling Program - Initial Reports. Vol. 168: 35-45(1997).
4. Devanathan Deva R, Prema S and SaraswathyJeyaendra: Proce. of Fifth International Congresson Traditional Asian Medicine. pp 10 (2002).
5. Gosh SN: Jour. Mat. Sci. Vol. 13: 1877-1886 (1978).6. Konwar R, Changmai R and Baruah GD: Indian
Jour. of Pure and Applied Phy. Vol. 42: 812-815(2004).
7. Kalsi PS: Spectroscopy of Organic Compounds.IInd ed. New Age International Ltd., New Delhi.pp 74 (1995).
8. Narasimhalu, KV and Lakshmana RJ:Spectrochim. Acta- A. Vol. 56: 1345-1353 (2000).
9. Petsch, Clerc, Seibel and Simon: Tables ofSpectral Data for Structure Determination ofOrganic Compounds, IInd ed. Springer – Verlag,Hong Kong (1989).
10. Prabakaran K, Balamurugan A and RajeswariS: Bull. Mater. Sci. Vol. 28(2): 115-119 (2005).
Table 1. Elemental composition of Palakarai Parpam.
Weight % Atomic % Sl.
No. Element and Symbol JVP1 JVP2 JVP3 JVP1 JVP2 JVP3
1 Calcium, C 75.07 93.56 80.02 83.03 96.68 78.38
2 Magnesium, Mg - 00.20 11.70 - 00.34 18.90
3 Iron, Fe 11.50 00.10 - 9.13 00.07 -
4 Zinc, Zn 06.06 - - 04.11 - -
5 Strontium, Sr 07.36 06.14 02.19 03.73 02.90 00.98
6 Barium, Ba - - 06.08 - - 01.74
008_2009
Address for correspondence: S. Joseph Vedhagiri, Research & PG Department of Physics, T.B.M.L. College,Porayar - 609307 Nagai District, Tamil Nadu (India). E-mail: [email protected]
J. Res. Educ. Indian Med., Jan. - March, 2012 Vol. XVIII (1) : 33-43 ISSN 0970-7700
HYPOGLYCAEMIC ACTIVITY OF INDIAN MEDICINAL PLANTSIN STREPTOZOTOCIN DIABETIC RATSE. N. SUNDARAM,1 K. P. SINGH2 AND P. UMAMAHESWARA REDDY3
Drug Standardisation Unit,1,2 Department of Zoology,3Osmania University, Hyderabad – 500007 Andhra Pradesh (India)
Abstract: In the present study, the hypoglycaemic effect of alcoholic extracts of M. charantia,A. marmelos and E. jambolana was studied in streptozotocin (STZ) induced diabetes. Rats weremade diabetic by intraperitoneal injection of STZ (30 mg/kg) in citrate buffer. On confirmation ofdiabetes after 48 hrs of injection, alcoholic extract of medicinal plant (250 or 500 mg/kg) orglibenclamide (300 μg/kg) administered orally to rats for 30 days. These three plants produced doseand duration dependent hypoglycaemia very similar to that of glibenclamide. At the end of onemonth, serum glucose levels of STZ diabetic rats with daily doses of 500 mg/kg of any one ofthe alcoholic extract were ‘more or less’ comparable to that of normal rats. The anti-diabetic effectof these plants might be due to enhanced insulin secretion from the viable β-cells of islets ofLangerhans as evidenced by presence of more viable β-cells and less necrotic changes in thepancreas of diabetic rats as compared to that of control diabetic rats. Thus, these plants appearto be better alternative for the diabetic patients who are prone to develop side effects with theregular use of synthetic hypoglycaemic drugs as these are also plants devoid of any untoward/toxiceffects.
Keywords: STZ diabetic rats, Hypoglycaemic effect, Histopathology, Momordica charantia,Aegle marmelos, Eugenia jambolana.
IntroductionIn the present era, diabetes mellitus is a
global problem. There are an estimated 143million people worldwide, suffering fromdiabetes.1 This number may probably double bythe year 2030.2 The diabetes enters in man’slife unnoticed as its symptoms remain silent formany years and is considered to be the motherof many diseases. Reports from the WorldHealth Organization (WHO) indicate that diabetesmellitus is one of the major killers of our time,with people in South - East Asia and WesternPacific being most at risk.3 Prolong andcontinuous use of currently available oralhypoglycaemic drugs in modern medicine notonly associated with side effects like anorexia,nausea, vomiting, flatulence, abdominaldiscomfort but their use is also contraindicatedin patients suffering from renal, hepatic, cardio-respiratory insufficiency, alcoholism and inadvanced age. Likewise, repeated injections of
insulin may lead complications like insulin allergy,insulin resistance and localized lipoatrophy at the siteof injections.4,5
On the contrary, long before the use ofinsulin, medicinal plants have been used sinceancient times by the physicians and laymen totreat not only the diabetes but also a great varietyof human diseases such as heart diseases, canceretc.6, 7 The text of Charaka, Sushruta, Vagbhattaand Ayurveda also elaborated in details the clinicalfeatures of diabetes mellitus, its complicationsand treatment with medicinal plants. Thetherapeutic approach of traditional medicines ismore holistic because multifactorial pathogenicityof diabetes demands multi - model therapeuticapproach.8 Considering the economic resourceconstraints, easy availability and cheapness andday to day use of M. charantia, A. marmelos andE. jambolana in diabetes mellitus by laymen sinceancient time. The present study was undertaken toelucidate the effectiveness of these three plants as
1. Research Officer (Endocrinology) 2. Research Officer (Pharmacology) 3. Professor
Sundaram, Singh and Reddy34
anti-diabetic drugs in streptozotocin (STZ) diabeticrats in a comparative way as compared to that ofglibenclamide, a standard oral hypoglycaemic drugused in clinical practice in order to provide ascientific basis for their rationale use.
Materials and MethodsAnimals
Albino rats of both sexes, weighing between175 - 200 g, were procured from National Centrefor Laboratory Animal Sciences, National Institutefor Nutrition, Hyderabad and housed (12/ 12 hrs,light/dark cycles, room temp. 22 ± 1.0oC.) inpolypropylene cages (47 x 34 x 20 cm) linedwith husk. The rats were acclimatized to thelaboratory conditions for approximately 10 daysbefore use and had free access to nutritiousbalanced diet and water through out the study.
Prepration of Alcoholic ExtractsMature and unripe fruits of Momordica
charantia, mature and half ripe fruits of Aeglemarmelos and fully ripe fruits of Eugeniajambolana were collected locally (Figures 1-8).The whole fruit including seeds of M. charantia,and seeds of E. jambolana were cut into smallpieces and pulp of the fruits of A. marmelos wereall air dried and made into course powder in anelectric grinder. The powder was filled in Soxhletapparatus and extracted with 95% alcohol in aratio of 1:4 until the alcohol extract coming outof the Soxhlet becomes completely colourless. Lateron, alcohol extract was concentrated byevaporating the alcohol in a distillation apparatusand lyophilized under vacuum. Lyophilizedmaterial was weighed and made into suspension(50 mg/ml) with distilled water before use.
Drugs and ChemicalsStreptozotocin (Sigma, USA), glibenclamide
(Aventis Pharma, Mumbai, India), readymade kits/reagents (M/S. Excel Diagnostic Pvt. Ltd.,Hyderabad, India), haematoxylin-eosin andaldehyde-fuchsin stains and all other chemicalsused in this study were of analytical grade.
Figure 1. Momordica charantia Linn.Figure 2. M. chrarantia (fruits)Figure 3. M. chrarantia (Twig with fruit)
Figure 6. Eugenia jambolana Lam.Figure 7. E. jambolana (Twigs with fruits)Figure 8. E. jambolana (fruits)
6
7 8
1
2 3
Figure 4. Aegle marmelos (L.) Corr.Figure 5. A. marmelos (Twigs with fruits)
4
5
6
Hypoglycaemic Activity of Medicinal Plants 35
Collection of Blood SamplesThe blood samples were collected from
overnight fasted rats by retro-orbital puncturethrough heparinized coated microcapillaries intononheparinized tubes before and after 48 hrs ofSTZ injection, on 11th day, 21st day and on 31st
day of administration of alcoholic extract ofmedicinal plants or glibenclamide. The serum wasseparated from clotted blood by decanting.
Estimation of Serum GlucoseThe concentration of glucose in serum was
determined by enzymatic method as describedby Trinder.9 In brief, 0.01 ml of serum samplewas added into test tube containing one ml ofglucose reagent. A parallel standard sample and ablank were also run simultaneously. The contentswere mixed well and incubated at 37oC for 10min. The absorbance of the serum samples andthe standard sample were measured onspectrophotometer (Systronic) at 505 nm aftercalibrating it against blank and calculated as:
Histopathological StudiesOn last day of the experiment, rats were
sacrificed. Pancreas, liver and kidneys weredissected out, examined macroscopically and fixedin 10% formalin. After processing the tissues forparaffin embedding,10 sections of the tissues(5μ thicknesses) were cut with a rotatorymicrotome and stained with haematoxylin-eosinand aldehyde-fuchsin stains.11 The sections wereexamined under binocular microscope forhistopathological changes.
Experimental ProtocolThe experimental protocol was approved
by the Institutional Animals Ethic Committee.
Measurement of Body WeightsThe body weights of the rats were taken
on the day before injection of STZ and thereafteron 11th, 21st and 31st day of administration ofalcoholic extract of medicinal plants orglibenclamide.
Induction of Diabetes MellitusIn overnight fasted rats, diabetes was
induced by a single intra-peritoneal injection (30mg/kg) of freshly prepared solution of STZ incitrate buffer of pH 4.5 at a rate of 1 ml/kg.12
Forty eight hrs after STZ administration, serumglucose concentration of each rat was estimated.Rats having serum glucose concentrationsbetween 250 -300 mg/100 ml, were considereddiabetic and included in the study.
Experimental DesignA total of 54 rats (six nondiabetics and
forty eight STZ diabetics) were used. Theanimals were divided into following nine groupsof six each: i) Nondiabetic (normal) rats withoutany treatment, ii) STZ diabetic rats administeredvehicle (normal saline), iii & iv) STZ diabeticrat administered orally 250 mg and 500 mg/kg/day respectively alcoholic extract of M.charantia fruit, v & vi) STZ diabetic ratadministered orally 250 mg and 500 mg/kg/dayrespectively alcoholic extract of A. marmelosfruit, vii & viii) STZ diabetic rat administeredorally 250 mg and 500 mg/kg/day respectivelyalcoholic extract of E. jambolana seeds andix) STZ diabetic rat administered orally 300μg/kg/day glibenclamide (in distilled water)for 30 days.13
Statistical AnalysisThe mean value and the standard error of
the mean of each group were calculated. Thelevel of significance of difference between thegroups were analysed by student’s-‘t’ test.14
Results1. Effect of Alcoholic Extracts of M.charantia, A. marmelos and E. jambolanaon the Body Weight of STZ Diabetic Rats
The average initial body weight of normalrats was 186.7 ± 3.57 g. There was a progressiveincrease in the body weights of normal rats.After one month period, the average body weightof normal rats was 201.2±4.6g (7.8%) (Table 1).On the other hand, STZ diabetic control ratsshowed a progressive decrease in their body
Glucose in serum (mg/100m l) = Absorbance of test sample x conc. of standard sample
Absorbance of standard sample
Sundaram, Singh and Reddy36
weights. The body weights of STZ diabetic controlrats decreased by 10.4% (165.0 ± 4.47 g) duringone month period from their initial body weightsof 184.7 ± 4.36 g taken before STZ injection(Table 1).
Alcoholic extracts of M. charantia, A.marmelos and E. jambolana showed a progressivedose and duration dependent increase in bodyweights of STZ diabetic rats which was apparentonly after 20 days of the treatment. On 31st day,M. charantia, A. marmelos and E. jambolana inthe daily doses of 250 mg/kg increased the bodyweights of STZ diabetic rats by 7.6% (201.7 ±3.33 g), 5.4% (195.8 ± 4.72 g) and 3.1% (190.8± 3.27 g) respectively while in daily doses of 500mg/kg, the respective increase in the body weightsof STZ diabetic rats was of the order of 13.3%(205.8 ± 3.96 g), 7.3% (195.8 ± 3.51 g) and5.5% (193.3 ± 1.67 g) when compared with that
of their respective initial body weights taken beforeSTZ injections (Table 1).
The increase in the body weights of STZdiabetic rats at the end of one month period withthree alcoholic extracts (500 mg/kg daily) andglibenclamide (300μg/kg daily) was statisticallysignificant and was comparable to each other.Of the three, M. charantia was comparativelymost effective (Table 1).2. Hypoglycaemic Activity of AlcoholicExtracts of M. charantia, A. marmelos andE. jambolana in STZ Diabetic Rats
The average serum glucose concentrationsof normal rats varied between 85.5 ± 3.67 mg/100 ml to 93.0 ± 2.77 mg/100 ml on differentdays of the experiments during one month period(Table 2).
Intraperitoneal injection of STZ (30 mg/kg)in 12 hrs fasted rats increased serum glucose
Table 1. Effect of alcoholic extracts of M. charantia, A. marmelos and E. jambolana on body weight of streptozotocindiabetic rats
Body weightMean ± S.E.M (g)
On days after initiation of the treatment
Treatment
Before STZ injection
On 11th Day On 21st Day On 31st Day
Percent change(+/-) @
Non diabetic (Control) 186.7 ± 3.57 190.8 ± 3.75NS
196.7 ± 4.94NS
201.2± 4.69a
7.8 (+)
STZ diabetic (Control) 184.2 ± 4.36 179.2 ± 4.36NS
167.5 ± 4.23a
165.0 ± 4.47a
10.4 (-)
M.charantia250mg500mg
187.5 ± 3.82
181.7 ± 3.33
188.3± 3.80 NS
183.3 ± 2.47 NS
196.7 ± 5.72 NS
198.3 ± 4.01b
201.7 ± 3.33 a205.8 ± 3.96 c
7.6 (+) 13.3 (+)
A.marmelos250mg500mg
185.8 ± 3.96
182.5 ± 2.14
185.8 ± 3.96 NS
183.3 ± 1.67 NS
187.5 ± 4.95 NS
195.0 ± 4.65 a
195.8 ± 4.72 NS
195.8 ± 3.51b
5.4 (+) 7.3 (+)
E.jambolana250mg500mg
185.0 ± 4.83
183.3 ± 3.33
185.8 ± 4.36 NS
185.0 ± 2.89 NS
189.2 ± 3.00 NS
190.8 ± 1.54 NS
190.8 ± 3.27 NS
193.3 ± 1.67a
3.1 (+) 5.5 (+)
Glibenclamide 300 g 184.2 ± 3.75 185.8 ± 3.52 NS
190.0 ± 2.89 NS
196.7 ± 3.57 a
6.8 (+)
Values NS - not significant (p> 0.05) and significant at p - value a < 0.05, b < 0.01 and c < 0.001 from the initial body weight taken before STZ injection. Values represent the Mean ± S.E.M. of six animals.@ Percent change (+ increase/- decrease) in the body weight relative to the initial body weight before STZ injection in the same group of rats Treatment was given 48 hrs after STZ injection and was considered as day 1 of the treatment.
Hypoglycaemic Activity of Medicinal Plants 37
concentrations to a great extent. In diabetic ratsserum glucose concentrations raised to 273.5 ±8.10 mg/100 ml (219.5%) to 290.3 ± 7.59 mg/100 ml (239.5%) from its initial normal values indifferent groups when measured after 48 hrs ofSTZ injection.
During one month of study, glucoseconcentrations in serum further increased by 19%(i.e. 326.0 ± 10.58 mg/100 ml) in diabetic controlrats (Table 2).
Alcoholic extracts of M. charantia, A.marmelos and E. jambolana showed a progressivedose and duration dependent decrease in serumglucose concentrations in STZ diabetic rats. Thedecrease in serum glucose concentrations observedon last day of the experiments (i.e. on 31st day) wasof the order of 49.7% (142.5 ± 8.48 mg/100 ml)with a daily oral doses of 250 mg/kg of M. charantiawhile in the daily doses of 500 mg/kg, it decreased
the serum glucose concentrations by 65.48% (100.0 ±7.30 mg/100 ml) from the initial values observed after48 hrs of STZ injection (Table 2).
Likewise, A. marmelos and E. jambolanaalso decreased serum glucose concentrations ofSTZ diabetic rats by 44.4% (161.3 ± 9.45 mg/100ml) and 36.3% (180.7 ± 10.18 mg/100 ml)respectively with the daily doses of 250 mg/kg and62.9% (106.8 ± 9.45 mg/100 ml) and 60.9% (110.5± 8.42 mg/100 ml) with the daily doses of 500mg/kg respectively. The hypoglycaemic effect ofthe three alcoholic extracts was statisticallysignificant and was comparable with that ofglibenclamide (300 μg/kg daily) which alsodecreased serum glucose concentrations by 62.9%(103.8 ± 7.03 mg/100 ml) on 31st day ofexperiment. Of the three, M. charantia wascomparatively more effective followed by A.marmelos and E. jambolana (Table 2).
Table 2. Antidiabetic activity of alcoholic extracts of M. charantia, A. marmelos and E.jambolana in streptozotocin diabetic rats.
Serum glucose concentration Mean ± S.E.M ( mg/100ml) On days after initiation of the treatment Treatment After 48 hrs. of
STZ injection On 11th day On 21st day On 31st day Percent change(+/-)@
Non diabetic (Control) 85.5 ± 3.67 87.7 ± 3.21 93.0 ± 2.77 91.5 ± 2.90
STZ diabetic (Control) 273.5 ± 8.10* 282.8 ± 9.51* 322.0 ± 18.06*
326.0 ± 10.58* 19.0 (+)
M.charantia 250mg 500mg
284.2 ± 7.51
289.7 ± 9.31
252.0 ± 7.43 a
236.7 ± 9.09 c
206.3 ± 10.53 c
173.8 ± 12.59 c
142.5 ± 8.48 c
100.0 ± 7.30 c
49.7 (-)
65.5 (-)
A.marmelos250mg500mg
290.3 ± 7.59
287.7 ± 8.79
265.7 ± 8.91 NS
251.5 ± 9.80 a
215.7 ± 6.28 c
198.0 ± 9.53 c
161.3 ± 9.45 c
106.8 ± 9.45 c
44.4 (-)
62.9 (-)
E.jambolana250mg500mg
283.8 ± 7.55
282.5 ± 7.13
280.3 ± 9.66 NS
230.0 ± 15.62a
235.8 ± 17.86 b
201.7 ± 9.45 c
180.7 ± 10.18 c
110.5 ± 8.42 c
36.3 (-)
60.9 (-)
Glibenclamide 300μg 279.7 ± 6.96 211.2 ± 22.44 a 117.3 ±
10.56 c103.8 ± 7.03 c 62.9 (-)
Values significant at p-value: - * < 0.001 from nondiabetic control, and a < 0.05, b < 0.01, c < 0.001and NS- Not significant (> 0.05) from STZ diabetic control of the same day Values represent the Mean ± S.E.M. of six animals. @ Percent change in the serum glucose conc. from its initial value observed after 48hrs of STZ injection in the same group. Treatment was given 48 hrs after STZ injection and was considered as day 1 of the treatment.
Sundaram, Singh and Reddy38
3. Effect of Alcoholic Extracts ofM. charantia, A. marmelos and E.jambolana on βββββ - Cells of islets ofLangerhans in Pancreas and on Liver andKidney Tissues in STZ Diabetic Rats
PancreasHistology of the pancreas of nondiabetic
rats showed dark stained granules of denselypacked viable β- cells of the islets of Langerhans.On the contrary, diffused necrotic changes wereseen in the pancreas of STZ diabetic rats.Occasional viable β- cells of islets of Langerhanscould be seen in few rats only.
Histopathological studies of pancreas alsorevealed that daily oral administration of alcoholicextracts of M. charantia, A. marmelos and E.jambolana in doses of 250 and 500 mg/kg andglibenclamide in doses of 300 μg/kg for a periodof 30 days significantly improved the histologicalarchitecture of the pancreas as evidenced by thepresence of more viable β - cells of islets ofLangerhans with less necrotic changes ascompared to that of untreated STZ diabeticcontrols. The degree of improvement in thearchitecture of β - cells of islets of Langerhansof STZ damaged pancreas was more with 500mg/kg dose than that of 250 mg/kg dose. Thenecrotic changes observed in pancreas of M.charantia group were less followed by A.marmelos, glibenclamide and E. jambolana.However, the presence of viable β - cells ofislets of Langerhans in pancreas of treated STZdiabetic rats was still less than that of normalrats (Figure 1).
Liver Histology of the liver of nondiabetic rat
showed normal architecture of parenchymal cells.Hepatic lobules with portal tracts and central veinswith hepatic sinusoids radially arranged the centralveins were visible. The hepatic veins were normalin size and portal tracts showed the collection oflymphocytes. On the other hand, histology of theliver of STZ diabetic rats showed hypertrophyand the distortion in the arrangement of the hepatic
cells around central veins, but such changes werevery less in the liver of STZ rats treated with anyone of the alcoholic extract of the plants andglibenclamide for 30 days (Figure 2).
KidneyThe histology of the kidney of nondiabetic
rat showed normal architecture of Bowman’scapsule. The basement membrane of glomerularcapsule was thin. There was sufficient spacebetween basement membrane and the tuft of theglomerulus (Figure 3). On the contrary, in STZdiabetic rats, there was a slight hypertrophy ofthe glomeruli and thickening of the basementmembrane of glomerular capsule, thereby,decreasing the free space between the two. Thewalls of the renal tubules were also thickeneddue to hyalinization, thereby, decreasing the lumenof the renal tubules during 30 days period. Suchchanges in the architecture of the kidney ofdiabetic rats administered with three alcoholicextracts (M. charantia, A. marmelos and E.jambolana) in doses of 500 mg/kg orglibenclamide in doses of 300 μg/kg for 30 dayswere very less as compared to STZ diabeticcontrol rats (Figure 3).
DiscussionDiabetes mellitus or ‘Madhumeham’ known
for centuries as a disease related to sweetness isa serious metabolic disorder with micro andmacro-vascular complications that results insignificant morbidity and mortality. The incidenceof its occurrence in the population is increasingday by day at an alarming rate due to urbanizationand modernization of civilization. The use of oralhypoglycaemic drugs and insulin which controlsthe blood glucose concentrations within normalrange, do little in alleviating late clinicalmanifestations15 of the disease. Hence, it becomesnecessary to look for new and if possible moreefficacious drug; therefore, the research for sucha hypoglycaemic drug has to be an area of activeresearch.16,17
More than 400 medicinal plants areavailable globally for the medication of thediabetes. Their therapeutic effectiveness varied
Hypoglycaemic Activity of Medicinal Plants 39
Figure 1(a) Pancreas of healthy rat showing densely packed β -cells of islets of Langerhans (x 360),Figure 1(b) Pancreas of STZ diabetic rat showing necrosis of β - cells of islets of Langerhans (x 360),Figure 1 (c, d, e, f ) Diabetic pancreas showing presence of β - cells of islets of Langerhans of varying degree withless necrotic changes (x 360) after treatment with M. charantia, A. marmelos, E. jambolana alcoholic extracts(500 mg) and glibenclamide (300 μg) respectively
Sundaram, Singh and Reddy40
Figure 2(a) Liver of healthy rat showing radially arranged normal parenchymal cells (x 360) around central vein.Figure 2(b) Liver of STZ diabetic rat showing distortion of usual arrangement of parenchymal cells (x 360).Figure 2(c, d, e & f) Showing protective effect on histology of liver by M. charantia, A. marmelos, E. jambolanaalcoholic extracts (500 mg) and glibenclamide (300 μg) respectively (x 360).
Hypoglycaemic Activity of Medicinal Plants 41
Figure 3(a) Kidney of healthy rat showing normal structure of Bowman’s capsule and renal tubules (x 360).Figure 3(b) Kidney of STZ diabetic rat showing expanded glomerulus of Bowman’s capsule and hyalinization of thewalls of renal tubules (x 360).Figure 3(c, d, e & f) Kidney of STZ diabetic rat showing protective effect on histology of kidney by M. charantia,A. marmelos, E. jambolana alcoholic extracts (500 mg) and glibenclamide (300 μg) respectively (x 360).
Sundaram, Singh and Reddy42
from one study to other depending upon themethodologies and the experimental modelsused.18,19 We have chosen M. charantia, A.marmelos and E. jambolana plants because theyare extensively grown and their fruits not onlybeen eaten either as a raw fruit (Bael, Jamun) oras a vegetable (Karela) by the public but havealso been used by the laymen for the treatmentof the diabetes in the ancient time.
There are reports that the body weights ofSTZ diabetic rats decreased as they consumedless food.20 In present study also, body weightsof STZ diabetic rats decreased by 10.4% at theend of one month from the initial body weightstaken before STZ injection. On the other handrats administered alcoholic extracts of all thethree plants showed dose and duration dependentincrease in the body weights which was apparentonly after 20 days of the treatment with 500 mg/kg dose. The restoration of the body weights indiabetic rats indicate that these rats properly utilizedthe glucose for their energy production whichmight be due to enhanced insulin secretion. Inaddition, M. charantia being a bitter might alsoacted as an appetizer hence more gain in the bodyweights when compared to that of normal rats.
The present results indicate that alcoholicextracts of M. charantia, A. marmelos and E.jambolana produced dose and durationdependent decrease (p<0.001) in serum glucoseconcentrations of STZ diabetic rats. At the endof one month study, the serum glucoseconcentrations observed in STZ diabetic rats withall three alcoholic extracts (in daily doses of 500mg/kg) and that of glibenclamide (300 μg/kg)were ‘more or less’ comparable with that ofserum glucose concentrations of normal rats.The present findings are in conformity with thefindings of earlier workers for theirhypoglycaemic effects.17,21,22 The order ofeffectiveness was M. charantia > A. marmelos= Glibenclamide > E. jambolana.
Present results showed that oraladministration of alcoholic extracts of all the threeplants and glibenclamide for one month hadprotective effect against the damage caused by
STZ as evidenced by the presence of more numberof viable β - cells of islets of Langerhans inpancreas and practically normal architecture of liverand kidney tissues on histological examination. Thepresent findings are in agreement with those ofearlier workers for their protective effect on thepancreas by M. charantia,23 A. marmelos 24 andE. jambolana 2 5 respectively. The appearance ofmore number of dark stained granules may be dueto activation of survival/ dormant β - cells whichwere not functional in normal pancreas. Someworkers, however, also pointed out the regenerationof the β - cells of islets of Langerhans withmedicinal plants.24,26
In brief, present result showed that theantidiabetic effect observed with all threemedicinal plants is due to enhanced insulinsecretion as evidenced by the presence of moreviable β - cells of islets of Langerhans in STZdamaged pancreas of treated rats which wasdose dependent.
AcknowledgementsAuthor (ENS) is grateful to Prof. (Dr) C.
Nayak, Director General, Central Council forResearch in Homoeopathy, New Delhi for grantingpermission to take up this study.
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burden of diabetes 1995-2025. Diabetes Care. 21,pp. 1414-1431 (1998).
2. Harris, M.I., Flegal, K.M., Cowie, C.C.,Eberhardt, M.S., Goldstein, D.E., Little, R.R.,Wiedmeyer, H.M. and Bryd-Holt, D.D:Prevalence of diabetes impaired fasting glucose,and glucose tolerance in U.S. adults. DiabetesCare. 21, pp. 518-524 (1998).
3. Firshein, R: Eliminating diabetes in the 21st
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5. Masharani, U: Current Medical Diagnosis andTreatment. In: Tierney, L.M.J.R., Mc Phee, S.J.and Papadakis, M.A. Lange Medical Books /
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11. Gomori, G: Aldehyde-Fuchsin: a new stain forelastic tissue. Am J Clin Path. 20, pp. 665-666(1950).
12. Siddique, O., Sun, Y., Lin, J.C. and Chain,Y.W: Facilitated transdermal transport of insulin.J Pharm Sci. 76, pp. 341-345 (1987).
13. Kamalakkannan, N. and Stanely MainzenPrince, P: Anti-diabetic and antioxidant activityof Aegle marmelos extract in streptozotocindiabetic rats. Indian J Exp Biol. 41,pp. 1285-1288 (2003).
14. Gupta, B.N: Statistics - Theory and Practice. In:Gupta, B.N. Sahitya Bhawan, Agra. pp. 186-584(1989).
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16. Pari, L. and Uma Maheswari, J: Hypoglycemiceffect of Musa sapientum L. in alloxan induced diabeticrats. J Ethnopharmacol. 68, pp. 321-325 (1999).
17. Stanely Mainzen Prince, P., Kamalakkannan,N. and Menon, V.P: Antidiabetic andantihyperlipidaemic effect of alcoholic Syzium
Address for correspondence: Dr. E. N. Sundaram, Research Officer (Endocrinology), Drug Standardisation Unit,Osmania Unversity Building No. 32, Street No. 4, Vikrampuri, Habsiguda, Hyderabad – 500007 A.P. (India).E-mail: [email protected]
cumini seeds in alloxan induced diabetic albino rats.J Ethnopharmacol. 91, pp. 209-213 (2004).
18. Chakravarthy, B.K., Gupta, S., Gambheer, S.S.and Gode, K.D: Pancreatic beta cell regeneration-a novel antidiabetic mechanism of Pterocarpusmarsupium (Roxb.). Indian J Pharmaco. 12,pp. 123-127 (1980).
19. Bopanna, K.N., Kannan, J., Sushma, G.,Balaraman, R. and Rathod, S.P: Antidiabeticand antihyperlipaemic effects of neem seed kernelpowder on alloxan diabetic rabbits. Indian JPharmacol. 29, pp. 62-167 (1997).
20. Sivajothi, V., Dey, A., Jayakar, B. andRajkapoor, B: Antihyperglycaemic property ofTragia cannabina in streptozotocin induceddiabetic rats. J Medicinal Food. 10, pp. 361-365(2007).
21. Upadhya, S., Shanbhag, K., Suneetha, G.,Naidu, M.B. and Upadhya, S.A: Study ofhypoglycaemic and antioxidant activity of Aeglemarmelos in alloxan induced diabetic rats. IndianJ Physiol Pharmacol. 48, pp. 476-480 (2004).
22. Viridi, J., Sivakami, S., Shahani, S., Suthar,A.C., Banavalikar, M.M. and Biyani, M.K:Antihyperglycemic effect of three extracts fromMomordica charantia. J Ethnopharmacol. 88,pp. 107-111 (2003).
23. Ahmed, I., Adeghate, E., Sharma, A.K., Pallot,D.J. and Singh, J: Effects of Momordicacharantia fruit juice on islet morphology in thepancreas of the streptozotocin-diabetic rats.Diabetes Res Clin Pract. 40, pp. 145-151 (1998).
24. Kamalakkannan, N. and Prince, P.S: Theeffect of Aegle marmelos fruit extract instreptozotocin diabetes: a histopathological study.J Herb Pharmacother. 5, pp. 87-96 (2005).
25. Grover, J.K., Vats, V., Rathi, S.S. and Dawar,R: Traditional Indian antidiabetic plantsattenuate progression of renal damage instreptozotocin induced diabetic mice. JEthnopharmacol. 76, pp. 233-238 (2001).
26. Chakravarthy, B. K., Gupta, S. and Gode, K.D: Functional beta cell regeneration in the isletsof pancreas in alloxan induced diabetic rats byepicatechin. Life Sci. 31, pp. 2693-2697 (1982).
030_2009
J. Res. Educ. Indian Med., Jan.-March, 2012 Vol. XVIII (1) : 45-50 ISSN 0970-7700
PHARMACOLOGICAL SCREENING OF CASSINE ALBENS (RETZ.)KOSTERM (CELASTRACEAE) FOR ANTIDEPRESSANT ANDANXIOLYTIC ACTIVITY IN RODENTSP. H. PATIL,* M. B. GAGARANI, K. R. PATIL AND S. J. SURANA
Department of Pharmacology, R. C. Patel Institute of Pharmaceutical Education and Research,Shirpur – 425405 Dhule, Maharashtra (India)
Abstract: Cassine albens (R.) Kosterm belongs to the family celastraceae which is the richsource of triterpenes and alkaloids. In present study hydroalcoholic extract of stem bark of Cassinealbens was investigated for antidepressant and anxiolytic activity in rodents. The dose dependantstudy was performed at the doses of 100, 200, 400 and 800 mg/kg, p.o. Cassine albens showedsignificant antidepressant activity in forced swim and tail suspension test. We observed significantanxiolytic effect at the dose of 400 and 800 mg/kg, p.o. in elevated plus maze and marble buryingtest in rodents. The present study substantiates folkloric use of Cassine albens in the treatmentof psychosomatic disorders.
Keywords: Cassine albens, Neurotransmitter, Antianxiety, Antidepressant.
IntroductionCassine albens (R.) Kosterm (Celastraceae)
(synonym: Elaeodendron glaucum or Cassineglauca) is found throughout India and Sri Lanka(Patil D.A., 2003). This plant is popularly knownas ‘Bhutakes’ at Toranmal, a hill station locatedin North Maharashtra, which is used in thetreatment of CNS related disorders (Patil andBhaskar, 2006). In folkloric system of medicine,it is used for the treatment of hysteria, syncopeand normal headache (Kirtikar and Basu, 2005).Fresh root bark of this plant is used for swellingand cold water extract is used as emetic whichmay be fatal in overdose. In Sonbhadra districtof Uttar Pradesh (India) this plant is used incholera, menstrual disorders and to promotesterility in women (Singh et al., 2002). A pasteprepared from root of this plant is used withcow’s milk in snake bite (Parinitha et al., 2005).Celastraceae family is rich source ofsesquiterpenoids (Aleksandra et al., 2007).Lupeol, octacosanol, friedelin, betulonic acid, 23-hydroxybetulin, β- sitosterol and its glucosidesare isolated from its leaves and stem bark. Thecardiac glycosides isolated from this plant arenamed as elaeodendroside A ,B, C, D, E, H, I, J
(Rastogi and Malhotra 2001; Anjaneyulu andNarayanrao, 1980; Shimada et al., 1985).Infusion of Cassine albens was used in headacheand in psychosomatic disorders by local tribesresiding in Nandurbar district of North Maharashtra(Patil and Bhaskar, 2006). Extensive review ofliterature revealed that the plant was not investigatedsystemically for its central nervous system activity.The Cassine albens is used in psychosomaticillness by certain tribes residing in the Satpuda hillregion of Maharashtra. Therefore Cassine albensseem to affect neuronal pathway involved in anxietyand depression. In the present study, we haveevaluated the effect of hydroalcoholic extract ofCassine albens for its antidepressant and anxiolyticactivity in various animal models.
Material and MethodsPlant material and extract preparations
Stem bark of Cassine albens was collectedfrom the Satpuda hill region (Village: Legapani;Taluka: Dhadgaon; Dist: Nandurbar) ofMaharashtra. The plant material was authenticatedby Dr. D. A. Patil, Department of Botany, SSVPSScience College, Dhule (Maharashtra). The stembark of Cassine albens was shade dried for
* Head, Department of Pharmacology
Patil, Gagarani and Surana46
15 days. The dried material was pulverized toobtain coarse to fine powder. The powder wassubjected to extraction with ethanol and water(1:1) by cold maceration for three days. Thematerial was filtered and the filtrate wasconcentrated. The concentrated material was airdried to obtain hydroalcoholic extract of Cassinealbens. This extract was used for pharmacologicalstudies in the form of suspension made in 1%carboxy methyl cellulose (CMC).Drugs
Fluoxetine and Diazepam were obtained asa gift sample from Cadila Pharmaceuticals Ltd.,Ahmedabad and SVIZERA Healthcare Ltd.,Mumbai (India) respectively.Animals and Treatment
All the experimental procedures wereapproved by Institutional Animal EthicalCommittee. Swiss albino mice (20-25 g) and Albinowistar rats (220-230 g) of either sex werepurchased from central animal house of RCPIPER,Shirpur, India. All animal were maintained undercontrol condition of temperature (22 ± 2º C) and12 hour light–dark cycle with free access of food(rodent lab standard diet) and water. Allexperiments were conducted in between 10.00AM to 5.00 PM in a noise free room. Animalswere divided into various groups, containing sixanimals in each group, the animals receiveddifferent treatments like vehicle (1% CMC),Fluoxetine (20 mg/kg p.o.) or Diazepam (1 mg/kg, i.p.) and hydroalcoholic extract of Cassinealbens at the doses of 100, 200, 400 and 800 mg/kg, p.o. of body weight daily for 7 days.Neurobehavioral profile screening
Neuropharmacological screening ofhydroalcoholic extract of Cassine albens(HCA) was studied using mice. Differentneuropharmacological test batteries were usedduring study as reported by Moscardo et al.(2007). The scoring of test was done as perIrwin scale (Turner, 1996).Spontaneous locomotor activity
One hour after the oral administration eachmouse was placed individually in digital activitycage (Besto Instrument) for about 10 minutes. At
the end of observation period mouse wasremoved and reading was recorded.Forced swim test (FST)
FST is the most widely used model forantidepressant studies (Herrera et al., 2006).All animals were dosed for 7 days, on the 6th
day all the animals were individually placed in atank made up of PVC (20 × 40 cm and 25 cmdeep) containing clean water at 25ºC for 15minutes (pretest session). 24 hour later, on the7th day, one hour after the oral administration ofdrug, each animal was subjected to 5 minutesswim test. During the test session the totalimmobility period was recorded. A rat wasconsidered to be immobile when it remainedfloating in the water without struggling. Afterthe end of observation period animal was takenout of cylinder then dried and returned to theirrespective home cages.Tail suspension test
In this test mouse was suspended on theedge of wire 50 cm above the floor by adhesivetape placed approximately 1 cm from the tip oftail. On the 7th day of drug treatment the totalduration of immobility induced by tail suspensionwas measured during 6 minute test session.Animal was considered to be immobile when itdid not show any movement of body and hangedpassively.Elevated plus maze
The EPM evokes the conflict between theneed to explore the novel area and need to avoidmore vulnerable (aversive) areas of elevated plusmaze (Wijeweera et al., 2006). Apparatusconsist of two open arms (50× 10 × 40 cm)with an open roof, arranged such that two openarms were opposite to each other; the apparatuswas elevated to 50 cm from the floor. Theapparatus was carefully cleaned after each testsession to remove any stress of odor. On 7th dayone hour after the oral administration of differentdoses of HCA, and 30 minutes after Diazepam(1 mg/kg, i.p.) administration, each rat was placedin Central Square of the maze facing towards theopen arm. During 5 minute test period, the numberof open and closed arm entries were recorded.
Pharmacological Screening of Cassine albens 47
Marble burying test in miceThe 5 cm layer of sawdust was placed in
polyethylene cage and 20 glass marbles wereevenly distributed on the sawdust in the cages.One hour after oral administration of drugs, animalwas placed individually in cage. During the 15minutes observation period, the total number ofmarbles buried was counted. Only those marblesto be taken into account which were buried two-third of its size (Matsushita et al., 2005;Laurent et al., 2006).
Statistical analysisThe statistical analysis was done by using
PRISM Graph pad (4.0) program. All values wereexpressed as Mean ± SEM. Data were analyzedby one way ANOVA followed by Dunnet’s ‘t’test for group comparison.
ResultsA) Neurobehavioral profile
Neurobehavioral profile of HCA tested atthe dose of 1000 mg/kg and 2000 mg/kg did notshow any significant alterations in animal behavior.
B) Spontaneous locomotor activityThe HCA at all the four doses studied was
unable to produce significant change in motoractivity. This suggests that antidepressant andanxiolytic activity is devoid of motor activity(Kulkarni and Sharma, 1991).
C) Effect of HCA on immobility time in Forced Swim Test (FST)
HCA at 200 mg/kg, 400 mg/kg and 800mg/kg doses significantly (p< 0.01) decreasedimmobility period as compared to the controlgroup. Fluoxetine (20 mg/kg) treated groupshowed more reduction in immobility time ascompared with control group (Table 1).
D) Effect of HCA on immobility time in Tail Suspension Test (TST)
HCA (400 mg/kg) significantly (p<0.01)decreased the immobility time as compared tocontrol group. However HCA (100 mg/kg, 200mg/kg and 800 mg/kg) did not show significantdecline in immobility time. Fluoxetine treated miceshowed greater decline in immobility time and
Table 1. Effect of hydroalcoholic extract of Cassine albens on immobility period in Forced Swim Test in rats.
Group Treatment Dose Immobility Period in FST(Sec)
I Control 10 ml/kg 240 ± 1.3
II Fluoxetine 20 mg/kg 170 ± 2.4**
III HCA 100 mg/kg 230 ± 3.3*
IV HCA 200 mg/kg 220 ± 5.7**
V HCA 400 mg/kg 180 ± 1.7**
VI HCA 800 mg/kg 200 ± 3.3**
Data presented as mean ± SEM. Statistical analysis: One way ANOVA followed by Dunnet’s ‘t’ test. *p < 0.05; **p< 0.01 as compared with control group.
Table 2. Effect of hydroalcoholic extract of Cassine albens on immobility period in Tail Suspension Test in mice.
Group Treatment Dose Immobility period in TST (Sec)
I Control 10 ml/kg 136 ± 6.0
II Fluoxetine 20 mg/kg 72 ± 1.6**
III HCA 100 mg/kg 139 ± 4.2
IV HCA 200 mg/kg 130 ± 5.4
V HCA 400 mg/kg 113 ± 2.5**
VI HCA 800 mg/kg 122 ± 3.4
Data presented as mean ± SEM. Statistical analysis: One way ANOVA followed by Dunnet’s ‘t’ test. **p< 0.01 as compared with control group
Patil, Gagarani and Surana48
showed significant (p<0.01) reduction inimmobility time with compared to control group(Table 2).
E) Effect of HCA in Elevated Plus Maze (EPM) Test
In the present study HCA at the doses of400 mg/kg, 800 mg/kg and Diazepam (1 mg/kg)showed significant increase in open arm entriesas compared to control group. HCA at 100 mg/kg and 200 mg/kg dose was failed to showanxiolytic activity. The increase in open arm entryas preference was observed at the oral dose of800 mg/kg (Table 3).
F) Effect in Marble Burying TestIn this test, HCA (400 mg/kg and 800 mg/
kg) significantly (p<0.05) reduced the numberof marbles buried as compared to control group.The Fluoxetine treated animals showedsignificantly (p<0.01) reduction in marble buryingbehavior as compared to control animals (Table 4).
DiscussionCelastraceae family is a rich source of
sesquiterpenoids of remarkably diverse structuresranging from small alkaloids and oligopeptide tovery complex macrocyclic arrays (Alexandra etal., 2007). In the present study different dosesof hydroalcoholic extract of Cassine albensshowed antidepressant and anxiolytic activity instress-evoked models. The major cause of mentaldisorders is the imbalance betweenneurotransmitter within the brain. Most ofantidepressant and anxiolytic compounds targetsneurochemical processes that occur during thesedisorders. Antidepressant compound acts throughpreventing the reuptake of noradrenalin and/or5- hydroxytryptamine at synaptic nerve endingand increases the availability of neurotransmitterin brain. MAOI inhibits enzyme monoamineoxidase and increase the availability ofnoradrenalin in brain (Goodman and Gilman,2001; Kasper et al., 2001). Currently, thetreatment of anxiety and depression is restricted
Table 3. Effect of hydroalcoholic extract of Cassine albens on number of entries in an open arm in
Elevated Plus Maze test in rats.
Group Treatment Dose Number of entries in open arm
I Control 10 ml/kg 0.71 ± 0.29
II Diazepam 01 mg/kg 3.0 ± 0.38**
III HCA 100 mg/kg 0.57 ± 0.20
IV HCA 200 mg/kg 0.43 ± 0.20
V HCA 400 mg/kg 2.1 ± 0.26**
VI HCA 800 mg/kg 1.9 ± 0.26*
Data presented as mean ± SEM. Statistical analysis: One way ANOVA followed by Dunnet’s ‘t’ test. *p < 0.05, **p< 0.01 as compared with control group
Table 4. Effect of hydro alcoholic extract of Cassine albens on number of Marbles Buried by mice.
Group Treatment Dose Number of marbles buried
I Control 10 ml/kg 6.3 ± 0.6
II Fluoxetine 20 mg/kg 1.4 ± 0.3**
III HCA 100 mg/kg 6.0 ± 0.8
IV HCA 200 mg/kg 4.6 ± 0.3
V HCA 400 mg/kg 4.0 ± 0.4*
VI HCA 800 mg/kg 4.1 ± 0.51*
Data presented as mean ± SEM. Statistical analysis: One way ANOVA followed by Dunnet’s ‘t’ test. *p < 0.05, **p< 0.01 as compared with control group
Pharmacological Screening of Cassine albens 49
to some typical classes such as Benzodiazepines,SSRI, TCA, MAOI, etc. Some atypical drugslike Buspirone came into existence but they arenot therapeutically as effective as conventionaldrugs. In the treatment of anxiety Benzodiazepinesare now slowly replaced by antidepressants whichare not only efficacious in depression but also inacute and long term treatment of anxiety. WhenCassine albens was studied at the doses of 100,200, 400 and 800 mg/kg for effect on locomotoractivity, we don’t observe any significant changein locomotor activity of animal. This suggeststhat the antidepressant activity of Cassine albensmay be devoid of locomotor activity (Dhingraand Kumar, 2007). These results confirmed theassumption that antidepressant like effect ofextract is specific. In forced swim test werecorded the immobility period of animal whenforced to swim. Among the doses studied Cassinealbens at the doses of 200 mg and 800 mg/kgshowed little activity. Whereas we observedsignificant activity at the oral dose of 400 mg/kgas compared with control group. Fluoxetine, aserotonin reuptake inhibitor was used as areference drug in this test. The effect ofhydroalcoholic extract of Cassine albens onimmobility time in FST was comparable withFluoxetine. Hence, it can be proposed thatantidepressant effect shown by Cassine albensmay involve serotoninergic pathway. Tailsuspension test induces a state of immobilityknown as behavior despair in animals and claimedto reproduce a condition similar to humandepression (Dhingra and Sharma, 2006). Thistest is based on the observation that animalsfollowing initial escape oriented movementsdevelop an immobile posture. The tail suspensiontest is less stressful and has greater sensitivity(Bhattamisara et al., 2008). In the presentstudy Cassine albens at a dose of 400 mg/kgshowed significant activity as compared tocontrol group. The elevated plus maze isconsidered to be etiologically valid animal model,because it uses natural stimuli that is the fear ofnovel open space and fear of balancing on arelatively narrow, raised platform that can induce
anxiety in humans (Grundmann et al., 2007).The validity of the EPM test for evaluation ofanxiolytic or anxiogenic effects of drugs hasbeen well documented (Kulkarni and Sharma,1991). Diazepam (1 mg/kg) was used as referencestandard in EPM test which significantly increasedthe number of entries in open arm. It was alsodemonstrated that the behavioral anxiety in animalon elevated plus maze was indirect correlationwith decrease the number of GABA andBenzodiazepines receptor in cerebral cortex inradioligand studies (Kulkarni and Sharma, 1991).In present study Cassine albens at 400 mg/kg and800 mg/kg showed increase in number of entriesin open arm. Hence it may postulate that theanxiolytic activity of hydroalcoholic extract mayinvolve GABAergic pathway. However detailedinvestigations are needed to confirm thismechanism. In marble burying test Cassine albensat the doses of 400 mg/kg and 800 mg/kg showeddecrease in number of marbles buried by animal.The marble burying test is a valid model forobsessive compulsive disorders which isconsidered as one of the type of anxiety. Anxiolyticagents such as diazepam have been shown toreduce burying, behavior indeed antidepressants;particularly SSRIs are effective in the treatmentof obsessive compulsive disorders.
ConclusionIn present study, hydroalcoholic extract
of Cassine albens shown significant activity instress evoked animal models of depression andanxiety in rodents. The results of present studysubstantiate the folk use of Cassine albens in thetreatment of central nervous system relatedailments. Further detailed investigations areneeded to elucidate the exact mechanism ofCassine albens behind its antidepressant andantianxiety effects.
References1. Aleksandra, S., Cuperly, D., White, A. J. and
Anthony, G. M: Total synthesis of the tricyclicskeleton of the natural Celastraceaesesquiterpenoids and related synthetic analogs.Tetrahedron. 63(26): 590-5917 (2007).
Patil, Gagarani and Surana50
2. Anjaneyulu, A. and Narayanrao, M:Elaeodendrol and elaeodendradiol new nor-triterpene from Elaeodendron glaucum,Phytochemistry. 19(6): 1163-1169 (1980).
3. Bhattamisra, S. B., Khanna, V. K., Agrawal,A.K., Singh, P.N. and Singh, S. K:Antidepressant activity of standardized extract ofMarsilea minnuta Linn. Journal ofEthnopharmacology. 117: 51-57 (2008).
4. Dhingra, D. and Kumar, V: Pharmacologicalevaluation for antidepressant –like activity ofAsparagus racemosus wild in mice.Pharmacologyonline. 3: 133-152 (2007).
5. Dhingra, D. and Sharma, A: Antidepressant –like activity of Glycyrriza glabra L. in mousemodel of immobility test. Progress in Neuro-psychopharmacology and Biological Psychiatry.30: 449-454 (2006).
6. Goodman, S. L. and Gilman, A: ThePharmacological Basis of Therapeutics. 10th
edition. Mcgrew Hill Publisher, Medical Division.304-312 (2001).
7. Grundmann, O., Nakajima, J., Seo, S. andButterweck, V: Anti-anxiety effect of Apocynumvenetum L. in the elevated plus maze. J ofEthnopharmacology. 110: 406-411 (2007).
8. Herrera-ruiz, M., Garcia-beltran, Y., Mora,S., Diaz-veliz, G., Viana, S. B., Tortoriello, J.and Ramirez, G: Antidepressant and anxiolyticeffect of hydroalcoholic extract from Salviaelegans. J of Ethnopharmacology. 107: 53-58(2006).
9. Kasper, Braundwald, Fauci, Houser and Longo:Harrison’s Principle of Internal Medicine. 16th
edition. Mcgrew Hill Medical Publishing Division.New York (2001).
10. Kirtikar, K. R. and Basu, B. D: Indian MedicinalPlant. International Book Publisher, Deharadun.Vol. I: 580-581 (2005).
11. Kulkarni, S. K. and Sharma, A. C: ElevatedPlus- Maze: A novel paychobehavioural tool tomeasure anxiety in rodents. Meth Find Exp ClinPharmacol. 13(8): 573-577 (1991).
12. Laurent, B. N., Yeter K. and Eric, P. M: Acombined marble burying – locomotor activity inmice : A practical screening test with sensitivity to
042_2008
Address for correspondence: Prof. P. H. Patil, Head, Department of Pharmacology, R. C. Patel Institute ofPharmaceutical Education and Research, Shirpur - 425405 Dhule, M.S. (India). E-mail: [email protected]
different classes of anxiolytics and antidepressant.European Journal of Pharmacology. 547: 106-115(2006).
13. Matsushita, M., Egashira, N., Harada, S. andOkano, R: Perospiron: a novel antipsychotic druginhibit marble- burying behavior via 5 HT1Areceptor in mice : implication for obsessivecompulsive disorders. J Pharmacol Sci. 99:154-159 (2005).
14. Moscardo, E., Maurin, A., Dorigatti, R.,Champeroux, P. and Richard, S: An optimizedmethodology for the neurobehavioral assessment inrodent. Journal of Pharmacological andToxicological Methods. 56(2): 239-255 (2007).
15. Parinitha, M., Srinivasa, B.H. and Shivanna,M. B: Medicinal plants wealth of local communitiesin some villages in Shimoga District of Karnataka,India. J Ethnopharmacol. 98: 307-312 (2005).
16. Patil, D. A: Flora and Fauna of Dhule and Nandurbardistricts (Maharashtra). Bishen Singh MahendrapalSingh Publisher, Deharadun. 139-140 (2003).
17. Patil, H. M. and Bhaskar, V. V: Medicinal usesof plant by tribal men of Nandurbar district inMaharashtra. Natural Product Radiance. 5(2):125-130 (2006).
18. Rastogi, R. M. and Malhotra, B. N: Compendiumof Indian Medicinal Plants, 1980-1984. CentralDrug Research Institute, Lucknow. 3: 146-147(2001).
19. Shimada, K., Kyuno, T., Uchida, I. and Nmbara,T: Structure of elaeodendrosides B, C, F, G, K andL: A series of cardiac glycoside isolated fromElaeodendron glaucum. Phytochemistry. 24(6):1345-1350 (1985).
20. Singh, A. K., Raghubanshi, A. S. and Singh, J.S: Medical ethnobotany of the tribals of Sonaghatiof Sonbhadra district, Uttar Pradesh, India. Journalof Ethnopharmacology. 81: 31-41 (2002).
21. Turner, R. A: Organization of Screening, Methodsin Pharmacology. Academic Press, Elsevier. Vol. 1:22-41 (1996).
22. Wijeweera, P., Arnason, J. T., Koszycki, D. andMerali, Z: Evaluation of anxiolytic properties ofGotukola- (Centella asiatica) extract and asiaticosidein rat behavioral models. Phytomedicine. 13:668-676 (2006).
J. Res. Educ. Indian Med., Jan. - March, 2012 Vol. XVIII (1) : 51-54 ISSN 0970-7700
SCREENING OF XIMENIA AMERICANA L.FOR IT’S ANTI-IFLAMMATORY ACTIVITYM. SIDDAIAH,1 K.N. JAYA VEERA,2 P. MALLIKARJUNA RAO,3K. YOGANANDA REDDY4 AND C. MADHUSUDHANA CHETTY1*
Annamacharya College of Pharmacy,1 Rajampet, Kadapa, Andhra Pradesh (India)Department of Chemistry,2 Jawaharlal Nehru Technological University, College of Engineering,Ananthapur, Andhra Pradesh (India)International Medical University,3 Sesama Center, Plaza Komenwel, Kulalmpur – 57000 (Malysia)International Science Tech. Research Institute,4 Anathapur, Andhra Pradesh (India)
Abstract: The anti-inflammatory activity of petroleum ether ethyl acetate and methanolicextracts of Ximenia americana L. was investigated in chemical models - using carrageenan inducedpaw oedema. Ximenia americana is the most common plant widely grown in tropic and sub tropicalclimatic conditions and is used in rheumatism and dropsy.1 The preliminary phytochemicalscreening of the plant shows the presence of sterols, terpenoids, alkaloids, tannin, saponin andphenolic compounds3 which are responsible for various pharmacological activities. The leaf extractof the Ximinia americana were used to evaluate anti-inflammatory activity. The result proved thatpetroleum ether and methanolic extract of the plant has anti-inflammatory activity.
Keywords: Ximenia americana, Ximenia spinosa salisb, Anti-inflammatory, Plethysmograph,Carrageenan, Formalin, Paw oedema.
Introduction For many centuries medical treatment has
relied to a large extent on the use of plants.Natural products in current use possess nearlyevery conceivable type of biological activity. Theusage of herbal drugs in various ailments isincreasing in the modern civilization. W.H.O.estimates that four billion people from all overthe world are using herbal medicines. Thediscovery of a vegetable, the active principle andit’s subsequent chemical characterization will helpin synthesis of analogues relating the essentialstructural features for improved therapeuticactivity.
For the present study Ximenia americanawas used. It is also called Ximenia spinosa salisb.The various plant parts like leaf is used asanalgesic, anti-inflammatory in dropsy etc.1 intraditional systems of medicine. The plant isknown to posses antitumer, antimalarial,antileprotic activity.1,2 But so far no report isavailable in the literature regarding the anti-inflammatory activity. Hence the following study
was carried out to evaluate scientifically the anti-inflammatory activity by using carrageenaninduced paw oedema method in albino rats andcompared with the standard drug Diclofenacsodium.
Materials and MethodsPlant Material
Fresh leaves of Ximenia americana wascollected form the surrounding villages of ChintalaDhornala Mandal Prakasham District A.P. (India)in the month of October 2006 and wastaxonomically identified by an eminent botanist,S.V. University and a Specimen Voucher No.1295 is kept for future reference at S.V.University. The plant leaves were washedthoroughly and shade dried and powdered inmechanical grinder and stored in air tightcontainers for further studies.
Preparation of ExtractsThe shade dried powder of Ximenia
americana was subjected to successive extraction
1. Research Scholar 2. Director 3. Senior Lecturer 4. Research Associate 1* Principal
Siddaiah et al.52
using solvents of increasing polarity likepetroleum ether, ethyl acetate and methanol usingsoxhlet apparatus. The solvent was removed afterthe completion of extraction under reducedpressure.
Animals usedWister albino rats (150-200g) were used
for the present study. The animals were kept inhealthy constant temperature (22±2°C), humidity(55%) and light-dark conditions (12/12 light/dark)and provided with standard pellet diet (HindustanLever) and water ad libitum. The experimentswere performed under the guidance of animal’sEthical committee of Annamacharya College ofPharmacy, Rajampet, Kadapa, Andhra Pradesh,India (Registration No: 122/ a/ CPCSE/ACP).
Chemicals and DrugsShade dried leaf powdered Ximenia
americana petroleum ether AR, ethyl acetatemethanolic AR and methanol (S.D. finechemicals), soxlet apparatus, carrageenan,Diclofenac sodium and plethysmograph wereused as received.
Phytochemical ScreeningPreliminary phytochemical screening of
petroleum ether, ethyl acetate and methanolextracts were tested for the presence ofcarbohydrates steroids flavonoids, tannins,alkaloids glycosides, phenolic compounds,terpinoids, saponins resins, proteins and aminoacids by using standard procedure as describedby Kandalwal (2003).4
Statistical AnalysisThe Experimental results were expressed
as the mean +SEM. N= 6 animals in each group.Statistical significant test for comparision wasdone by using unpaired ‘t’ test.
Acute toxicity studyWister albino rats weighing 200-250gm
were choosen for the present study. They weredivided into 6 groups of six animals each andthen they were fasted for 24 hours prior to
treatment with all the extracts of Ximeniaamericana leaf. There after the test extract wasadministered orally as suspension in tween 80(3ml of 1% solution) to different groups inincreasing dose level of 10,100, 1000,4000, 6000mg/kg body weight.
The animals were then observedcontinuously for three hours for generalbehavioral nemological, anatomical profiles,atoxia, diarrhea or diuresis and for every 30minutes for next three hours and finally deathafter 24 hours.
Anti-inflammatory activityCarrageenan induced paw oedema
Anti-inflammatory activity of petroleumether ethyl acetate and methanolic extracts wasdetermined by carrageenan induced paw oedemausing plethysmograph.5 Albino rats of either sexweighing 150-200gm, were divided into elevengroups and each group consists six animals.Group I served as control and received 3ml oftween 80 in normal saline. Group XI served asstandard and received 10mg/kg of body wt. ofDiclofenac sodium orally. Group III, IV, V VI,VII, VIII, IX and X, served as test and received200, 400 and 600 mg/kg of body wt. of petether ethyl acetate and methanolic extractsrespectively. Tween 80 in normal saline,Diclofenac sodium, extracts were administeredone hour before the carrageenan administration.6Carrageenan 1% w/v, in the normal saline wasinjected into sub-plantar region of the left hindpaw of all groups of animals and right hind pawserved as control. The volume of hind oedemawas measured by plethysmograph at after meanincrease in paw volume and % inhibition ofinflammation were calculated. The percentinhibition of inflammation produced by theextracts of Ximenia americana was comparedwith the standard. Percentage inhibition wascalculated by using the following formula.
Percentage of inhibiton = 100 (1-Vt/Vo)
Where Vt=volume of the extract treatedanimals: Vo= volume of the control (saline).
Anti-inflammatory Activity of Ximenia americana 53
ResultsPhytochemical screening
Preliminary phytochemical screening ofpetroleum ether and methanol extracts indicatesthe presence of alkaloids, carbohydrates,flavonoids, glycosides, terpinoids, steroids,tannins. Amino acids and resins were absent.The results were presented in the Table 1.
Acute toxicity studyIn acute toxicity study no mortality was
observed during the 24 hours period at the doses
tested and the animals showed no stereotypicalsymptoms associated with toxicity, such asconvulsions, ataxia, diarrhea or increased diuresisand the results were shown in Table 2.
Carrageenan induced paw oedemaIn carrageenan induced paw oedema
method,5 the oral administration of petroleumether ethyl acetate and methanolic extract ofXimenia americana at dose of 600 mg/kgproduced significant reduction in paw volumewhen compared with control. The maximumeffect was seen in the oral dose of 600 mg/kgb.w which showed significant. The anti-inflammatory activity in this dose of the testdrug was comparable to standard, Diclofenacsodium (10mg/kg). The maximum anti-inflammatory effect was observed at 4th hour inall the doses of test drug.
The yield value was maximum with ethylacetate extract and methanolic and less with pet.
Table 1. Results of qualitative tests for phytoconstituents of
Ximenia americana leaf.
Sl.
No
Tests Pet.
Ether
Ethyl
Acetate
Methanol
Alkaloids 1
a) Dragendorff’s
test
b) Hager’s test
c) Wagner’s test
d) Mayer’s test
-ve
-ve
-ve
-ve
-ve
-ve
-ve
-ve
+ve
ve
-ve
+ve
Carbohydrates 2
a) Anthrone test
b) Benedict’s test
c) Fehling’s test
d) Molisch’s test
+ve
+ve
+ve
+ve
+ve
+ve
+ve
+ve
++ve
++ve
++ve
++ve
3 Flavanoids
a) Shinoda’s test +ve +ve ++ve
Glycosides 4
a) Molisch test -ve +ve ++ve
Triterpenoids 5
a) Liebermann –
Burchard test
+ve +ve ++ve
6 Resins -ve -ve
7 Saponins -ve +ve -ve
Steroids 8
a) Liebermann
Burchard’s test
b) Salkowski
reaction
+ve
+ve
+ve
+ve
++ve
9 Tannins +ve +ve ++ve
Proteins 10
Biuret test +ve +ve ++ve
11 Amino acids -ve -ve -ve
++++vvee == PPrreesseenntt ,, ++ == TTrraacceess,, --vvee == aabbsseenntt
Table 2. Toxicity study of pet. ether, ethyl acetate and
methanolic extract of Ximenia americana leaf.
Number of
Animals
Sl.
No.
Treatment Dose
mg/kg
body weight * ** ***
% LD50
Value
1 Control 1 % Tween
80
6 6 0 0 ---
2 MEXMA 10 6 6 0 0 ---
3 MEXMA 100 6 6 0 0 ---
4 MEXMA 1000 6 6 0 0 ---
5 MEXMA 4000 6 6 0 0 ---
6 MEXMA 6000 6 6 0 0
7 EEXMA 10 6 6 0 0 ---
8 EEXMA 100 6 6 0 0 ---
9 EEXMA 1000 6 6 0 0 ---
10 EEXMA 4000 6 6 0 0 ---
11 EEXMA 6000 6 6 0 0
12 PEXMA 10 6 6 0 0 ---
13 PEXMA 100 6 6 0 0 ---
14 PEXMA 1000 6 6 0 0 ---
15 PEXMA 4000 6 6 0 0 ---
16 PEXMA 6000 6 6 0 0 >6.0gm/kg
B.W
* = No of Animals; ** = No.of Survived; *** = No.of Death; % = % of Mortality
Siddaiah et al.54
ether extract. Pharmacological studies of Ximeniaamericana revealed that the % inhibition ofinflammation for both methanolic and ethylacetate extracts were remarkable i.e. 60% and60% at 4th hour respectively. The comparisonbetween petroleum ether, ethyl acetate andmethanolic extracts with the Diclofenac sodiumare represented in Table 3.
DiscussionAfter the observations of the results
obtained on carrageenan induced rat paw oedema.
It was concluded that the Ximenia americanapossess considerable anti-inflammatory activity inall extracts. Our pharmacological studies explainsthe use of Ximenia americana leaf extract as ananti-inflammatory agent. This gives valuableinformation regarding the treatment of anti-inflammatory condition with lesser side effects andtoxicities, which are encountered in conventialpain killer drugs. The common adverse effects ofconventional pain killer are ulcer, hepatotoxicityand nephrotoxicity on prolonged use in conditionlike arthritis etc. These adverse effects can beminimized by using the extracts of Ximeniaamericana. Inhibition of carrageenan induced pawoedema in rats is one of the most suitable test toscreen anti arthritic and anti-inflammatory agentsas it closely resembles human arthritis.
References1. Ogunleye DS and Ibitoye SF: Trop. J. Pharm.
Res. 2(2): 239-241 December (2003).2. Madhava Chetty K, Sivaji K and Tulasi Rao
K: Flowering Plants of Chittoor District A.P. India.1st edition. Student Offset Printers, Tirupathi.pp 85 (2008).
3. VSSC. E. YME, Berger MR. PMID: 16005923(Pub. Med-indexed for MEDLINE)
4. Khandelwal KR: Practical Pharmacognsoy.10th edition. Nirali Prakashan, Pune, India (2003).
5. William M Carey, Jeevan Manibabu D,Venkata Raju N and Krishna Mohan G: J. ofPharmacy and Chemistry. Vol. 2(3): 133-138(2003).
6. Shetty SC, Bhagat VC, Kore KJ and SheteRV: Indian Drugs. 45(3) March (2008).
7. The Wealth of India: Vol. XI X-2 CSIR,pp 6 – 8 (1976).
8. Malaya Gupta, Upal Kanti Mazumder,Ramanathan, Sampath Kumar and ThangavelSiva Kumar: Iranian J. of Pharmacology andTherapeutics (IJPT). Vol. 2(2) (2003).
9. Paradesi Goldee, Gadgoli Charya, VaidyaMadhava D, et al.: Pharmacologyonline.1: 111-116 (2008).
10. Goverdan P and Bobbale Diwakar: Ethno.Botanical Leaflets. 13: 65-72 (2009).
11. Yasodha Krishna Janapati, Jayaveera K N,et al. : J. Pharm. and Chemistry. Vol. 2: 156–160July (2008).
Table 3. Anti-inflammatory activity of the petroleum ether,
ethyl acetate, methanolic extract Ximenia americana
in carrageenan induced paw oedema method.
Group Treatment
Design
Dose
(mg/kg
/b.w/p.o.)
Oedema
volume(ml)
Inhibition
(%)
I Control
(Tween 80,
1%)
…… 0.15
± 0.010
……
II PEEXMA 200 0.12
± 0.007
**
20.00
III ...... 400 0.09
± 0.003*
40.00
IV ...... 600 0.07 ±
0.012 *
53.33
V EAEXMA 200 0.12 ±
0.004**
13.34
VI ...... 400 0.09 ±
0.014*
40.75
VII ...... 600 0.06 ±
0.005 *
60.00
VIII MEEXMA 200 0.12 ±
0.010**
20.00
IX ...... 400 0.08 ±
0.006*
46.66
X ...... 600 0.06 ±
0.011 *
60.00
XI Diclofinac
sodium
100 0.05 ±
0.010*
66.67
PEEXMA= Petroleum Eather Extract Ximenia americana; EAEXMA = Ethyl
Acetate Extract Ximenia americana; MEEXMA = Methanolic Extract Ximenia
americana; * p < 0.001 compared to control; ** p < 0.01 compared to control.
Values are mean ± SEM of six animals in each group. Data was analysed by
unpaired ‘t’ test.
Address for correspondence: Dr. M. Siddaiah, Annamacharya College of Pharmacy, Rajampet, Kadapa,Andhra Pradesh (India). E-mail: [email protected]_2009
J. Res. Educ. Indian Med., Jan. - March, 2012 Vol. XVIII (1) : 55-59 ISSN 0970-7700
IN-VITRO ANTIOXIDANT CAPACITY OF GRADED DOSES OFMETHANOLIC EXTRACT FROM LUFFA CYLINDRICA (L) SEEDSK. NAGARAJAN,1 SATYAJIT DUTTA,2 SUMIT DAS,2 SURABHI SINGHAL,3PALLAVI SAXENA,4 AVIJIT MAZUMDER5 AND L. K.GHOSH6
Division of Bio-Medicinal Chemistry, R&D Laboratory, Department of Pharmacy,1,2,4
Department of Microbiology,3 IIMT College of Medical Sciences, ‘O’ Pocket, Ganga Nagar,Mawana Road, Meerut - 250001 U.P. (India)School of Pharmacy,5 Technology and Management, NMIMS University, Mumbai - 400053 M.S. (India)Department of Pharmaceutical Technology,6 Jadavpur University, Kolkata - 700032 W.B. (India)
Abstract: Luffa cylindrica (L) (Family: Cucurbitaceae) commonly called sponge gourd planthave antibacterial, antifungal, anti-HIV and antiasthmatic activity. The seed powder was extractedwith methanol under 60-80°C by continuous hot percolation process using Soxhlet apparatus andthe solvent was removed by distillation under reduced pressure and the residue was stored indesiccator. The total antioxidant activity of the extract was determined with phosphomolybdenummethod using α-tocopherol as the standard. Among the graded doses (1 μg/ml, 2 μg/ml, 3 μg/mland 4 μg/ml) selected for the study, the maximum total antioxidant potency was observed withhigher dose of seed extract of Luffa cylindrica (L) at a concentration of 3 μg/ml which is slightlygreater than the standard α-tocopherol. The antioxidant activity was statistically significant at 1%and 5% level of significance, which clearly indicates the validation for the biological workaccomplished.
Keywords: Luffa cylindrica, Antioxidant, α-tocopherol, Seed extracts.
IntroductionThere is an increased evidence for the
participation of free radicals in the etiology ofvarious diseases like cancer, diabetes,cardiovascular diseases, autoimmune disorders,neurodegenerative diseases, aging, etc.(Bandopadhyay et al., 1999). An antioxidant is amolecule capable of slowing or preventing theoxidation of other molecules which scavenge thefree radicals and prevent the damage caused bythem. They can greatly reduce the damage due tooxidants by neutralizing the free radicals beforethey can attack the cells and prevent damage tolipids, proteins, enzymes, carbohydrates and DNA(Fang et al., 2002). Oxidation is a chemicalreaction that transfers electrons from a substanceto an oxidizing agent. Oxidation reactions canproduce free radicals, which start chain reactionsthat damage cells (Umamaheswari andChatterjee, 2008). Antioxidants terminate these
chain reactions by removing free radicalintermediates and inhibit other oxidation reactionsby being oxidized themselves. As a result,antioxidants are often reducing agents such asthiols, ascorbic acid or polyphenols.
Natural plants have been used in thetreatment of various diseases from the time ofimmemorial the use of plants as a source ofmedicine lies deep in the history of mankind. Awide range of antioxidants from both natural andsynthetic origin has been proposed for use in thetreatment of various human diseases (Cuzzocreaet al., 2001). Loofa is derived from the cucumberand marrow family and originates from America(Mazali and Alves, 2005). Luffa [Luffa cylindrica(L) Rome syn L. aegyptiaca Mill] (Family:Cucurbitaceae) commonly called sponge gourd,loofa, vegetable sponge, bath sponge or dish clothgourd, plant have antibacterial, antifungal, anti-HIV,antiasthmatic, immunomodulatory and antitumor
1. Assistant Professor 2. Lecturer 3. Assistant Professor and Head 4. Research Scholar 5 & 6.. Professor
Nagarajan et al.56
activitiy (Ng et al., 1992). In oriental medicine, L.cylindrica has effect on the treatment of fever,enteritis and swell etc. The extracts from vinesalive are used as an ingredient in cosmetics andmedicine (Lee and Yoo, 2006). Immature fruit isused as vegetables, which is good for diabetes (Balet al., 2004). When extracted and analysed, theseed of L. cylindrica contained 40% oil which isused as oil sources and oil cake obtained is usedas fertilizer and feed (Lee and Yoo, 2006). It hasbeen suggested as an immobilization matrix forplant, algal, bacterial and yeast cells (Iqbal andZafar, 1993 a, b; Iqbal and Zafar, 1994).
The fruits of Luffa cylindrica (L) has beenalready tested for their antioxidant activity withthe radical scavenging effect on the 1,1-diphenyl-2-picrylhydrazyl radical (Du et al., 2006). Thus,this study was designed to evaluate the in-vitrototal antioxidant activity of methanolic extract ofLuffa cylindrica (L) seeds.
Materials and MethodsCollection of plant material
Luffa cylindrica (L) was collected fromthe road sides of Meerut, Uttar Pradesh, India,in the month of January, 2009. The seeds weredried under shade with occasional shifting andthen powdered with a mechanical grinder andstored in an airtight container.
Identification of the plantThe plant was identified and authenticated
by the botanist and a voucher specimen wasdeposited to Dr. Surabhi Singhal, Department ofMicrobiology, IIMT College of Medical Sciences,Meerut, Uttar Pradesh, India (voucher specimenno. IIMT/BD/01/04/2009/02/Tech.2005).
Preliminary phytochemical screeningThe powdered material were subjected to
preliminary phytochemical qualitative screeningby using different organic solvents for thepresence or absence of various primary orsecondary metabolites like alkaloids, glycosides,steroids, terpenoids, flavanoids, carbohydrates
and saponins following standard procedures(Kokate, 2005; Harborne, 1984).
Phytochemical extraction About 120 g of seed powder of Luffa
cylindrica was extracted with 500 mL. methanolunder 60-80°C by continuous hot percolationprocess using Soxhlet apparatus until the solventbecame colorless. After completion of theextraction, the extracts were filtered andevaporated to dryness by Rotary Vacuumevaporator (Buchi Rota vapour, Switzerland) andthe residue was stored in desiccator (Keiichi etal., 1990). The extract was purified by preparativeTLC by using 60% ethyl acetate and 40% hexaneas mobile phase.
Chemicals and solvents usedThe chemicals and solvents used for the
experimental work were procured from E.Merck, India. Silica gel G (30–70 mesh) usedfor chromatography (TLC) was obtained fromE. Merck. Standard α-tocopherol was procuredfrom Cadila Pharmaceuticals, Ahmedabad, India,as a gift sample. All other chemicals and solventsused in the study were obtained commerciallyand were of analytical grade.
Total antioxidant efficacyThe total antioxidant activity of the extract
was determined by phosphomolybdenum methodusing α-tocopherol as the standard(Guddadarangavvanahally et al., 2004;AsokKumar et al., 2009; Jayaprakasha et al.,2002).
The assay is based on the reduction ofMo(VI)-Mo(V) by the extract and a subsequentformation of a green phosphate / Mo(V) complexat acid PH. Different graded doses were preparedwith the extracts (1 μg/ml, 2 μg/ml, 3 μg/ml and4 μg/ml) solution and each was combined with1.0 ml of reagent (0.6 M H2SO4, 218 mM sodiumphosphate and 4 mM ammonium molybdate). Thetubes were capped and incubated in a boilingwater bath at 95°C for 90 minutes. After thesamples had cooled to room temperature, the
Antioxidant Efficacy of Luffa cylindrica Seeds 57
maximal absorption was measured at 695 nmagainst the blank solution in PhotoelectricColorimeter (Systronics-113, India) (Hecker andUllrich, 1989).
ResultsPhytochemical investigation
The results of the preliminary phytochemicalscreening of Luffa cylindrica seeds are expressedin Table 1. From the results of Table 1, it wasconcluded that the methanolic extracts having moreactive constituents like terpenoids and steroids thanthe other active constituents. For the terpenoids,
methanolic seed extracts give highly active resultfor Rochan test whereas for steroids, it givesmoderately active result for Salkovaski test. Noneother organic extracts have shown positive resultfor terpenoids and steroids. Terpenoids present inthe seed extract was further confirmed with TLCanalysis having an hRf value of 84 with 60% ethylacetate : n-hexane and iodine as visualizing reagent.
Total antioxident capacityThe results of the various graded doses of
Luffa cylindrica seed extracts and its totalantioxidant capacity are expressed in Table 2.
Table 1. Preliminary Phytochemical screening of Luffa cylindrica Seeds.
Solvents Alkaloids (Hager’s test)
Glycosides(Borntrager’s
test)
Steroids (Salkovaski
test)
Terpenoids(Rochan test)
Flavonoids(Shinoda test)
Carbohydrates
(Benedict’s
test)
Saponins (Foam test)
Petroleum ether - - - - - - -
Chloroform ++ - - - - ++ -
Benzene ++ - - - - - -
n-Butanol - - - - - + -
Ethanol - - - - - + -
Ethyl acetate + - - - - - -
Methanol - - + ++ - - -
Water - - - - - - ++
++ Highly active; + Moderately active; - Not present
Table 2. Total Antioxidant Capacity of Methanolic extracts of Luffa cylindrica Seeds.
Sl.
No.
Sample
Accession
Number
Sample
Concentration
Used
Absorbance
at 695 nm
Standard Accession
Number &
Concentration Used
for Standard
Absorbance
at 695 nm
Total Antioxidant
Capacity ( g
Vitamin E
equivalent/mg)
1 LCSE1 1 g/ml 0.98 T1 (0.6 g/ml) 0.30 0.0009 IU
2 LCSE2 2 g/ml 1.01 T2 (1.2 g/ml) 0.34 0.0018 IU
3 LCSE3 3 g/ml 1.13 T3 (1.8 g/ml) 0.70 0.0027 IU
4 LCSE4 4 g/ml 1.10 T4 (2.4 g/ml) 0.24 0.0036 IU
LCSE1: Luffa cylindrica Seed Extract (1 g/ml); T1: -tocopherol (standard) (0.6 g/ml);
LCSE2: Luffa cylindrica Seed Extract (2 g/ml); T2: -tocopherol (standard) (1.2 g/ml);
LCSE3: Luffa cylindrica Seed Extract (3 g/ml); T3: -tocopherol (standard) (1.8 g/ml);
LCSE4: Luffa cylindrica Seed Extract (4 g/ml); T4: -tocopherol (standard) (2.4 g/ml);
IU: International Unit, 1mg of Tocopherol= 1.5 IU
Nagarajan et al.58
The maximum total antioxidant potency wasobserved with higher dose of seed extract of Luffacylindrica (L) at a concentration of 3 μg/ml whichis slightly greater than the standard α-tocopherol.
The antioxidant capacity ofphosphomolybdate method is expressed as thenumber of equivalents of α-tocopherol amongthe extracts tested the dose at which theconcentration contains 0.0009 IU, 0.0018 IU,0.0027 IU, 0.0036 IU Vitamin E equivalent/mg.Hence the seed extract (3 μg/ml) was found tohave more pronounced antioxidant potency in-vitro, than the standard drug α-tocopherol used.The antioxidant activity were statisticallysignificant at 1% and 5% level of significance(calculated ‘t’=29.567; calculated ‘F’=20.5104by ANOVA), which clearly indicates thevalidation for the biological work accomplished.
DiscussionThe total antioxidant capacity assessed in
the methanolic seed extract of Luffa cylindricawas proved to be significant therapeutically in-vitro than the standard α-tocopherol with theabove mentioned results of Table 2. This is mainlydue to the presence of terpenoid as predominantbio-active constituent in the plant part with thefindings from qualitative chemical analysis andTLC.
To be discussed as health promoting orbiofunctional, the significant impact of a testedextract was either on human metabolism or onwell-defined and appropriate biomarkers (Wagnerand Elmadfa, 2003). Three main ways ofantioxidant action of carotenoid type terpenoidshave been detected until now (i.e., quenching ofsinglet oxygen, hydrogen transfer, or electrontransfer). Low-density lipoprotein oxidation isbelieved to play an important role in thedevelopment of atherosclerosis disorders(Grassmann, 2005). Several secondary plantmetabolites have been tested for and therefore ahigh resistance of LDL against oxidation mayprevent atherogenesis and accompanying theirability to prevent oxidation of LDL and manyphenolic terpenoids as well as carotenoids have
been shown to enhance LDL oxidation resistance(Milde et al., 2007). Hence the probablemechanism of terpenoid bio constituent in theseeds of Luffa cylindrica may be due to theirability to prevent the oxidation of LDL inperipheral tissues, which has to furtherextensively investigated for cardio-protectiveeffects only with clinical in-vivo studies in nearfuture.
AcknowledgementsThe authors are very much thankful to
Rtn. Yogesh Mohanji Gupta, Chairman, IIMTGroup of Colleges, Meerut and Rtn. AbhinavAgarwaal, Secretary General, IIMT Group ofColleges, Meerut U.P. (India) for their constantencouragement and continuous supportthroughout the project work.
References1. AsokKumar, K., UmaMaheswari, M.,
Sivashanmugam, A. T., SubhadraDevi, V. N.and Subhashiniand Ravi, T. K: Free radicalscavenging and antioxidant activities of Glinusoppositifolius (carpet weed) using different in vitroassay systems. Pharmaceutical Biology. Vol.47(6): 474-482 (2009).
2. Bal, K. E., Bal, Y. and Lallam, A: Grossmorphology and absorption capacity of cell-fibersfrom the fibrous vascular system of Loofah (Luffacylindrica). Textile Res. J. Vol. 74: 241- 247 (2004).
3. Bandyopadhyay, U., Das, A. and Bannerjee, R.K: Reactive oxygen species: Oxygen damage andpathogenesis. Curr. Sci. Vol. 77(5): 658-666 (1999).
4. Cuzzocrea, S., Riley, D.P., Caputi, A.P. andSalvemini, D: Antioxidant therapy: A newpharmacological approach in shock, inflammationand ischemia or reperfusion injury. Pharmacol.Rev. Vol. 53: 135-159 (2001).
5. Du, Q., Xu, Y., Li, L., Zhao, Y., Jerz, G. andWinterhalter, P: Antioxidant constituents in thefruits of Luffa cylindrica (L.) Roem. J. Agric.Food Chem. Vol. 54(12): 4186-4190 (2006).
6. Fang, Y., Yang, S. and Wu, G: Free radicals,antioxidants and nutrition. Nutrition. Vol.18: 872-879 (2002).
7. Grassmann, J: Terpenoids as plant antioxidants.Vitam Horm. Vol. 72: 505-535 (2005).
8. Guddadarangavvanahally, K., Jayaprakasha, Rao,L. J. and Kunnumpurath, K. S: Antioxidant
Antioxidant Efficacy of Luffa cylindrica Seeds 59
activities of flavidin in different in vitro model systems.Bioorganic and Medicinal Chemistry. Vol. 12(19):5141-5146 (2004).
9. Harborne, J.B: Phytochemical Methods. Chapmanand Hall, London, New York (1984).
10. Hecker, M. and Ullrich, V: On the mechanism ofprostacyclin and thromboxane A2 biosynthesis. J.Biol. Chem. Vol. 264(1): 141-150 (1989).
11. Iqbal, M. and Zafar, S. I: The use of fibrous networkof matured dried fruit of Luffa aegyptiaca asimmobilizing agent. Biotechnol. Tech. Vol.7: 15-18 (1993a).
12. Iqbal, M. and Zafar, S. I: Vegetable sponge: A newimmobilizing medium for plant cells. Biotechnol. Tech.Vol. 7: 323-324 (1993b).
13. Iqbal, M. and Zafar, S. I: Vegetable sponge as amatrix to immobilize microbes: a trial study for hyphalfungi, yeast and bacteria. Lett. Appl. Microbiol. Vol.18: 214-217 (1994).
14. Jayaprakasha, G. K., Jena, B. S. and Negi, P.S: Antioxidant activities of polyphenol containingextracts from citrus. J. Naturforsch. Vol. 57C:828-835 (2002).
15. Keiichi, W., Yuji, M. and Gunki, F: Isolation andpartial characterization of three protein synthesisinhibitory proteins from the seeds of Luffa of threeprotein synthesis inhibitory proteins from the seedsof Luffa cylindrica. Agric. Biol. Chem. Vol. 54(8):2085-2092 (1990).
16. Kokate, C.K: Practical Pharmacognosy .Vallabh Prakashan, New Delhi (2005).
17. Lee, S. and Yoo, J. G: (WO/2006/019205) Methodfor preparing transformed Luffa cylindrica Roem(World Intellectual property organization) http://w w w . w i p o . i n t / p c t d b / e n /wo.jsp?IA=KR2004002745&DISPLAY=STATUS.(2006).
18. Mazali, I. O. and Alves, O. L: Morphosynthesis:high fidelity inorganic replica of the fibrousnetwork of loofa sponge (Luffa cylindrica). An.Acad. Bras. Cien. Vol. 77(1): 25-31 (2005).
19. Milde, J., Elstner, E. F. and Grassmann, J:Synergistic effects of phenolics and carotenoidson human low-density lipoprotein oxidation. Mol.Nutr. Food Res. Vol. 51(8): 956-961 (2007).
20. Ng, T. B. and Chan, W. Y: Proteins withabortifacient, ribosome inactivating,immunomodulatory, antitumor and anti-AIDSavtivities from Cucurbitaceae plants. GeneralPharmacology. Vol. 23(4): 575-590 (1992).
21. Umamaheswari, M. and Chatterjee, T. K:In-vitro antioxidant activities of the fractions ofCocciniagrandis L. leaf extract. African Journalof Traditional, Complementary and AlternativeMedicines. Vol. 5(1): 61 – 73 (2008).
22. Wagner, Karl-Heinz and Elmadfa, I: Biologicalrelevance of terpenoids overview focusing onMono-, Di- and Tetraterpenes. Annals of Nutritionand Metabolism. Vol. 47: 95-106 (2003).
Address for correspondence: Dr. K. Nagarajan, Assistant Professor, Division of Bio-Medicinal Chemistry, R&DLaboratory, Department of Pharmacy, IIMT College of Medical Sciences, ‘O’ Pocket, Ganga Nagar, MawanaRoad, Meerut - 250001 U.P. (India). E-mail: [email protected], [email protected]
035_2009
J. Res. Educ. Indian Med., Jan. - March, 2012 Vol. XVIII (1) : 61-63 ISSN 0970-7700
ANTIBACTERIAL ACTIVITY OF ALCOHOLIC EXTRACT OFALOE VERA L. BY DISC DIFFUSION METHODG.. S. NITURE,1 M. K. PATIL,2 A. G. KARPE3 AND A.V. BHONSLE4
Department of Microbiology,1,4 Department of Pharmacology,2College of Veterinary and Animal Sciences, Udgir - 413517 District Latur, Maharashtra (India)
Abstract: The study was carried out to assess antibacterial activity of 50% alcoholic coldextract of Aloe vera against pathogenic strains of E.coli, Staphylococcus aureus, Streptococcuspyogenes, Pseudomonas aeruginosa using extract impregnated disc by disc diffusion method. Thepresent study revealed that 50% alcoholic cold extract of Aloe vera L. (37.52 + 0.77 mg/ disc) waseffective against all the bacteria by disc diffusion method. The study concluded that 50% alcoholiccold extract of Aloe vera L. had significant antibacterial activity but lesser than reference antibioticciprofloxacin.
Keywords: Aloe vera L., Antibacterial activity, Medicinal Plants.
IntroductionAloe vera (Family – Lilliaceae) commonly
known as Grithkumari, Korphad is found in semiwild dry parts of India. Aloe vera possessantibacterial, anthelmentic, purgative, stomachic,cooling, bitter, carminative and digestiveproperties. It is useful in the treatment of eyediseases, tumours, enlargement and inflammationof spleen, liver complaints, vomiting, bronchitis,fever, skin diseases, jaundice, chronic ulcers,muscular pain and constipation (Anjaria, 2002;Rajpal, 2002). The study on antibacterial activityis meager, therefore the present investigation isundertaken to screen the antibacterial propertyof Aloe vera extract.
Materials and Methods1. Preparation of extract
Aloe vera L. leaves was used. The gel wassubjected to drying and the powder was subjectedfor preparation of 50% alcoholic cold extract asper method described by Rosenthaler (1930).
2. Preparation of extract impregnated discThe sterile blank disc was obtained from M/
s. HiMedia Laboratory Ltd. Mumbai, India. Extract
1. M. V. Sc. Scholar, Veterinary Microbiology 2. Assistant Professor, Veterinary Pharmacology3. Associate Dean, KNP College of Veterinary Sciences, Shirwal - 412801 District Satara, Maharashtra (India)4. Assistant Professor, Veterinary Microbiology
impregnated discs were prepared using dissolvedextracts in the 50% alcoholic solvent andimpregnated onto the disc, until the discs get fullyTable 1. Amount of alcoholic extract of Aloe vera
impregnated on to the discs.
Weight (mg) Extract
Blank
discs*
Extract
impregnated
discs*
Extract in
each disc
Cold 65.00
65.00
65.00
327.00
330.00
326.00
37.43
37.86
37.28
Mean
+ SE
65.00
+0.00
327.67
+1.20
37.52
+0.77
* Weight of 7 discs at each time
saturated and was air dried. The extract impregnateddiscs were collectively weighed before and afterimpregnation of the extract. The amounts of theextract actually absorbed onto the disc wererecorded as shown in Table 1.
3. Test organismThe typed pathogenic bacterial culture of
Escherichia coli (MTCC 723), Staphylococcusaureus (MTCC 96), Streptococcus pyogenes(MTCC 442) and Pseudomonas aeruginosa
Niture et al.62
of 50% alcoholic cold extract was 37.52 + 0.77.Table 2 depicts zone of inhibition by 50% alcoholiccold extract of Aloe vera against test bacteria. It isevident that 50% alcohol discs did not have anyantibacterial activities which were used as control.50% alcohol Aloe vera extract discs were effectiveon the test bacteria. Ciprofloxacin discs which wereused as control positive revealed significantantibacterial activity on all the test bacteria.
ConclusionFifty percent alcoholic Aloe vera dry gel
extract exhibited antibacterial activity by discdiffusion method against all test bacteria, whereas50% alcohol control base disc (which was usedto check the residual effect of alcohol) was foundineffective against all test bacteria. Fifty percentalcoholic Aloe vera dry gel extract showedComparatively lesser antibacterial activity thanCiprofloxacin. After further study, dose ofCiprofloxacin may be reduced to half in thetreatment of diseases caused by S. aureus,S. pyogenes, E. coli and P. aeruginosa by usingAloe vera extract preparation, thereby promotinguse of herbal drugs and reducing side effects ofchemical drug on animal body.
AcknowledgementsThe authors are thankful to Associate Dean,
College of Veterinary and Animal Sciences, Udgir,M.S. (India) for providing necessary facilities toundertake the work.
(MTCC 741) were obtained from Microbial TypedCulture Collection (MTCC), Institute of MicrobialTechnology, Chandigarh (India). The pathogenicbacterial cultures were sub cultured and maintainedon nutrient agar (MM 012) and in nutrient broth(M 088).
4. Determination of antibacterial activityThe antibacterial effectiveness of 50%
alcoholic cold extract of Aloe vera L. wasdetermined by disc diffusion method (Baueret al., 1966).
Disc Diffusion TestEach of the four bacterial culture viz.,
Escherichia coli Staphylococcus aureus,Streptococcus pyogenes, Pseudomonas aeruginosawas grown in nutrient broth at 37oC for overnightincubation. The bacterial culture was diluted10-3, 10-4, 10-5, 10-6 using sterile nutrient broth.Each dilution of four bacteria was plated onPetri- plates aseptically. The extract impregnateddiscs Aloe vera L. were placed in Petri-platescontaining diluted four bacterial cultures onnutrient agar with the help of sterile forceps andincubated at 37oC. After 24 hours of incubationthe Petri-plates were examined for zone ofinhibition, measured in mm scale as shown inTable 2. The data of this investigation werestatistically analyzed by Student-t-test (Snecdorand Cochran, 1968).
Results and DiscussionThe weight of the extract adsorbed on
each disc is depicted in Table 1. The adsorption
Table 2. Zone of inhibition by 50% alcoholic cold extract of Aloe vera L. against test bacteria.
Sensitivity pattern Sr.
No. Extract discs Staphylococcus
aureus
Stereptococcus
pyogenes
E.coli Pseudomonas
aeruginosa
1 50 % Alcohol No zone No zone No zone No zone
2 50 % alcohol Aloe
vera extract discs ++ ++ ++ ++
3 Ciprofloxacin ++++ ++++ ++++ ++++
+: Less sensitive (Zone < 10 mm) ++: Sensitive (Zone between 10 – 15 mm)
+++: Moderately sensitive (Zone between 15 – 20 mm) ++++: Highly sensitive (Zone > 20 mm)
Antibacterial Activity of Aloe vera 63
References1. Anjaria, J: Inventory of Traditional Medicinal
Practices in India. Ministry of Agriculture, Govt.of India. pp. 194 (2002).
2. Bauer, A.W., Kirby, M.M., Sherris, T.C. andTruck, M: Antibiotic susceptibility testing bystandardized single disc method. Am J. Clin.Pathol. 45: 493-496 (1966).
3. Nadkarni, K.M. and Nadkarni, A.K: MedicinalPlants Indian Materia Medica. Vol. 1. Popular
Prakashan Private Limited, Bombay, India. pp. 28(1976).
4. Rajpal, V: Standardization of Botanical Testingand Extraction Method of Medicinal Herbs.Eastern Publisher, New Delhi. pp. 11-20 (2002).
5. Rosenthaler, L: The Chemical Investigation ofPlants. 1st Edn. Bell and Sons, London. pp. 36 (1930).
6. Snedecor, G.W. and Cochran, W.G: StatisticalMethods. Oxford and IBH Publication Co.,Calcutta. pp. 534 (1968).
Address for correspondence: Dr. G. S. Niture, Niture Niwas, Near H.S. College, Dam Road, Udgir - 413517Dist. Latur, Maharashtra (India). E-mail: [email protected]
031_2009
Conferences and Forthcoming Events JREIM: ISSN 0970-770064
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Theme: LIFESTYLE DISORDERS - A CHALLENGE
Date : 4th February 2012Vanue : Shri Shri Ravi Shanker Vidya Mandir, Una.
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