Journal Leaf Extract of Allium Stracheyi

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Journal of Applied Sciences Research, 6(2): 139-143, 2010 © 2010, INSInet Publication Anti-inflammatory and Analgesic Potential of Leaf Extract of Allium Stracheyi 1 Shashi Ranjan, 1 Vikash S. Jadon, 1 Nitish Sharma, 1 Kamini Singh, 1 Varsha Parcha, 1Sanjay Gupta and 2J.P. Bhatt Sardar Bhagwan Singh Post Graduate Institute of Biomedical Sciences and Research, Balawala, Dehradun, Uttarakhand, India, 248161 2 Department of Zoology and Biotechnology, HNB Garhwal University, Srinagar, Uttarakhand
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  Journal of Applied Sciences Research, 6(2): 139-143, 2010© 2010, INSInet Publication Anti-inflammatory and Analgesic Potential of Leaf Extract of   Allium Stracheyi  Shashi Ranjan, Vikash S. Jadon, Nitish Sharma, Kamini Singh, Varsha Parcha, Sanjay Gupta 111111 and J.P. Bhatt 2 Sardar Bhagwan Singh Post Graduate Institute of Biomedical Sciences and Research, Balawala, 1 Dehradun, Uttarakhand, India, 248161Department of Zoology and Biotechnology, HNB Garhwal University, Srinagar, Uttarakhand, India 2 Abstract:  Allium stracheyi, a medicinal plant found at the height of 2500-3000 meters of AlpineHimalayas of Uttarakhand, India is used as a spice and in ethano-medicine by local people. In the presentstudy,  Allium stracheyi leaves were explored for their anti-inflammatory and analgesic potential onexperimental model and compared to standard drugs. The results showed that methanolic extract hassignificant reduction in inflammation i.e. 61% (100mg/kg) (p< 0.5) as compared to the standard drugDiclofenac sodium suspension in 0.1% Tween 80 (10 mg/kg body weight) as well as the highest analgesic potential i.e. 64.62% (100mg/kg) as compared to aspirin 68.62 % at 25 mg/kg body weight. Thus,methanolic extract of the plant can be fully explored for its anti-inflammatory and analgesic potentialwhich has not been reported so far. The plant extract showed a relative low toxicity hence justifies thefolkloric use of plant by the local people in Western Himalayan region for curing inflammation and painful conditions. Key words:  Allium stracheyi , anti-inflammatory activity, analgesic activity, Phytochemical screening INTRODUCTION The importance of medicinal plants in traditionalhealthcare practices, providing clues to new areas of research and in biodiversity conservation is now wellrecognized. The use of traditional medicine andmedicinal plants in most developing countries, as anormative basis for the maintenance of good health has been widely observed. However, information on theuses for plants for medicine is lacking from manyinterior areas of Himalayan regions in India. Utilizationof plants for medicinal purposes in India has beendocumented long back in ancient literature. However,organized studies in this direction were initiated in1956 and off late such studies are gaining recognitionand popularity due to loss of traditional knowledge anddeclining plant population.Drugs which are in use presently for themanagement of pain and inflammatory conditions areeither narcotics or non-narcotics (NSAIDS) and presentwell known side and toxic effects. On the contrary [1,2,3] herbal medicines with good absorption, less toxicityand easy availability have been used since long. It is [4] therefore essential that efforts should be made tointroduce new medicinal plants to develop cheaper andeffective drugs. Plants represent still a large [5,6] untapped source of structurally novel compounds thatmight serve as lead for the development of noveldrug. [7]  Allium stracheyi (Jambu)   is a perennial herb,flowers are white in color about 35-40 centimeters inheight and is traditionally being used by the local people as spice for flavoring. It is mainly found at theheight of 2500-3000 meters of Alpine Himalayas of Uttarakhand, India near moist rock, dry rock and steepslope with a strong preference of sunny site. Edible plant part used includes flowers, leaves, root and bulb.The leaf and bulb parts of this plant are used locally inthe alleviation of inflammation and painful conditions. [8] Leaves and inflorescences are also used as seasoningagents. Although no specific mention of medicinal useshas been for this species, member of this genus are ingeneral very healthy additions to the diet. They containsulphur rich compounds with antioxidant, anti-inflammatory, and antimicrobial properties. Thus, in the present study attempts were made to investigate itsanti-inflammatory and analgesic potential with a viewof justifying the use of this plant in treatment of inflammatory disease and analgesic locally. MATERIALS AND METHODSCollection of Plant Materials: Whole plant of   Allium stracheyi was collected from various parts of  Corresponding Author: Shashi Ranjan, Sardar Bhagwan Singh Post Graduate Institute of Biomedical Sciences and Research,Balawala, Dehradun, Uttarakhand, India, 248161E-mail: shashiranjan16@gmail.com, Mobile: +91-9997581583139   J. Appl. Sci. Res., 6(2): 139-143, 2010 Uttarakhand, India during August to September 2008.The seed samples were submitted for conservation at National Gene bank, National Bureau of Plant GeneticResources, Pusa Campus, New Delhi, India and thenational identity number (SR-01-IC 567645) wasobtained. The leaves of this plant were dried under shade at 27 C - 30 C for 15 to 30 days, after which °° the leaves of the plant were chopped and grounded intocoarse powder. Preparation of Plant Extract: The coarse powder of material (200gm) was successively extracted withsolvents in order of increasing polarity like petroleumether, chloroform, methanol and water using hot soxhletextractor. The resulting extracts were concentrated byvacuum evaporator keeping the maximum temperature48 C to 50 C. °° Phytochemical Screening: The presence of various phytochemical constituents in the extract wasdetermined using standard screening tests. [9] Experimental Animals: Wistar rats (150-200g) andSwiss Albino Mice (25-40g) of either sex were usedfor this study. The animals were obtained from theanimal house of the Department of Pharmacy, Sardar Bhagwan Singh (P.G) Institute of Biomedical Sciencesand Research, Dehradun, Uttarakhand, India. Theanimals were maintained under standard environmentalcondition and had free access to food and water beforeadministration of plant extract 50 Acute Toxicity study (LD): The intraperitoneal (i.p)acute toxicity of the Methanolic extract was evaluatedin Swiss Albino Mice by method described byLorke. The method involved the determination of  [10]50 LD value in biphasic manner. The animals werestarved of feed but allowed access to water 24 hours prior to the study. In the initial investigatory step(phase 1), a range of doses of the extract producing thetoxic effects was established. This was done byintraperitoneal administration of widely differing dosesof the extract (50, 100, 500, 1000, 1200mg/kg i.p) tofour groups of mice (of four each). Based on the resultobtained, a phase II investigatory step was done bygiving more specific doses (350, 400, 450, 500 mg/kgi.p) to four other groups of mice.The mice were observed for 24 hours for such behavioral signs as nervousness, excitement, dullness, 50 ataxia or death. The LDwas estimated from the   geometric mean of the dosethat caused 100% mortalityand the dose which caused no lethality. Anti-inflammatory Activity: Anti-inflammatoryactivity of   Allium stracheyi was evaluated bycarrageenan-induced rat paw edema method. Twenty [11] five albino rats of either sex were taken and dividedinto 5 groups, each group contained 5 rats. Group I    – received Tween 80 solution (0.1%) in a volume of 10ml/kg body weight, Group II- received Diclofenacsodium suspension in 0.1% Tween 80 (10 mg/kg bodyweight), Group III- received Petroleum ether extractsuspension in 0.1 % Tween 80 (100mg/kg bodyweight), Group IV - received aqueous extractsuspension in 0.1 % Tween 80 (100mg/kg bodyweight)   Group V    – received methanol extractsuspension in 0.1% Tween 80 (100mg/kg bodyweight) in a volume of 10ml/kg, half hour after carrageenan (0.1 ml of 0.1 %) administration in the sub plantar region of left hind paw of each rat. They weredeprived of water during experiment to ensure uniformhydration and to minimize variability in oedematousresponse (Winter et al. 1962). The measurement of the paw volume (cm) was done on the principle of  3 volume displacement using LETICA digitalPlethysmometer. The readings were taken before and at30 min intervals after the injection of carrageenan for a period of 4 hrs. The edema at each time wascalculated in relation to the paw volume before theinjection of the carrageenan.The anti- inflammatory activity was determined asthe percentage of inhibition of inflammation after itwas induced by carrageenan by taking volume of inflammation in control group as 100%. The percentageinhibition was calculated by using the formula:% Inhibition =Mean paw inflammation of control – Mean paw inflammation of test / Mean pawinflammation of control x 100. Acetic Acid-induce Writhing in Mice: The analgesicinvestigation was carried out according to themethod. The mice were divided into six different [12] groups (of four mice each). They were differently pre-treated with the different extract (100mg/kg i.p), aspirin(100mg/kg i.p) and Control normal saline (10ml/kg i.p)30, 60, 90 and 120 min after the treatment, 1% aceticacid solution was administered to the mice(1ml/100gm, i.p). They were placed in a transparentobservation box. Five minutes after the administrationof acetic acid the number of abdominal constrictions(writhes) made within 20 minutes of every mouse wascounted. The results of the treatment groups werecompared with those of normal saline pre-treatedcontrol. The percentage of the writhes was calculatedas:% of writhes = (Test mean/Control mean) x 100.140   J. Appl. Sci. Res., 6(2): 139-143, 2010 Statistical Analysis: The calculation of the averageedema for the anti-inflammation and percentagewrithing reflex for anti-nociception were based on theexpression of numerical data as mean ± SEM. Thestatistical significance between groups were analyzedusing two-way analysis of variance (ANOVA),followed by students t-test. p values < 0.5 were takento be significant. Results:Phytochemical Screening: The results of qualitativeanalysis indicated that petroleum ether and chloroformextract is rich in steroids while methanolic and aqueousextract is rich in alkaloid and saponins (Table 1). Anti-inflammatory Activity: In the study the pro-inflammatory effect was observed for methanol, petroleum ether and aqueous decoction against thestandard Diclofenac sodium. Out of the three extractsMethanolic extract showed significant percent reduction(61%) (p<0.5) as compared to the diclofinac sodiumthat showed 72% inhibition of paw volume (Figure 1). Non-significant inhibition was observed for petroleumether extract and aqueous extract with 46% and 20%inhibition when the dose administered was 100mg/kg. Acetic Acid-induced Writhing: The result representedin Figure 2, shows that all the extracts of   Allium stracheyi at the doses of 100mg/kg and aspirin25mg/kg exhibited significant (p<0.5) inhibition of thecontrol writhes at the rate of 68.62%, 55%, 64.62%and 38.47 for aspirin, petroleum ether extract,methanolic extract and aqueous extract respectivelywhen compared to that of control.In addition, extracts of   Allium stracheyi at theabove mentioned doses, potentiated analgesic activityof aspirin shown by further decreasing the writhingresponse when given in combination. Statistical analysisshowed that Methanolic extract has highest analgesic potential than petroleum ether extract of 64.62% of inhibition as compared to the standard aspirin that gave68.62% inhibition. 50 Acute Toxicity Study (LD): Mice treated with doses $ 500 mg/kg i.p. were dull, panted, showed occasionalabdominal stretching and died within 24h of treatment.The adverse signs and deaths were however, not seen 50 at doses below 500mg/kg i.p. The LD of Methanolicextract of   Allium stracheyi was estimated to be 445.5mg/kg i.p. Discussion: The presence of alkaloids andorganosulphur compounds, which has been indicated inanalgesic and anti-inflammatory activities, supports thetraditional and local use of   Allium stracheyi . The 50 revelation of the median lethal dose LD of theMethanolic extract of   A. stracheyi to 445.5 mg/kg i.pis probably an indicator that the extract may be 50 relatively safe where as estimated LDvalue > 1g isconsidered safe. This is of importance because a [10] cumulative toxic effect could not occur if the extract istaken over time. In the present study, the abdominalconstriction (Writhing) model adopted is thought to partly involve local peritoneal receptors. The [13, 14, 15] ability of the methanolic extract to cause a significantreduction in the number of acetic-acid induced writhesin mice probably suggests an anti-nociceptive property.The use of abdominal constriction (writhing) model isknown to be very sensitive when compared with other models such as tail flick model. This study also [14, 16] showed that anti-nociceptive effect of the aqueous andchloroform extract showed very less effect in sameduration of time as compared to methanolic extract.The result obtained complimented the earlier investigation that methanolic extract of   Allium speciescontains copious amount of flavonoids, organosulphur compounds.Previous study on different  Allium [17] species also shows that these flavonoids andorganosulphur compounds are potent analgesic and anti-inflammatory agents. These compounds prevent theformation of pro-inflammatory messengers. This is inline with some other reports that some metabolites areas potent or even more potent in some activities as the parent compound.The carragenan induced edema model showed theanti-inflammatory property of the extract. Although themechanisms of action for the anti-nociception and anti-inflammation are not yet elucidated, the combination of  both effects can be taken advantage of, therapeutically.Significantly (p<0.5) high anti-inflammatory activity of methanolic extract (100 mg/kg body weight) of   A. stracheyi may be due to inhibition of the mediators of inflammation such as histamine, serotonin and prostaglandin. Thus, the research work justified thetraditional use of the plant as a spice in foodsupplements and in the treatment of pro-inflammatorydisease. Further studies will be carried out on pharmacodynamics pattern to establish the mechanismof the action of the plant extracts. ACKNOWLEDGMENTS The authors wish to acknowledge the financialsupport of The Director, Uttarakhand State Council of Science and Technology, Dehradun, Uttarakhand, India.The authors would also like to express their gratitudeto The Management, Sardar Bhagwan Singh PGInstitute of Biomedical Sciences & Research, Balawala,Dehradun, India, for providing research facilities toconduct the work.141   J. Appl. Sci. Res., 6(2): 139-143, 2010 Table 1: Phytochemical analysis of different extracts of Allium stracheyiTest performedPetroleum ether extractChloroform extractMethanol extractAqueous extract1.Test for steroids---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------i.Salkowski test(+)(+)(-)(-)---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ii.Gilberman-Buchard's test(+)(+)(-)(-)---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------2.Test for alkaloids---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------i.Wagner's test(-)(-)(+)(+)---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ii.Hager's test(-)(-)(+)(+)---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------3.Test for phenolic and flavonoid compounds---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------i.Vanilin-HCL test(-)(-)(+)(+)---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ii.Ferric chloride test(+)(+)(+)(-)---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------iii.Zinc hydrochloric acid reduction test(-)(-)(-)(-)---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------4.Test for tannins(-)(-)(+)(+)---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------5.Test for saponins(-)(-)(+)(+) Fig. 1: Anti-inflammatory activity of different extract of   Allium    stracheyi in Carrageenan induced paw edema inrat compared to the same point in control group (Normal saline)  Fig. 2: Analgesic effect of different extract of   Allium    stracheyi (100mg/kg, ip) & Aspirin (25mg/kg ip) in 1%acetic acid induced paw licking in rat compared to the same point in control (Saline)142
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